6727030
Genetic Dissection of Functional Contributions of Specific Potassium Channel Subunits in Habituation of an Escape Circuit inDrosophila
Potassium channels have been implicated in central roles in activity-dependent neural plasticity. The giant fiber escape pathway ofDrosophila has been established as a model for analyzing habituation and its modification by memory mutations in an identified circuit. Several genes in Drosophila encoding K+ channel subunits have been characterized, permitting examination of the contributions of specific channel subunits to simple conditioning in an identified circuit that is amenable to genetic analysis. Our results show that mutations altering each of four K+ channel subunits (Sh,slo, eag, and Hk) have distinct effects on habituation at least as strong as those ofdunce and rutabaga, memory mutants with defective cAMP metabolism (Engel and Wu, 1996). Habituation, spontaneous recovery, and dishabituation of the electrically stimulated long-latency giant fiber pathway response were shown in each mutant type. Mutations of Sh (voltage-gated) andslo (Ca2+-gated) subunits enhanced and slowed habituation, respectively. However, mutations ofeag and Hk subunits, which confer K+-current modulation, had even more extreme phenotypes, again enhancing and slowing habituation, respectively. In double mutants, Sh mutations moderated the strong phenotypes of eag and Hk, suggesting that their modulatory functions are best expressed in the presence of intactSh subunits. Nonactivity-dependent responses (refractory period and latency) at two stages of the circuit were altered only in some mutants and do not account for modifications of habituation. Furthermore, failures of the long-latency response during habituation, which normally occur in labile connections in the brain, could be induced in the thoracic circuit stage in Hk mutants. Our work indicates that different K+ channel subunits play distinct roles in activity-dependent neural plasticity and thus can be incorporated along with second messenger “memory” loci to enrich the genetic analysis of learning and memory.
26284561
Diversity and ubiquity of K channels
Abstract The following developments since this review was prepared and submitted (February, 1987) are important to the theme and conclusions made here: 1. Apamin block of Ca-activated K channels has now been observed in neurons of at least two mammalian brain areas: in neurosecretory neurons in the rat superoptic nucleus of the hypothalamus [Bourque C.W. and Brown D.A. (1987) Neurosci Lett.82, 185] and in neurons in the guinea-pig mammillary bodies (Alonso A., personal communication). In hippocampal neurons, a Ca-activated K channel which may be responsible for the slow AHP has been identified in single channel recordings. However, this channel has unique properties and it is not blocked by Apamin. This is another example that suggests that more than one subclass of Ca-activated K channel may mediate slow AHPs. Therefore, the term IAHP channel used in Table 5 for the Apamin-sensitive channel may be incorrect. 2. cDNAs for the “high threshold” “A” channel encoded in the Shaker gene locus in Drosophila have been cloned [Kamb A., Iverson L.E. and Tanouye M.A. (1987) Cell50, 405; Papazian D.M., Schwarz T.L., Tempel B.L., Jan Y.N. and Jan L.Y. (1987) Science237, 794]. The primary transcript of this gene undergoes extensive differential splicing generating a large diversity of products (Kamb A., Tseng-Crank J. and Tanouye M.A. Cell, submitted). At least three of these express “A” currents in Xenopus oocytes (Iverson L.E., Davidson N., Lester H., Tanouye M. and Rudy B. Proc. Natn. Acad. Sci., U.S.A., submitted; Timpe L.C., Schwarz T.L., Tempel B.L., Papazian D.M. Jan Y.N. and Jan L.Y. Nature, in press), which differ in inactivation kinetics. In all cases, however, the voltage-dependence of activation and inactivation is similar among the various clones and distinct from the “low threshold” “A” channels (Table 3). It is possible then that the Shaker “A” channel is a subclass of “A” channels (see Table 4) that then diversifies into distinct types. Knowledge of the molecular relationship between the Shaker “A” channels, the “low-threshold” “A” channels (Table 3) and other voltage-dependent K channels may eventually help in establishing a hierarchy of K channel diversification into classes, subclasses and types as suggested here. 3. G protein subunits may also activate K channels directly in the CNS [Andrade R., Malenka R.A. and Nicoll R.A. (1986) Science234, 1261]. 4. A book on ion-channel modulation, with many examples of modulation of K channels was published: Kaczmareck L.K. and Levitan I.B. (1987) Neuromodulation: The Biochemical Control of Neuronal Excitability. Oxford University Press, New York.
