Neurogenetic studies of ion channels in Drosophila.
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Amino Acid Sequence↗
Biomedical subjects
C F Wu
Publications and source records attributed to C F Wu.
Interactions of membrane excitability mutations affecting potassium and sodium currents in the flight and giant fiber escape systems of Drosophila.
We have studied the influence of the K(+)-current mutations eag and Sh and the Na(+)-current mutation napts upon two well-defined neural circuits that underlie flight and an escape response in Drosophila, recording from dorsal longitudinal and tergotrochanteral muscles. Mutations of Sh and eag affected refractory period and following frequency, but not latency, of the jump-and-flight escape response. The napts mutation altered these 3 physiological parameters of the "jump" (TTM), but not the "flight" (DLM), branch, suggesting differences in the vulnerability of different circuit components to the mutation. In contrast to their interaction in some other systems, napts did not counteract the effects of eag and Sh upon these physiological parameters in eag Sh; nap triple mutants. In eag Sh double mutants, in which multiple K+ currents may be diminished, flight muscles showed abnormal rhythmic activity not associated with flight, and some flies also had an abnormal wings-down posture. The low-frequency spikes probably originated in the flight muscle motoneurons, but the coordination between muscle fibers during this "non-flight activity" was distinct from flight. Nevertheless, in spite of the presence of this non-flight activity in resting eag Sh flies, those animals with normal wing posture were also able to fly, with a normal pattern of muscle activity. This suggests that in these mutants, the DLM motoneuron circuit is able to switch between two patterns of output, non-flight activity and flight.(ABSTRACT TRUNCATED AT 250 WORDS)
Action Potentials↗
Synaptic plasticity in Drosophila memory and hyperexcitable mutants: role of cAMP cascade.
Activity-dependent synaptic plasticity has been implicated in the refinement and modification of neural circuits during development and learning. Previous studies show that activity-induced facilitation and potentiation are disrupted at larval neuromuscular junctions in the memory mutants dunce (dnc) and rutabaga (rut) of Drosophila. The diminished learning-memory capacity and synaptic transmission plasticity have been associated with altered cAMP levels since dnc affects the cAMP-specific phosphodiesterase and rut affects adenylate cyclase. In this study, the morphology of larval motor axon terminals was examined by anti-HRP immunohistochemistry. It was found that the numbers of terminal varicosities and branches were increased in dnc mutants, which have elevated cAMP concentrations. Such increase was suppressed in dnc rut double mutants by rut mutations, which reduce cAMP synthesis. More profuse projections of larval motor axons have also been reported in double-mutant combinations of ether à go-go (eag) and Shaker (Sh) alleles, which display greatly enhanced nerve activity as a result of reduction in different K+ currents. Therefore, we examined combinations of dnc and rut with eag and Sh mutations to explore the possible relation between activity- and cAMP-induced morphological changes. We found that the expanded projections in dnc were further enhanced in double mutants of dnc with either eag or Sh, an effect that could again be suppressed by rut. The results provide evidence for altered plasticity of synaptic morphology in memory mutants dnc and rut and suggest a role of cAMP cascade in mediating activity-dependent synaptic plasticity.
3',5'-Cyclic-AMP Phosphodiesterases↗
Alteration of four identified K+ currents in Drosophila muscle by mutations in eag.
Voltage-clamp analysis of Drosophila larval muscle revealed that ether à go-go (eag) mutations affected all identified potassium currents, including those specifically eliminated by mutations in the Shaker or slowpoke gene. Together with DNA sequence analysis, the results suggest that the eag locus encodes a subunit common to different potassium channels. Thus, combinatorial assembly of polypeptides from different genes may contribute to potassium channel diversity.
4-Aminopyridine↗
Altered synaptic plasticity in Drosophila memory mutants with a defective cyclic AMP cascade.
