Voltage-gated potassium currents in cultured ovine oligodendrocytes

Cultured oligodendrocytes (OLGs) develop processes and form myelin following attachment to a substratum. We applied the whole-cell voltage-clamp technique to identify and characterize the ionic currents of OLGs in culture. Within 2 d after attachment, OLGs extended processes and began to express an outward current that represents a composite response of an inactivating/transient component and a non-inactivating component. The current had a reversal potential of -66 mV and was sensitive to potassium channel blockers. After 4-5 d in culture, the transient component was less prominent, often accompanied by an increase in noninactivating or steady-state outward current. In addition, there was an increase in inward rectifier current. Four of 7 cells that failed to develop processes exhibited only linear high-resistance membranes. We conclude that cultured OLGs express 3 voltage-gated potassium conductances: (1) a transient outward current, (2) a noninactivating outward current, and (3) an inward rectifier current. The sequential appearance of the several currents may relate, at least in part, to process formation.     

Serotonin Induces a Cyclic AMP-Mediated Outwardly Rectifying Slow K+ Current in a Single Identified Snail Neuron

In the F2 neuron of the parietal ganglion of the snail Helix aspersa either bath or iontophoretic application of serotonin (5-HT) induces an outward current. This current has a long latency (10 - 60 s) and a slow time course, a 500 ms iontophoretic application of 5-HT evoking a response lasting 3 - 5 min. This slow outward current reverses at -80 mV, a value equal to EK. After doubling the extracellular K+ concentration the reversal potential of the 5-HT response is shifted by 19 mV, as predicted by the Nernst equation. The I-V curves reveal that the 5-HT-induced slow outward current is outwardly rectifying. This 5-HT response is blocked by intracellular Cs+ and tetraethylammonium (TEA+) and by extracellular TEA+ and Ba2+, but is not affected by the removal of extracellular Ca2+ or the intracellular injection of ethyleneglycol-bis-(beta-amino-ethylether)-N,N,N',N'-tetra-acetic acid (EGTA). These results indicate that the outwardly rectifying slow outward current induced by 5-HT in the F2 neuron is carried by K+ and is Ca2+-independent. In the single isolated F2 neuron, 5-HT induces a 2.5-fold stimulation of the adenylate cyclase activity. In addition, both the intracellular injection of 3',5'-adenosine monophosphate (cAMP) and the application of forskolin mimic the effect of 5-HT on the F2 neuron. The phosphodiesterase inhibitor isobutylmethylxanthine also induces a slow outward current and potentiates the 5-HT response. The intracellular injection of a synthetic 20-residue peptide inhibitor of the cAMP-dependent protein kinase blocks the slow outward K+ current induced by 5-HT. These results show that in the F2 neuron, 5-HT elicits a slow K+ current via the stimulation of adenylate cyclase, an increase in intracellular cAMP and the activation of the cAMP-dependent kinase which probably phosphorylates a population of outwardly rectifying K+ channels or some protein/s associated with these channels.     

Chemokine modulation of high-conductance Ca(2+)-sensitive K(+) currents in microglia from human hippocampi

During acute pathological processes, microglia transform into an activated state characterized by a defined morphology and current profile, and are recruited to injury sites by chemokines. No information is available on the ion channels and the mode of action of chemokines in microglia in brain slices from humans with a chronic pathology. Thus, patch-clamp recordings of microglia were performed in hippocampal slices from seven patients who underwent surgery for pharmaco-resistant epilepsy. Cells were identified as microglia by positive labelling with fluorescein-conjugated tomato lectin before recording. All the recorded cells had an ameboid morphology characteristic of activated microglia. However, they had a high input resistance (3.6 G omega), a zero-current resting potential of -16 mV, and lacked Na+ currents, inwardly rectifying and delayed rectifying K+ currents such as non-activated microglia. Importantly, recorded cells expressed Ca2+-sensitive outward currents that activated at 0 mV with non-buffered intracellular Ca2+ and were sensitive to 1 mm tetraethylammonium (TEA). The estimated single-channel conductances were 187 pS in cell-attached and 149 pS in outside-out patches, similar to those of high-conductance Ca2+-dependent K+ channels. The chemokine MIP1-alpha increased whole-cell outward current amplitudes measured at +60 mV by a factor of 3.3. Thus, microglia in hippocampi from epileptic patients express high-conductance Ca2+-dependent K+ channels that are modulated by the chemokine MIP1-alpha. This modulation may contribute to the migratory effect of MIP1-alpha on microglia.     