96149557
Ionic channels of excitable membranes
PART I Classical biophysics of the squid giant axon The superfamily of voltage-gated channels Voltage-gated calcium channels Potassium channels and chloride channels Ligand-gated channels of fast chemical synapses Modulation, slow synaptic action, and second messengers Sensory transduction and excitable cells Calcium dynamics, epithelial transport, and intercellular coupling PART II Elementary properties of ions in solution Elementary properties of pores Counting channels Structure of channel proteins Selective permeability: Independence Selective permeability: Saturation and binding Classical mechanisms of block Structure-function studies of permeation and block Gating mechanisms: Kinetic thinking Gating: Voltage sensing and inactivation Modification of gating in voltage-sensitive channels Cell biology and channels Evolution and origins
9466423
Modulation of ion currents and regulation of transmitter release in short-term synaptic plasticity: The rise and fall of the action potential
Up and down-regulation of calcium and potassium conductances are associated with several forms of short-term synaptic modulation. Detailed investigation of synaptic plasticity in the marine gastropodAplysia, and in other mollusks, indicates that synaptic transmission can be influenced in a number of ways by modulatory neurotransmitters acting through several second-messenger cascades. Modulation at the synapse itself occurs by means of the regulation of calcium current as well as through effects on processes directly involved in transmitter mobilization and exocytosis. Modulation of potassium current plays a major role in controlling neuronal excitability and may contribute to a lesser extent to the regulation of transmitter release through actions on the resting potential and on action potential configuration.
15322725
Presynaptic facilitation revisited: state and time dependence
The mechanisms underlying short-term presynaptic facilitation, the enhancement of transmitter release from sensory neurons in Aplysia, induced by serotonin (5-HT), can be divided into two categories: (1) changes in ionic conductances leading to spike broadening and enhancement of Ca2+ influx; and (2) actions on the machinery for transmitter release that are independent of spike broadening and the resulting increases in Ca2+ influx. Spike broadening and the associated enhancement of excitability are induced by the modulation of K+ conductances in the sensory neuron. The cellular mechanisms that contribute to the enhancement of release that is independent of spike broadening are not known and may involve vesicle mobilization or other steps in exocytotic release. These two facilitatory actions of 5-HT are mediated by at least two second-messenger-activated protein kinase systems, protein kinase A (PKA) and protein kinase C (PKC). These two second-messenger cascades overlap in their contributions to synaptic facilitation. However, their relative contributions to enhancement of transmitter release are not simply synergistic but are state- and time-dependent. The state dependence is a reflection of the synapse's previous history of activity. When the synapse is rested (and not depressed), a brief pulse of 5-HT (lasting from 10 sec to 5 min) produces its actions primarily through PKA via both spike broadening-dependent and -independent mechanisms. The broadening primarily involves the modulation of a voltage-dependent K+ current, IKV, with a small contribution by a voltage-independent K+ current, IKS. By contrast, the enhancement of excitability is mediated primarily by the modulation of IKS. As the synapse becomes depressed with repeated activity, the contribution of PKC becomes progressively more important. As is the case with PKA, PKC produces its action both by broadening the spike via modulation of IKV and by a spike broadening-independent mechanism. In addition to being state-dependent, the mechanisms of facilitation are time-dependent. There are differences in the response to 5-HT when it is given briefly to produce short-term facilitation or when the exposure is prolonged. When exposure is brief (< or = 5 min), PKA dominates. When exposure is prolonged (10-20 min), PKC becomes dominant as it is with depressed synapses. Thus, synaptic plasticity appears to be expressed in several overlapping time domains, and the transition between very short-term facilitation and various intermediate duration phases seems to involve interactive processes between the kinases.
924307
The behavior of four neurological mutants of Drosophila.
INGLE gene changes offer an efficient and attractive way to study the genetic control of behavior. Drosophila, with its numerous technical advantages, would seem to provide a fruitful approach in working out the complexities of neurological control. The four neurological mutants discovered serendipitously and described in this report present just such an opportunity. The phenotype common to all four is a rapid shaking of the legs following etherization. Because the mutants appeared, among the progeny of four different males of the original thirty treated, they represent four independent mutational events. Subsequent study has disclosed that three separate gene loci are involved, all on the X-chromosome. This shaking phenotype has been described previously in D. melanogaster by CATSCH (1944) who localized the dominant gene at 58 on the X-chromosome and named the mutant, Shaker, because of its resemblance to the sex-linked dominant in D. funebris (LUERS 1936). Since then three other occurrences of the Shaker phenotype in D. melanogaster have been reported, ( NOVITSKI 1949; FAHMY and FAHMY 1959) all produced by a gene change at the original Shaker locus. To our knowledge, however, these stocks no longer exist nor were the studies carried beyond the description of the phenotype and the localization of the gene.