Synaptic transmission was examined in Drosophila mutants deficient in memory function. These mutants, dunce and rutabaga, are defective in different steps of the cyclic adenosine 3',5'-monophosphate (cAMP) cascade. In both dunce and rutabaga larvae, voltage-clamp analysis of neuromuscular transmission revealed impaired synaptic facilitation and post-tetanic potentiation as well as abnormal responses to direct application of dibutyryl cAMP. In addition, the calcium dependence of transmitter release was shifted in dunce. The results suggest that the cAMP cascade plays a role in synaptic facilitation and potentiation and indicate that synaptic plasticity is altered in Drosophila memory mutants.
Action Potentials↗
Distinctions in growth cone morphology and motility between monopolar and multipolar neurons in Drosophila CNS cultures.
Growth cones play a central role in determining neurite extension, pathfinding and branching, and in establishing synaptic connections. This paper describes an initial characterization of growth cone morphology and behavior in dissociated larval central nervous system (CNS) cultures of Drosophila. Contrast-enhanced video images of growth cones in monopolar and multipolar neurons were characterized by employing morphometric parameters such as the number and length of filopodia, and the area and roundness of the lamellipodia. Behavior of growth cones was analyzed by a motility index and boundary flow plots originally devised for measuring motility in other cellular systems. We found that separate CNS regions yielded cultures of different major cell types with distinct neuritic patterns that could be correlated with the morphology and motility of the associated growth cones. Monopolar neurons were the major cell type in brain cultures, whereas multipolar neurons were predominant in ventral ganglion cultures. Moreover, the growth cones of monopolar neurons, which are likely to be associated with the axonal processes, differed from those of multipolar neurons, which might be related to dendritic terminals. Growth cones in monopolar neurons had larger lamellipodia of less erratic shape accompanied by fewer and shorter filopodia, and, when active, displayed much higher motility and less directionality in motion. Alternatively, these morphological and behavioral distinctions between monopolar and multipolar neurons may result from intrinsic differences in membrane adhesion and intracellular transport properties.
Animals↗
Dual muscarinic and nicotinic action on a motor program in Drosophila.
The effect of cholinergic agonists and antagonists on the central pattern generator of the pharyngeal muscles has been studied in third instar larvae of Drosophila. The pharyngeal muscles are a group of rhythmically active fibers involved in feeding. Bath application of the cholinergic agonists carbachol, muscarine, pilocarpine, and acetylcholine (ACh) to a semiintact preparation including the pharyngeal muscles and the central nervous system (CNS), initiated long-lasting endogenous-like bursting activity in the muscles. The muscarinic antagonists, atropine and scopolamine, blocked these responses as well as endogenous activity. Perfusion with nicotine elicited a short, tonic response that was marginally blocked by mecamylamine but not by curare, alpha-bungarotoxin, hexamethonium, or the muscarinic antagonists. This is the first time that a response to cholinergic drugs has been examined in Drosophila. The pharyngeal muscle preparation may prove to be a valuable system for studying mutations of cholinergic metabolism, receptors, and second messengers.
Acetylcholine↗
Single-channel K+ currents in Drosophila muscle and their pharmacological block.
Four types of nonvoltage-activated potassium channels in the body-wall muscles of Drosophila third instar larvae have been identified by the patch-clamp technique. Using the inside-out configuration, tetraethylammonium (TEA), Ba2+, and quinidine were applied to the cytoplasmic face of muscle membranes during steady-state channel activation. The four channels could be readily distinguished on the basis of their pharmacological sensitivities and physiological properties. The KST channel was the only type that was activated by stretch. It had a high unitary conductance (100 pS in symmetrical 130/130 mM KCl solution), was blocked by TEA (Kd approximately 35 mM), and was the most sensitive to Ba2+ (complete block at 10(-4) M). A Ca(2+)-activated potassium channel, KCF.72 pS (130/130) mM KCl), was gated open at greater than 10(-8) m Ca2+, was the least sensitive to Ba2+ Kd of approximately 3 mM) and TEA (Kd of approximately 100 mM), and was not affected by quinidine. K2 was a small conductance channel of 11 pS (130/2 KCl, pipette/bath), and was very sensitive to quinidine, being substantially blocked at 0.1 mM. It also exhibited a half block at approximately 0.3 mM Ba2+ and approximately 25 mM TEA. A fourth channel type, K3, was the most sensitive to TEA (half block less than 1 mM). It displayed a partial block to Ba2+ at 10 mM, but no block by 0.1 mM quinidine. The blocking effects of TEA, Ba2+ and quinidine were reversible in all channels studied. The actions of TEA and Ba2+ appeared qualitatively different: in all four channels, TEA reduced the apparent unitary conductance, whereas Ba2+ decreased channel open probability.