Channel-mediated and carrier-mediated uptake of K+ into cultured ovine oligodendrocytes

Uptake of radioactive K+ by mature ovine oligodendrocytes (OLGs) maintained in primary culture was measured under steady-state conditions, i.e., in cells maintained in a normal tissue culture medium (5.4 mM K+), and in cells after depletion of intracellular K+ to less than 15% of its normal value by pre-incubation in K(+)-free medium. The latter value is dominated by an active, carrier-mediated uptake (although it may include some diffusional uptake), whereas the former, in addition to active uptake, also reflects passive K+ diffusion through ion selective channels and possible self-exchange between extracellular and intracellular K+, which may be carrier-mediated. The total uptake rate was 144 +/- 10 nmol/min/mg protein, and the uptake after K+ depletion was 60 +/- 2 nmol/min/mg protein, much lower rates than previously observed in astrocytes. The uptake into K(+)-depleted cells was inhibited by about 80% in the presence of ouabain (1 mM) and about 30% in the presence of furosemide (2 mM). Activators of protein kinase C (phorbol esters) and cAMP-dependent protein kinase (forskolin) have been shown to alter the myelinogenic metabolism as well as outward K+ current in cultured OLGs. The present study demonstrates that K+ homeostasis in OLGs is modulated through similar second messenger pathways. Active uptake was inhibited by about 60% in the presence of active phorbol esters (100 nM) but was not affected by forskolin (100 nM). Forskolin likewise had no effect on total uptake, whereas phorbol esters caused a much larger inhibition than expected from their effect on carrier-mediated uptake alone, suggesting that channel-mediated uptake was also reduced.(ABSTRACT TRUNCATED AT 250 WORDS)     

Neural control of the heart: developmental changes in ionic conductances in mammalian intrinsic cardiac neurons

The expression and properties of ionic channels were investigated in dissociated neurons from neonatal and adult rat intracardiac ganglia. Changes in the hyperpolarization-activated and ATP-sensitive K+ conductances during postnatal development and their role in neuronal excitability were examined. The hyperpolarization-activated nonselective cation current, Ih, was observed in all neurons studied and displayed slow time-dependent rectification. An inwardly rectifying K+ current, IK(IR), was present in a population of neurons from adult but not neonatal rats and was sensitive to block by extracellular Ba2+ Using the perforated-patch recording configuration, an ATP-sensitive K+ (KATP) conductance was identified in > or = 50% of intracardiac neurons from adult rats. Levcromakalim evoked membrane hyperpolarization, which was inhibited by the sulphonylurea drugs, glibenclamide and tolbutamide. Exposure to hypoxic conditions also activated a membrane current similar to that induced by levcromakalim and was inhibited by glibenclamide. Changes in the complement of ion channels during postnatal development may underlie observed differences in the function of intracardiac ganglion neurons during maturation. Furthermore, activation of hyperpolarization-activated and KATP channels in mammalian intracardiac neurons may play a role in neural regulation of the mature heart and cardiac function during ischaemia-reperfusion.     

A subpopulation of bone marrow-derived macrophage-like cells shares a unique ion channel pattern with microglia.

Rat microglia share a number of antigenic, functional, and morphological similarities with macrophages from other tissues, but are characterized by a distinctly different pattern of ion channels in the cellular membrane (Kettenmann et al., J Neurosci Res 26:278-287, 1990). Macrophages typically express outward and inward K+ currents. In contrast, microglia lack outward currents and only show inwardly rectifying K+ currents, regardless of the isolation or cultivation method employed for microglia. In this study we demonstrate that a subpopulation of bone marrow-derived macrophage-like cells possesses inward rectifier K+ currents, but no outward currents and thus with regard to the electrophysiological characteristics closely resembles microglia. A second population of bone marrow-derived macrophage-like cells shows the usual channel pattern described for other body macrophages. Our results strengthen the hypothesis that in the bone marrow distinct pools of precursor cells exist, possibly reflecting an early differential lineage determination for body and brain macrophages, i.e., microglia.     

Cultured microglial cells have a distinct pattern of membrane channels different from peritoneal macrophages.