5401514
Neurogenetic studies of ion channels in Drosophila.
The enormous information handling capacity of nervous systems depends on the proper function of a rich variety of neurons and other excitable cells that display distinctive signal processing characteristics. This physiological complexity reflects cellular differences in the expression and regulation of ion channels. Genetic dissection has been successfully applied in elucidation of mechanisms underlying many biological processes in model systems. Drosophila has been a favored organism for such an approach to complex problems in higher organisms, such as function and development of the nervous system (Benzer, 1973; Pak and Pinto, 1976).
26111943
Voltage‐gated K+ channels of the mammalian brain
Research on voltage‐gated K+ channels of the mammalian brain has seen a flood of new data in the last 2 years. A genetic approach, based on the Shaker mutation of Drosophila, led to cDNA clones for mammalian voltage‐gated K+ channels. K+ channel proteins were detected independently and purified with the help of channel specific toxins. From these studies the structure of two families of mammalian K+ channels emerged. One family is defined molecularly by the sequence homology of its members, the other by binding sites for the snake toxin dendrotoxin. The two families have several members in common. The voltage‐gated K+ channels of mammalian brain are oligomers of glycosilated peptides of 65–95 kDa. The primary structure of these subunits is characterized by six to eight potential transmembrane regions, including the S4 region, the voltage‐sensor of the channels. Associated with at least some K+ channels are 38‐ and 42‐kDa peptides of unknown function. The channels give rise to non‐ or slow‐inactivating K+ currents that are regulated through phosphorylation by both cAMP‐dependent and an endogenous kinase.—Rehm, H.; Tempel, B. L. Voltage‐gated K+ channels of the mammalian brain. FASEB J. 5: 164–170; 1991.
4038041
An essential ‘set’ of K+ channels conserved in flies, mice and humans
The molecular genetic approach to studying K+ channels has revealed that at least four subfamilies of voltage-gated K+ channels originally discovered in Drosophila are conserved in mice and humans. This conservation of the K+ channel subfamilies Shaker, Shal, Shab, and Shaw suggests that not only the broad outlines of membrane electrical properties but also many molecular details as well evolved in the parent species ancestral to both invertebrate and vertebrate life. Shaker, Shal, Shab, and Shaw K+ channels have similar structures, but appear to be independent channel systems: when co-expressed in Xenopus oocytes, all four function independently. These four K+ channel subfamilies may be part of an essential 'set' of excitable channels required by most nervous systems. The task now remaining is to understand the functions of each member of the set.
10313347
Mutational and pharmacological alterations of neuronal membrane function disrupt conditioning in Drosophila.
Neuronal membrane channels of Drosophila melanogaster were altered either genetically or pharmacologically in order to investigate the role of specific ionic currents in the acquisition and retention of a conditioned behavior. Conditioning could not be detected for Shaker mutants, in which the fast transient potassium current (IA) is altered; a second potassium channel mutant, eag (ether a go-go) is conditioned like wild type, but the retention period is abnormally short. The napts mutant (no action potential, temperature sensitive), in which nerve excitability is reduced, also expresses normal acquisition and a shortened period of retention. Double mutants of Sh5 and napts as well as Sh5 treated with tetrodotoxin, are essentially normal with respect to acquisition; in both cases these flies remain retention-defective. These experiments therefore reveal a behavioral phenotype of Drosophila mutants in which the primary physiological defect seems to be in the functioning of specific neuronal ionic channels.
43394914
Drosophila mutations that alter ionic conduction disrupt acquisition and retention of a conditioned odor avoidance response.
We have previously found that genetic alterations in neuronal ion channel function have specific effects on learning and memory of a conditioned courtship behavior in Drosophila. Using a behavioral assay of conditioned odor avoidance, we report here that shaker mutants, in which potassium-channel function is abnormal, are defective in both their rate and maximum level of acquisition when compared to wild-type flies. napts (no action potential, temperature sensitive) mutants, in which nerve excitability is decreased due to faulty sodium-channel function, can achieve a normal (wild-type) level of learning, but the rate of acquisition is reduced. Neither the shaker nor the napts mutations affect memory decay following rest periods greater than 30 min. However, memory may decay more rapidly in both shaker and napts flies during the initial 30-min period following training. These results suggest that neuronal sodium- and potassium-channel function may be of general importance to the processes of acquisition and short-term memory in Drosophila.