Animals↗
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.
4-Aminopyridine↗
Clinical features and comprehensive treatment of late stage radiation skin injuries.
105 patients with late stage radiation skin injury were treated from 1970 to 1986, using drugs combined with surgical intervention according to the clinical features of the injury. Drug therapy consisted of topical application of urea, antibiotics, trypsin and elastase, and systematic use of alpha 2-Macroglobulin. Of 62 patients receiving medication, 55 (88.7%) were completely or basically treated and 7 failed. Of 51 lesions of 43 patients receiving surgical treatment, 47 healed by first intention. In 4 patients, necrosis of partial and/or peripheral skin flaps healed after renewed repair.
Adolescent↗
Giant Drosophila neurons differentiated from cytokinesis-arrested embryonic neuroblasts.
The relative contributions of the intrinsic and extrinsic factors in determining neuronal differentiation are not fully understood yet. We found that isolated neuroblasts from Drosophila gastrulae were able to differentiate neuron-specific properties in culture even when cell divisions were inhibited. The resultant giant multinucleated neurons displayed thickened neurites with a variety of distinct branching patterns. Neuronal antigens were expressed as in normal cultured neurons, and action potentials could be evoked by current injection within two days after plating. These results indicate that the factors for initiating specific differentiation programs for basic neuronal form and function are present in a neuroblast already. The cells of increased sizes in this culture system are more accessible to physiological and cell biological analyses and could facilitate future studies of the Drosophila nervous system.
Action Potentials↗
Mutational and gene dosage analysis of calcium-activated potassium channels in Drosophila: correlation of micro- and macroscopic currents.
In Drosophila, two Ca2(+)-activated K+ currents, ICF and ICS, have previously been distinguished in conventional voltage clamp experiments. The slowpoke (slo) mutation eliminates ICF specifically. We report that in patch clamp recordings a single-channel Ca2(+)-activated K+ current is readily distinguished from other channel activities in normal larval muscle membrane, whereas no such current is observed in slo muscles. This single-channel current thus correlates with the macroscopic ICF. No obvious differences in amplitude or properties were detected between normal (+/+) and heterozygous (slo/+) ICF channels in whole-cell voltage clamp recordings or single-channel patch clamp recordings. These results are consistent with the hypothesis that slo is a structural gene for the ICF channels only under certain conditions. The selective effect of the slo mutation may reflect a defect in a regulatory mechanism that is specific for the functioning of the ICF channel protein.
Animals↗
Properties of potassium currents and their role in membrane excitability in Drosophila larval muscle fibers.