Microglia are the source of the resident macrophages of the brain and thus belong to one of the most reactive cell types in cerebral tissue. They are attributed to have an important role in a number of pathological conditions, such as multiple sclerosis, viral infections like AIDS, and in lethal or sublethal injuries of neurons where the blood-brain barrier is left intact (Streit et al., 1988; McGeer et al., 1988; Gendelman et al., 1989). Microglia share a number of macrophage characteristics but so far lack a distinguishing positive marker. In this study it is shown that microglia are distinguished from other macrophages by a unique pattern of ion channels. We compared membrane currents of microglial cells with those from peritoneal macrophages cultured under identical conditions. Although in macrophages a delayed outward K+ current was previously described (Randriamampita and Trautmann, 1987), microglial cells lacked any specific outward current. Instead, these cells were characterized by large inwardly rectifying currents, activated by hyperpolarizing voltage steps. The reversal potential in different K+ gradients and the sensitivity of the current to to Ba2+, TEA, and 4-AP indicates that this current is K+ selective. In single-channel recordings, a 30 pS K+ selective channel similar to the classical inward rectifier K+ channel was observed. Thus, the expression of membrane channels served not only to distinguish microglia from other cells inside and outside the brain, e.g., blood macrophages, but also suggests a unique functional state of this cell population.     

Heterogeneity of potassium currents in cultured oligodendrocytes

In the present study we have analyzed the membrane currents of mature oligodendrocytes in cultures from dissociated fetal mouse cerebral hemispheres and explant cultures from fetal mouse spinal cord. Both types of oligodendrocytes showed large voltage-dependent, but time-independent inward and outward currents that were partially blocked by Ba2+. In addition, time- and voltage-dependent inward and outward currents were observed in a minority of oligodendrocytes from spinal cord. All voltage-dependent currents were completely blocked by Ba2+, and inward currents were completely blocked by Cs+, suggesting that they are mediated by K+ channels. Current-voltage curves of mouse spinal cord oligodendrocytes varied from being linear to outwardly or inwardly rectifying. In contrast, oligodendrocytes cultured from mouse brain always showed an inward rectification of the current voltage relation and a lack of time-dependent currents. It thus appears that mature oligodendrocytes in explant cultures of mouse spinal cord, in contrast to oligodendrocytes from dissociated brain, consist of different cell populations that are distinguished by their expression or active state of K+ channels.     

Potassium channel activity in sarcolemmal vesicles formed from skeletal muscle fibres of normal and dystrophic mice

Sarcolemmal vesicles were produced from adult mouse extensor digitorum longus muscle (EDL) by treating swollen muscle fibres with collagenase. Vesicles formed from dystrophic (C57BL/6J dy/dy) and phenotypically normal animals were patch clamped and the single channel activity was recorded. Three types of K+ channel were observed in excised patches taken from normal and dystrophic muscle. A large conductance (300 pS) Ca2(+)-dependent K+ channel (KCa) was the most frequently observed of the K+ channels in both types of muscle preparation. In a number of patches taken from dystrophic muscle the open probability-voltage relationship for the KCa channel was markedly different from that in normal muscle, suggesting a possible reduction in Ca2+ sensitivity. An ATP-sensitive K+ channel (90 pS) was common to both normal and dystrophic muscle vesicles and was present in a large number of patches. An inwardly rectifying K+ channel (40 pS) was also observed in both types of sarcolemmal vesicles. The properties of all three K+ channels types were broadly consistent with other observations of skeletal muscle K+ channels, though all had higher conductances than had previously been noted in other species.     

Ion channel expression by astrocytes in situ: comparison of different CNS regions

Patch-clamp recordings were obtained in brain slices from 283 rat astrocytes. The expression of voltage-activated whole-cell currents was compared in four different CNS regions (hippocampus, cerebral cortex, spinal cord, and cerebellum). Our data show that CNS astrocytes do not show significant regional differences in their ion channel complement. With the exception of cerebellar Bergmann glial cells, essentially all astrocytes express a combination of delayed rectifying outward K(+) currents, transient A-type K(+) currents, and small Na(+) currents. Developmentally, an increasing percentage of astrocytes and Bergmann glial cells express inwardly rectifying K(+) currents. We did not observe cells that were passive, i.e., lacking voltage-activated currents. A few cells that appeared "passive" in initial recordings showed voltage-activated K(+) currents after off-line leak subtraction. The heterogeneity observed in the ion channel complement was found to be identical when cell-to-cell variations observed within a given CNS region and between various CNS regions were compared, suggesting a common and fairly stereotypical complement of ion channels in CNS astrocytes. Ion channel expression in Bergmann glial cells differed from that of all other CNS regions studied. These cells typically showed very low input resistances attributable to a significant time- and voltage-independent resting K(+) conductance. However, as with electrophysiologically "passive"-appearing astrocytes, Bergmann glial cells showed expression of delayed rectifying K(+) currents after off-line leak subtraction. Inwardly rectifying K(+) currents were observed in Bergmann glial cells after postnatal day 17. Collectively, our data suggest that all astrocytes contain voltage-gated ion channels that display a common pattern of expression during development.     