15026608
Voltage clamp analysis of membrane currents in larval muscle fibers of Drosophila: alteration of potassium currents in Shaker mutants
The body wall muscles in Drosophila larvae are suitable for voltage clamp analysis of changes in membrane excitability caused by mutations. Both inward and outward ionic currents are present in these muscle fibers. The inward current is mediated by voltage-dependent Ca2+ channels. In Ca2+-free saline, the inward current is eliminated. The remaining outward K+ currents consist of two distinct components, an early transient IA and a delayed steady IK, which are separable by differences in the rate and voltage dependence of activation and inactivation. The steady-state and kinetic properties of the activation and inactivation processes of these two currents are analyzed. The results provide a basis for quantitative analysis of altered membrane currents in behavioral mutants of Drosophila. Previous studies indicate that mutations in the Shaker (Sh) locus alter excitability in both nerve and muscle in Drosophila. Our results support the idea that the channels mediating IA are molecularly distinct from those mediating IK. All Sh mutations studied specifically affect IA without changing the properties of the calcium current and IK. In certain alleles (ShKS133, Sh102, and ShM) IA is eliminated, permitting detailed studies of IK in isolation of IA. Studies of the alleles that do not eliminate IA provide additional information of the channels. In one such allele, Sh5, voltage dependence of IA activation is shifted to more positive potentials. This is accompanied by a less pronounced shift in the voltage dependence of inactivation. These results suggest that Sh5 mutation affects the voltage-sensitive mechanism of both activation and inactivation processes and that these two processes are not controlled by independent parts of the channel. Furthermore, the differential effects of these alleles on different excitable membranes imply that other genes take part in the control of IA. The effects of Sh5 on muscle depend on developmental stage. In larval muscle, Sh5 reduces the amplitude of IA because of the shift in the current-voltage (I-V) relation. In contrast, in adult Sh5 muscles, IA is reported to be normal in amplitude but shows abnormally rapid inactivation (Salkoff, L., and R. Wyman (1981) Nature 293: 228–230). A different allele, ShrK0120, causes a clear defect in nerve excitability, but analysis of IA in ShrK0120 larval muscle reveals I-V relations, inactivation, and recovery from inactivation similar to those seen in normal fibers. We suggest a possible mechanism of combinations of multiple interacting genes participating in the control of potassium channels to account for the presence of a variety of potassium channels in different excitable membranes.
33857184
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.
11317087
A component of calcium-activated potassium channels encoded by the Drosophila slo locus.
Calcium-activated potassium channels mediate many biologically important functions in electrically excitable cells. Despite recent progress in the molecular analysis of voltage-activated K+ channels, Ca(2+)-activated K+ channels have not been similarly characterized. The Drosophila slowpoke (slo) locus, mutations of which specifically abolish a Ca(2+)-activated K+ current in muscles and neurons, provides an opportunity for molecular characterization of these channels. Genomic and complementary DNA clones from the slo locus were isolated and sequenced. The polypeptide predicted by slo is similar to voltage-activated K+ channel polypeptides in discrete domains known to be essential for function. Thus, these results indicate that slo encodes a structural component of Ca(2+)-activated K+ channels.
1473716
Abnormal action potentials associated with the Shaker complex locus of Drosophila.
Intracellular recordings of action potentials were made from the cervical giant axon in Shaker (Sh) mutants and normal Drosophila. The mutants showed abnormally long delays in repolarization. The defect is not due to abnormal Ca(2+) channels, because it persists in the presence of Co(2+), a Ca(2+)-channel blocker. On the other hand, the K(+)-channel blocker 4-aminopyridine causes a similar effect in normal animals, suggesting that the Sh mutant may have abnormal K(+) conductance. Gene-dosage analysis of Sh shows that the defect is not due to underproduction of an otherwise normal molecule; it may be due to an abnormal molecule produced by the mutated gene. Gel electrophoresis failed to detect an abnormal protein, suggesting that, if Sh codes for a nervous system protein, it is rare. Genetic analysis of the Sh locus indicates three regions. Mutations or chromosome breaks in the two flanking regions cause Sh mutant physiology; the central region shows a "haplolethal effect"-i.e., heterozygous females are lethal.
40107598
A Drosophila mutation that eliminates a calcium-dependent potassium current.
A mutation of Drosophila, slowpoke (slo), specifically abolishes a Ca2+-dependent K+ current, IC, from dorsal longitudinal flight muscles of adult flies. Other K+ currents remain normal, providing evidence that IC is mediated by a molecularly distinguishable set of channels. The pharmacological properties of IC are similar to those of Ca2+-dependent currents in some vertebrate cells. The muscle action potential was significantly lengthened in slo flies, indicating that IC plays the major role in its repolarization.