The larval muscle fibers of Drosophila show four outward K+ currents in addition to the inward Ca2+ current in voltage-clamp recordings. The Shaker (Sh) and the slowpoke (slo) mutations, respectively, eliminate the voltage-activated fast K+ current (IA) and the Ca2(+)-activated fast K+ current (ICF). Quinidine specifically blocks the voltage-activated delayed K+ current (IK) at micromolar concentrations. We used Sh, slo and quinidine to remove specifically one or more K+ currents, so as to study physiological properties of these currents not previously characterized, and to examine their role in membrane excitability. A linear relationship was observed between the peak ICF and the peak ICa at different membrane potentials. ICF inactivated considerably during a 140 ms pulse to +20 mV. Recovery from inactivation was not complete for up to 2 s at the holding potential of -50 mV, which is much slower than the recovery of Ca2+ current from inactivation. In addition to IA and ICF, two delayed K+ currents are also observed in these fibers, the voltage-activated IK and the Ca2(+)-activated ICS. Near the end of a 500 ms depolarizing pulse, both IA and ICF are inactivated. Ca2(+)-free and 20 mmol l-1 Ca2+ saline were used to examine the tail currents of the remaining IK and ICS. The tail currents of ICS were slower than those of IK and reversed between -30 and -50 mV in different fibers. We further studied the dose-dependence of the blockade of IK by quinidine, which did not indicate a simple one-to-one binding mechanism. Current-clamp recordings from normal, Sh, slo and the double-mutant Sh;slo fibers suggested that ICF plays a stronger role than IA in repolarization of the larval muscle membrane. Elimination of ICF facilitates the occurrence of action potentials. Further elimination of IK prolonged the action potentials to several hundred milliseconds.
Animals↗
Allelic interactions at the shibire locus of Drosophila: effects on behavior.
This report describes a detailed behavioral study of four shi alleles, shits1, shits2, shits4, and shiST139, and their heteroallelic combinations. Flies of different heteroallelic combinations (shi/shi) were less sensitive to high temperature than the corresponding homozygotes. Among them, shits2/shits4 showed a striking reduction in temperature sensitivity. In contrast, different shi/+ heterozygotes were more severe in temperature sensitivity than the hemizygotes, Df/+. The hemizygous combinations of shi alleles over deficiency (shi/Df) were not completely lethal, with shits2 conferring distinctively higher and shits4 lower viability. A novel behavior, bang sensitivity, was also found in shi/Df. The results of allelic interactions suggest that the shi mutations examined appear to be antimorphic and that the shi gene products are likely to function in multimeric form.
Alleles↗
Reversible inhibition of endocytosis in cultured neurons from the Drosophila temperature-sensitive mutant shibirets1.
The Drosophila mutant, shibirets1 (shits1), is paralyzed at restrictive temperatures (greater than 29 degrees C) by a reversible block in synaptic transmission. Heat pulses deplete synaptic vesicles in nerve terminals and inhibit endocytic internalization of plasma membrane in garland cells and oocytes. In dissociated cultures of larval central nervous system (CNS), a temperature-sensitive defect is also expressed in shits1 neurons: at 30 degrees C, growth cone formation is retarded and neurite outgrowth is arrested. We now report that we have examined constitutive endocytosis in Drosophila CNS culture and have demonstrated directly an endocytic defect in shits1 neurons. At the permissive temperature, 20-22 degrees C, both shits1 and wild-type neurons actively endocytosed fluorescein-labelled dextran (40 KD, 5%) or horseradish peroxidase (HRP, 1%). Within 5 min, HRP was seen in vesicles, cup-shaped bodies, tubules and multivesicular bodies in neurites and cell bodies. In contrast, endocytosis was inhibited in cultures derived from the temperature-sensitive paralytic shits1 by a 15 min heat pulse (30 degrees C). Even after 30 min of HRP exposure at 30 degrees C, HRP-containing membranes were absent from almost all shits1 neurites; a minority of cell bodies had a few HRP-containing vesicles. The temperature-dependent block in endocytosis was readily reversed at 20 degrees C. Interestingly, the block was overcome by high concentration of external cations: shits1 neurons in culture actively took up HRP in numerous vesicles at 30 degrees C if 18 mM Ca2+ or Mg2+ was added to the medium. Our results support the notion that membrane recycling plays a critical role in regulating neurite outgrowth. This study also provides baseline information for further mutational analysis of the mechanism underlying the membrane cycling process in cultured neurons.
Animals↗
Morphological plasticity of motor axons in Drosophila mutants with altered excitability.