The developmental expression of K+ channels in retinal glial cells is associated with a decrease of osmotic cell swelling

A major function of glial cells is the control of osmotic and ionic homeostasis, mediated by K+ and water movements predominantly through inwardly rectifying K+ (Kir) and aquaporin water channels. It has been suggested that K+ currents through Kir channels are implicated in the regulation of glial cell volume. Here, we investigated whether the developmental increase in Kir channel expression in Müller glial cells of the rat retina is associated with an alteration of cell volume regulation under anisoosmotic conditions. Around the time of eye opening at postnatal day (P) 15, developing retinal glial cells fully alter the profile of their membrane conductances, from a current pattern with prominent fast transient K+ and Na+ currents to a pattern of noninactivating currents through Kir and delayed rectifier K+ channels. Concomitantly, aquaporins-1 and -4 are expressed in the developing retina. This is accompanied by a conspicuous alteration of the swelling characteristics of cells; somata of immature glial cells in early postnatal retinas (P5-P15) swell under hypotonic stress but no swelling is inducible in mature cells at P18 and thereafter. However, glial cells at all developmental stages swell when their Kir channels are blocked by Ba2+. The postnatal maturation of Kir channel currents and volume regulation in retinal glial cells is delayed by visual deprivation. The data suggest that Kir channels are crucially involved in osmotic volume homeostasis of mature glial cells, and that the absence of Kir channels in immature cells is a major cause of their insufficient volume regulation.     

Oligodendrocyte-substratum adhesion activates the synthesis of specific lipid species involved in cell signaling.

Ovine oligodendrocytes (OLGs) undergo biochemical and morphological changes following attachment to polylysine. Autoradiographs of two-dimensional thin-layer chromatograms of [14C]Gal-labeled OLG cultures revealed that attachment of OLGs to a polylysine substratum and their subsequent morphological differentiation is accompanied by an increased synthesis of multiple forms of galactosylceramide, sulfogalactosylceramide, and both sulfogalactosyl- and galactosyl-diglycerides, together with an array of complex sialoglycosphingolipids, predominantly GM2 ganglioside. As previously reported, overall lipid synthesis measured by [14C]acetate incorporation into glycerophosphatides, sphingomyelin, and neutral lipids also increased dramatically for up to 60 days (last time point examined) following OLG-substratum adhesion, reflecting membrane growth. Attachment was associated with a rapid augmentation in the synthesis of ethanolamine plasmalogen from 12 to 27% within 24 hr to reach a 35% plateau at 30 days and remain constant thereafter. In contrast, the plasmalogen content of phosphatidylcholine remained constant at 3-5%. This rapid increase in lipid synthesis (especially in the ethanolamine plasmalogen content following attachment) closely paralleled increased diacylglycerol (DAG) production and protein kinase C-dependent phosphorylation of both myelin basic protein and 2',3'-cyclic nucleotide phosphohydrolase. Labeling studies indicated that the major source of [3H]arachidonate-labeled DAG following attachment was from phosphatidylinositol turnover (and to a lesser extent phosphatidylcholine) rather than polyphosphoinositides or plasmalogens. Enhanced lipid synthesis is not only required for the production of membranes in these myelin-producing cells but is also a source of second messengers required in the posttranslational modification of key myelin and cellular proteins.     

Phosphorylation of myelin basic protein in intact oligodendrocytes: inhibition by galactosylsphingosine and cyclic AMP