870795
Analysis of repolarization of presynaptic motor terminals in Drosophila larvae using potassium-channel-blocking drugs and mutations.
In Drosophila melanogaster muscles and neuronal cell bodies at least four different potassium currents have been identified whose activity shapes the electrical properties of these cells. Potassium currents also control repolarization of presynaptic terminals and, therefore, exert a major effect on transmitter release and synaptic plasticity. However, because of the small size of presynaptic terminals in Drosophila, it has not been possible to analyze the potassium currents they express. As a first approach to characterizing the ionic currents present at presynaptic motor terminals of Drosophila larvae, we recorded synaptic currents at the neuromuscular junction. From the alterations in evoked synaptic currents caused by various drugs and by mutations known to affect potassium currents in other tissues, we suggest that the repolarizing mechanism in presynaptic terminals consists of at least four distinct currents. One is affected by aminopyridines or Sh mutations, a second component is affected by the slo mutation, a third is sensitive to quinidine and one or more additional components are blocked by tetraethylammonium. Depolarization depends on a presynaptic calcium current, which displays only slight voltage-dependent inactivation. Because the mechanism of repolarization exerts a major effect on synaptic activity, this analysis provides a framework for further genetic and molecular dissection of the basic processes involved in the regulation of transmitter release.
16463809
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.
10571566
Heteromultimeric Interactions among K+ Channel Subunits from Shaker and eag Families in Xenopus Oocytes
Heteromultimeric interactions of K+ channel subunits across different families have been thought to contribute to the functional diversity of ionic currents, as suggested by previous genetic evidence. We present here direct demonstration in Xenopus oocytes that subunits from distinct eag and Shaker families functionally interact, most likely as heteromultimeric channels. Coexpression with eag accelerates the inactivation and slows the recovery from inactivation of the transient Shaker current. Site-directed mutagenesis indicates that the eag carboxyl terminus is crucial for this interaction, exerting effects preferentially on N-type inactivation. Many members of the eag and Shaker families have now been identified and their human homologs implicated in cardiac and neurological disorders. Studies on channel subunit interactions may prove important in understanding the disease pattern and the complex functions of the brain.
33085047
A distinct potassium channel polypeptide encoded by the Drosophila eag locus
Many of the signaling properties of neurons and other electrically excitable cells are determined by a diverse family of potassium channels. A number of genes that encode potassium channel polypeptides have been cloned from various organisms on the basis of their sequence similarity to the Drosophila Shaker (Sh) locus. As an alternative strategy, a molecular analysis of other Drosophila genes that were defined by mutations that perturb potassium channel function was undertaken. Sequence analysis of complementary DNA from the ether a go-go (eag) locus revealed that it encodes a structural component of potassium channels that is related to but is distinct from all identified potassium channel polypeptides.
9899730
Association and colocalization of K+ channel alpha- and beta-subunit polypeptides in rat brain
Recent cloning of auxiliary subunits associated with voltage-gated ion channels and their subsequent coexpression with the channel forming alpha-subunits has revealed that the expression level, gating and conductance properties of the expressed channels can be profoundly affected by the presence of an auxiliary subunit polypeptide. In the present study, we raised antibodies against the beta-subunit associated with the bovine dendrotoxin sensitive K(+)-channel complex and used these antibodies to characterize the related beta-subunit polypeptides in rat brain. The anti-beta-subunit antibodies displayed a specific reaction on immunoblots of rat brain membranes with a major 38 kDa polypeptide, and a minor 41 kDa polypeptide, which correspond closely to the predicted sizes of the Kv beta 2 and Kv beta 1 beta-subunit polypeptides, respectively, recently cloned from rat brain. Reciprocal coimmunoprecipitation experiments revealed that the beta-subunit polypeptides are associated with Kv1.2 and Kv1.4, but not Kv2.1, alpha- subunits. Immunohistochemical staining revealed that the beta-subunit polypeptides were widely distributed in adult rat brain. Moreover, the cellular distribution of beta-subunit immunoreactivity corresponded closely with immunoreactivity for Kv1.2, and to a lesser extent Kv1.4, but not with Kv2.1. These results suggest that neuronal mechanisms may exist to direct the selective interaction of K+ channel alpha- and beta- subunit polypeptides, and that the properties of K+ channels in specific subcellular domains may be regulated by the formation of heteromultimeric K+ channel complexes containing specific combinations of alpha- and beta-subunits.