An anatomical and electrophysiological study of Drosophila mutants has been made to determine the effect of altered electrical activity on the development and maintenance of larval neuromuscular junctions. We examined motor axon terminals of (1) hyperexcitable mutants Shaker (Sh), ether a go-go (eag), Hyperkinetic (Hk), and Duplication of para+ (Dp para+); and (2) mutants with reduced excitability, no action potential (napts) and paralytic (parats 1). Nerve terminals innervating larval body-wall muscles were visualized by using anti-HRP immunocytochemistry, which specifically stains neurons in insect species. In wild-type larvae, motor axon terminals were distributed in a stereotypic fashion. However, in combinations of eag and Sh alleles, the basic pattern of innervation was altered. There was an increase in both the number of higher-order axonal branches over the muscles and the number of varicosities on the neurites. A similar phenomenon was found in the double mutant Hk eag and, to a lesser extent, in Dp para+ and Dp para+ Sh mutants. It is known that at permissive temperature the napts, but not parats 1, mutation decreases excitability of larval motor axons and suppresses the behavioral phenotypes of Sh, eag, and Hk. In the mutant napts (reared at permissive temperature), a slight decrease in the extent of branching was observed. Yet, when combined with eag Sh, napts completely reversed the morphological abnormality in eag Sh mutants. No such reversion was observed in parats 1 eag Sh mutants. The endogenous patterns of electrical activity at the neuromuscular junction were analyzed by extracellular recordings in a semi-intact larval preparation. Recordings from wild-type body-wall muscles revealed rhythmic bursts of spikes. In eag Sh mutants, this rhythmic activity was accompanied by or superimposed on periods of strong tonic activity. This abnormal pattern of activity could be partially suppressed by napts in combination with eag Sh.
Animals↗
A voltage-clamp analysis of gene-dosage effects of the Shaker locus on larval muscle potassium currents in Drosophila.
Mutations of the Shaker (Sh) locus of Drosophila reduce, eliminate, or otherwise alter a transient potassium current, IA, in muscle. Recent molecular studies indicate that the Sh locus produces several proteins by alternative splicing, but the relationships of the variety of Sh gene products to IA channels in the various excitable membranes still remain to be determined. In Drosophila, many enzymes have been shown to exhibit gene-dosage effects; their amounts vary in direct proportion to the number of structural genes present. We describe a physiological isolation of IA in larval muscle which allowed precise quantification of gene-dosage effects on IA in Sh heterozygotes and aneuploids. We found that doubling the number of Sh genes in aneuploids increased IA to twice that of normal, consistent with the notion that the Sh locus encodes the entire IA channel in larval muscle. We further examined heterozygous combinations of different Sh mutations for evidence of interactions among Sh gene products within the IA channel, which may yield clues to the possible subunit composition of the channel. Combinations among 5 Sh mutations plus their normal counterpart followed a simple gene-dosage effect; in each case the resulting IA was about the average of the homozygous currents, compatible with the notion of additive contributions from 2 independent populations of IA channels. Two additional Sh mutations caused pronounced departures from the simple dosage effect; the amplitude of IA in heterozygotes was significantly smaller than that expected from gene dosage, a strong dominant effect attributable to interactions among protein subunits. These contrasting observations may be accounted for by certain hetero- or homo-multimeric arrangements of Sh products in the IA channel.
Aneuploidy↗
Complete separation of four potassium currents in Drosophila.
A number of voltage-activated and Ca2+ activated K+ currents are known to coexist and play a major role in a wide variety of cellular processes including neuromuscular phenomena. Separation of these currents is important for analyzing their individual functional roles and for understanding whether or not they are mediated by entirely different channels. In Drosophila, we have now been able to manipulate four different K+ currents, individually and in combination with one another, by a combined use of mutations and pharmacological agents. This allows analysis of the physiological and molecular properties of different K+ channels and of the role of individual currents in membrane excitability.
Animals↗