We have previously shown that cyclic AMP (cAMP) inhibits the protein kinase C (PKC)-mediated phosphorylation of myelin basic protein (MBP) in cultured oligodendrocytes (OLGs). Recently, it has been demonstrated that the long chain base sphingosine inhibits PKC by competing PKC effectors (diacylglycerol and phorbol esters) for a binding site on the kinase (Hannun and Bell: Science 235: 670-674, 1987). In this report we define further the mechanism by which cAMP inhibits MBP phosphorylation by comparing the effects of cAMP with that of galactosylsphingosine (psychosine), a potential catabolite of galactocerebroside, the major OLG glycosphingolipid. We identify the consequences of psychosine treatment and PKC down-regulation on OLG morphology and electrophysiology and discuss their relevance. Our results in intact ovine oligodendrocytes are consistent with a mechanism in which cAMP inhibits MBP phosphorylation by interfering with the release of diacylglycerol (DAG) from phosphatidylinositol. First, the effects of cAMP on MBP phosphorylation are reversed with exogenous TPA; and second, cAMP inhibits the incorporation of 1-[14C]arachidonate into DAG and specifically inhibits the turnover (as judged by 32PO4 3-incorporation) of phosphatidylinositol. Psychosine inhibits MBP phosphorylation, and its action can be reversed by TPA suggesting a mechanism of inhibition similar to that described for other systems. In addition, psychosine has profound effects on OLG morphology; it disintegrates OLG processes while leaving the cell soma intact. Stable hyperpolarized resting potentials were obtained following psychosine treatment, but there was a 66% decrease in membrane capacitance indicating a significant decrement in membrane surface area. The morphological changes induced by psychosine are reversible and can be eliminated by removing the drug but not by the addition of TPA. Whether inhibition of PKC by psychosine plays any role in process dissolution remains an unanswered question. However, current evidence suggests that a PKC-independent mechanism may be at play. This investigation in conjunction with our previous work emphasizes a role for the interregulation of protein kinase A (PKA) and PKC in the control of OLG somal vs. myelin components. This may have significant implications for central nervous system myelin assembly.     

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.     

Modulation of ionic currents in Aplysia motor neuron B15 by serotonin, neuropeptides, and second messengers

Both 5-HT and the 9 amino acid neuropeptide SCPb modulate 3 ionic currents in B15, enhancing a voltage-dependent inward sodium current, decreasing an outward potassium current and increasing an inward rectifying potassium current. In contrast, FMRFamide decreases a voltage-dependent inward sodium current and increases an outward potassium current. We have also investigated the roles of several second-messenger systems that may be mediating the effects of these modulators. Bath application of membrane permeable analogs of cAMP enhance the voltage-dependent inward sodium current and both 5-HT and SCPb increase cAMP levels in B15, suggesting that cAMP may be mediating part of the observed effects of these transmitters on B15. Experiments with phorbol ester, a protein kinase inhibitor, and a phospholipase inhibitor suggest that the phospholipase C/protein kinase C cascade may decrease an outward potassium current. Thus, 5-HT and SCPb may activate multiple second-messenger systems to modulate 3 ionic currents in B15. Additional studies suggest that a cascade involving arachidonic acid may be involved in mediating part of the FMRFamide responses in B15. These studies are beginning to define molecular mechanisms whereby a neuron differentially modulates multiple ionic currents in response to distinct chemical messengers.     

Role of glial K(+) channels in ontogeny and gliosis: a hypothesis based upon studies on Müller cells

The electrophysiological properties of Müller cells, the principal glial cells of the retina, are determined by several types of K(+) conductances. Both the absolute and the relative activities of the individual types of K(+) channels undergo important changes in the course of ontogenetic development and during gliosis. Although immature Müller cells express inwardly rectifying K(+) (K(IR)) currents at a very low density, the membrane of normal mature Müller cells is predominated by the K(IR) conductance. The K(IR) channels mediate spatial buffering K(+) currents and maintain a stable hyperpolarized membrane potential necessary for various glial-neuronal interactions. During "conservative" (i.e., non-proliferative) reactive gliosis, the K(IR) conductance of Müller cells is moderately reduced and the cell membrane is slightly depolarized; however, when gliotic Müller cells become proliferative, their K(IR) conductances are dramatically down-regulated; this is accompanied by an increased activity of Ca(2+)-activated K(+) channels and by a conspicuous unstability of their membrane potential. The resultant variations of the membrane potential may increase the activity of depolarization-activated K(+), Na(+) and Ca(2+) channels. It is concluded that in respect to their K(+) current pattern, mature Müller cells pass through a process of dedifferentiation before proliferative activity is initiated.     