3008341
Tissue-specific expression of a Drosophila calcium-activated potassium channel
The Drosophila slowpoke (slo) gene encodes a subunit of a CAK channel homologous to the vertebrate BK channel. We have examined slo expression throughout development. It is expressed in muscle cells, neurons of the CNS and PNS, mushroom bodies, a limited number of cells in embryonic and larval midgut and in epithelial-derived tracheal cells. The promoter has been cloned and shown to direct expression in the same pattern as the endogenous gene in both neural and epithelial- derived cells. During pupariation and embryogenesis, slo is expressed in muscles many hours prior to the appearance of functional channels.
18487572
Tissue-specific alternative splicing of hybrid Shaker/lacZ genes correlates with kinetic differences in Shaker K+ currents in vivo
Alternative splicing of the Shaker (Sh) locus of Drosophila generates several transcripts with divergent 5' and 3' domains that produce kinetically distinct K+ currents in Xenopus oocytes. Although suggestive that alternative splicing may be involved in generating K+ channel diversity, clear tissue-specific differences in the distribution of particular Sh gene products have not been demonstrated. Using lacZ as a reporter gene for accurate splicing of variable Sh3' domains, we observe differences in beta-galactosidase expression patterns in transgenic animals that indicate both temporal and spatial regulation of 3' splice choice. The differences in 3'splice choice can account for variation in recovery kinetics of Sh-encoded K+ currents recorded in adult flies. The results indicate that tissue-specific expression of functionally distinct Sh K+ channels is regulated, in part, at the level of pre-mRNA splicing and implicate sequences in or around the 3' splice sites in regulating the choice of 3' domain.
3098989
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.
13241262
Co-assembly of potassium channel subunits in Drosophila: the combinatorial hypothesis revisited.
A diversity of voltage-gated K+ channels has been identified which participates in the regulation of membrane excitability and neural plasticity. After decades of intensive molecular, genetic, pharmacological and electrophysiological studies, the structure and function of K+ channels have been elucidated in great detail. In particular, impressive progress has been made in recent years from studies in Drosophila and the Xenopus oocyte expression system. This review will focus on the variety and physiological functions of K+ channels found in Drosophila. Also discussed is the possible role of coassembly of different combinations of channel subunits in the generation of K+ channel diversity. Various lines of evidence from in situ experiments in Drosophila and heterologous channel expression in Xenopus oocytes will be reviewed.
21801704
Multiple forms of non-associative plasticity in Aplysia: a behavioural, cellular and pharmacological analysis.
A complete understanding of the cellular mechanisms underlying the formation of associations between stimuli, as occurs during classical conditioning, requires an understanding of the non-associative effects of the individual stimuli. The siphon withdrawal reflex of Aplysia exhibits both non-associative and associative learning when a tactile stimulus to the siphon serves as a conditioned stimulus, and tail shock serves as an unconditioned stimulus. In this chapter we describe experiments which examine the non-associative effects of tail shock at three different levels of analysis. At a behavioural level we found that the magnitude, and even the sign of reflex modulation induced by tail shock depended critically on three parameters: (i) the state of the reflex (habituated or non-habituated); (ii) the strength of the tail shock, and (iii) the time of testing after tail shock. Specifically, when non-habituated responses produced by water jet stimuli to the siphon were examined, tail shock produced transient inhibition 90 s later; facilitation of non-habituated responses (sensitization) only emerged after a considerable delay of 20-30 min. When habituated responses were examined, tail shock produced immediate facilitation (dishabituation); the amount of facilitation was inversely related to the strength of tail shock, with stronger shock producing no dishabituation. At a cellular level it was found that the complex excitatory postsynaptic potential (EPSP) in siphon motor neurons produced by water jet stimuli to the siphon provides a reliable cellular correlate of several of the non-associative effects of tail shock that we observe behaviourally. When non-decremented complex EPSPS were examined, strong tail shock produced transient inhibition at a test 90 s after shock.(ABSTRACT TRUNCATED AT 250 WORDS)
46552011
Habituation of an invertebrate escape reflex due to modulation by higher centers rather than local events.
Learning is widely thought to result from altered potency of synapses within the neural pathways that mediate the learned behavior. Support for this belief, which pervades current physiological and computational thinking, comes especially from the analysis of cases of simple learning in invertebrates. Here, evidence is presented that in one such case, habituation of crayfish escape, the learning is more due to onset of tonic descending inhibition than to the intrinsic depression of circuit synapses to which it was previously attributed. Thus, the altered performance seems to depend at least as much on events in higher centers as on local plasticity.