Kir potassium channel subunit expression in retinal glial cells: implications for spatial potassium buffering

To understand the role of different K(+) channel subtypes in glial cell-mediated spatial buffering of extracellular K(+), immunohistochemical localization of inwardly rectifying K(+) channel subunits (Kir2.1, Kir2.2, Kir2.3, Kir4.1, and Kir5.1) was performed in the retina of the mouse. Stainings were found for the weakly inward-rectifying K(+) channel subunit Kir4.1 and for the strongly inward-rectifying K(+) channel subunit Kir2.1. The most prominent labeling of the Kir4.1 protein was found in the endfoot membranes of Müller glial cells facing the vitreous body and surrounding retinal blood vessels. Discrete punctate label was observed throughout all retinal layers and at the outer limiting membrane. By contrast, Kir2.1 immunoreactivity was located predominantly in the membrane domains of Müller cells that contact retinal neurons, i.e., along the two stem processes, over the soma, and in the side branches extending into the synaptic layers. The results suggest a model in which the glial cell-mediated transport of extracellular K(+) away from excited neurons is mediated by the cooperation of different Kir channel subtypes. Weakly rectifying Kir channels (Kir4.1) are expressed predominantly in membrane domains where K(+) currents leave the glial cells and enter extracellular "sinks," whereas K(+) influxes from neuronal "sources" into glial cells are mediated mainly by strongly rectifying Kir channels (Kir 2.1). The expression of strongly rectifying Kir channels along the "cables" for spatial buffering currents may prevent an unwarranted outward leak of K(+), and, thus, avoid disturbances of neuronal information processing.     

Voltage-gated ionic currents in an identified modulatory cell type controlling molluscan feeding

An important modulatory cell type, found in all molluscan feeding networks, was investigated using two-electrode voltage- and current-clamp methods. In the cerebral giant cells of Lymnaea, a transient inward Na+ current was identified with activation at -58 +/- 2 mV. It was sensitive to tetrodotoxin only in high concentrations (approximately 50% block at 100 microm), a characteristic of Na+ channels in many molluscan neurons. A much smaller low-threshold persistent Na+ current (activation at < -90 mV) was also identified. Two purely voltage-sensitive outward K+ currents were also found: (i) a transient A-current type which was activated at -59 +/- 4 mV and blocked by 4-aminopyridine; (ii) a sustained tetraethylammonium-sensitive delayed rectifier current which was activated at -47 +/- 2 mV. There was also evidence that a third, Ca2+-activated, K+ channel made a contribution to the total outward current. No inwardly rectifying currents were found. Two Ca2+ currents were characterized: (i) a transient low-voltage (-65 +/- 2 mV) activated T-type current, which was blocked in NiCl2 (2 mm) and was completely inactivated at approximately -50 mV; (ii) A sustained high voltage (-40 +/- 1 mV) activated current, which was blocked in CdCl2 (100 microm) but not in omega-conotoxin GVIA (10 microm), omega-agatoxin IVA (500 nm) or nifedipine (10 microm). This current was enhanced in Ba2+ saline. Current-clamp experiments revealed how these different current types could define the membrane potential and firing properties of the cerebral giant cells, which are important in shaping the wide-acting modulatory influence of this neuron on the rest of the feeding network.     

Expression of ion channels and mutational effects in giant Drosophila neurons differentiated from cell division-arrested embryonic neuroblasts.

A culture system of "giant" Drosophila neurons derived from cytokinesis-arrested embryonic neuroblasts was developed to overcome the technical difficulties usually encountered in studying small Drosophila neurons. Cytochalasin B-treated neuroblasts differentiated into giant multinucleated cells that displayed neuronal morphology and neuron-specific markers (Wu et al., 1990). Here, we report that these giant neurons express different excitability patterns and membrane channels similar to those reported in excitable tissues of Drosophila. Individual neurons exhibited distinct all-or-none or graded voltage responses upon current injection. Both current- and voltage-clamp recordings could be performed on the same neuron because of the large cell size, thus making it possible to elucidate the functional role of the individual types of channels. By using pharmacological agents and ion substitution, the following currents were identified in these giant neurons: inward Na+ and Ca2+ currents and outward voltage-activated (the A-type and delayed rectifier) and Ca(2+)-activated K+ currents. In addition, we found a tetrodotoxin (TTX)-sensitive, Na(+)-dependent outward K+ current and a persistent component of an inward Na+ current, which have not been reported in Drosophila previously. This culture system can be used to analyze the mutational perturbations in ion channels and the resultant alterations in membrane excitability. Neurons from the mutant slowpoke (slo), which is known to lack a component of the Ca(2+)-activated K+ currents in muscles, exhibited prolonged action potentials associated with defects in the Ca(2+)-activated K+ current. This abnormality appeared to be more severe in the neurites than in the soma.