82477205
The Drosophila Giant Fiber System
Normal functioning of the nervous system depends on the formation of vast numbers of specific connections between neurons. During development, each of the thousands, millions, or billions of cells in a nervous system connects with a specific set of target cells. We currently have no knowledge of the molecular basis of this specificity. The long-term goal of our work is to identify genes that are directly involved in neural connectivity and then to use this knowledge to identify the gene products necessary for proper connectivity. It will be a major advance in neuroscience if the class (or classes) of molecules involved in nerve-cell recognition and connection can be identified.
40868928
Conduction in the giant nerve fiber pathway in temperature-sensitive paralytic mutants of Drosophila.
To study electrogenic conduction in neurons of the cervical giant nerve fiber (CGF) pathway in Drosophila adults carrying temperature-sensitive paralytic mutations that affect sodium channels, we recorded dorsal longitudinal muscle (DLM) responses evoked by electrical stimulation of the brain. In the mutants tipE, napts and parats, conduction in certain neurons presynaptic to the CGF failed at about the same temperature at which paralysis occurred in each mutant. Conduction in the CGF and neurons postsynaptic to it remained active in all mutants even at very elevated temperatures. In contrast, analysis of sei mutants showed enhanced spontaneous activity at elevated temperatures in at least some neurons of the CGF pathway. The implications of these results with respect to the normal in vivo functions of these genes in neuronal signalling are considered.
262334
Giant fiber activation of an intrinsic muscle in the mesothoracic leg of Drosophila melanogaster.
Cinematographic analysis reveals that an important component of the light-elicited escape response of Drosophila melanogaster is the extension of the femur-tibia joint of the mesothoracic leg. During the jumping phase of the response, this extension works synergistically with extension of the femur. Femur extension is generated by contraction of the tergotrochanteral muscle (TTM), one of four previously described escape response muscles. Femur-tibia joint extension in the mesothoracic leg has been thought to be controlled by contraction of the tibial levator (TLM), an intrinsic leg muscle. We investigated the activation of the TLM during the escape response. Electrical stimulation of the giant fiber interneuron that mediates the escape response results in activation of the TLM with a latency of 1.46 +/- 0.02 ms. The TLM is innervated by a motor neuron (TLMn) with a large cell body in the mesothoracic ganglion. The TLMn has extensive arborizations in the lateral mesothoracic leg neuromere and has a prominent medially directed neurite. To investigate possible presynaptic inputs activating the TLMn during the escape response, we analyzed the muscle responses of two mutants, giant fiber A1 and bendless. Our analysis suggests that the TLMn is activated by a novel pathway.
7118983
Influence of general anesthetics on a specific neural pathway in Drosophila melanogaster.
The neural pathway that governs an escape response of Drosophila to sudden changes in light intensity can be artificially induced by electrical stimulation of the brain and monitored by electrical recording from the effector muscles. We have refined previous work in this system to permit reliable ascertainment of two kinds of response: (i) a short-latency response that follows from direct excitation of a giant fiber neuron in the interior of the fly brain and (ii) a long-latency response in which electrical stimulation triggers neurons in the optic ganglia that ultimately impinge on the giant fiber neuron. The general anesthetic halothane is reported here to have very different potencies in inhibiting these two responses. The long-latency response is obliterated at concentrations similar to those that cause gross behavioral effects in adult flies, whereas the short-latency response is only partially inhibited at doses that are 10-fold higher. Three other volatile anesthetic agents show a similar pattern. Thus, as in higher organisms, the Drosophila nervous system is differentiated into components of high and low sensitivity to general anesthetics. Moreover, this work shows that one of the sensitive components of the nervous system lies in the optic lobe and is readily assayed by its effect on downstream systems; it should provide a focus for exploring the effects of genetic alteration of anesthetic sensitivity.
16890937
Habituation and dishabituation of a cleaning reflex in normal and mutant Drosophila
Upon tactile stimulation of its thoracic bristle(s), Drosophila cleans with a patterned set of leg movements the field covered by the stimulated bristles. We demonstrate that this cleaning reflex undergoes habituation and dishabituation. Repeated monotonous stimulation of the bristles by controlled air puffs leads to decrement, and finally to disappearance, of leg response. Spontaneous recovery of the response takes place in a time-dependent manner. Restoration of response can also be obtained by application of a high-frequency stimulus directed to other bristles. A mutant, rut, which is defective in learning and in adenylate cyclase activity, can habituate and dishabituate, but habituation is abnormally short-lived. As opposed to both nonassociative and associative learning paradigms used in Drosophila to date, the cleaning reflex lends itself to some aspects of cellular analysis, since single sensory neurons that mediate the input and motor neurons that mediate the behavioral output are identifiable. The modified reflex should therefore be useful in establishing the effects of single gene mutations that affect behavioral plasticity on the development and properties of identified neurons that contribute to discrete modifiable behaviors.
2090992
Neurogenetic analysis of potassium currents in Drosophila: synergistic effects on neuromuscular transmission in double mutants.
Recombinational dissection of the ShrK0120 strain of Drosophila melanogaster demonstrated that its extremely prolonged neuromuscular transmission, a defect qualitatively different from other Sh alleles, results from a synergistic interaction with a second site mutation. This new mutation caused spontaneous repetitive firing in motor axons and increased transmitter release. Complementation tests showed that it is allelic to eag. Combining either eag allele with various Sh alleles reproduced the same extreme defect observed in the original ShrK0120 strain. Thus, from their effects on neuromuscular transmission no qualitative difference among Sh alleles is apparent while Sh, eag, and eag Sh double mutant individuals can be uniquely distinguished. Pharmacological experiments indicated that Sh affects a membrane component sensitive to the potassium channel blockers 4-AP and TEA while eag affects a component sensitive to TEA but not 4-AP. We suggest that eag and Sh preferentially disrupt different potassium currents explaining their synergistic interaction.
24296278
Interactions of membrane excitability mutations affecting potassium and sodium currents in the flight and giant fiber escape systems of Drosophila
SummaryWe have studied the influence of the K+-current mutations eag and Sh and the Na+-current mutation naptsupon 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 naptsmutation 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, naptsdid 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. In eag Sh; nap triple mutants, the non-flight activity and abnormal wing posture were absent, indicating that a reduction of Na+ current counteracts the hyperexcitable influence of the K+-current mutations in this circuit.
19764983
Cobalt-coupled neurons of a giant fibre system in Diptera
SummaryCertain intact nerve cells in flies can be filled with cobalt from presynaptic or postsynaptic neurons. This cobalt coupling is best demonstrated in giant fibre systems where the phenomenon was originally termed ‘transsynaptic staining’. Fine structural analysis of silver-intensified, cobalt-coupled neurons indicates that the passage of cobalt ions occurs at gap junctions that are accompanied by conventional chemical synapses. Cobalt-coupled systems in dipterous insects are uniquely identifiable and can always be detected between the same kinds of neurons. The visualization of cobalt-coupled neurons allows the identification of functional pathways linking the brain to motor neuropils.
24521696
Morphological identification of the motor neurons innervating the dorsal longitudinal flight muscle of Drosophila melanogaster
The dorsal longitudinal flight muscle (DLM) of Drosophila is composed of six muscle fibers (DLM1‐6 from ventral to dorsal) and innervated by five motor neurons (MNs), DLM1‐4 being innervated singly by MN1‐4, and DLM5 and 6 being jointly innervated by MN5. This study identifies and describes the five motor neurons that innervate these six muscle fibers. Horseradish peroxidase (HRP) applied intracellularly to single identified DLM fibers resulted in the labeling of the single motor neuron innervating that muscle fiber by retrograde transsynaptic transport. This method allowed positive identification of the motor neuron innervating a particular muscle fiber, since only the innervating neuron was labeled. The axonal pathway, soma, and dendritic distribution of each labeled motor neuron was traced in the thoracic ganglion, and their relative positions were determined.
31087008
Alteration of potassium channel gating: Molecular analysis of the drosophila Sh5
 mutation
The Drosophila Shaker (Sh) gene encodes a family of voltage-gated K+ channels. Mutant alleles of Sh alter the currents expressed from these channels in a variety of ways. To identify the molecular basis of these alterations, Sh transcript sequences were amplified using the polymerase chain reaction after reverse transcription of mutant RNA. Amplified products from each mutant were cloned and sequenced. Two alleles, Sh102 and Sh5, had single base substitutions in the central conserved region shared by all Sh channels. RNA synthesized in vitro from a cDNA construct carrying the Sh5 mutation was injected into Xenopus oocytes. Currents expressed by the mutant RNA were altered in their voltage dependence of activation and inactivation, similar to the alterations in Sh currents recorded from different preparations of Sh5 fly tissue. The changes in current properties and the location of the mutation are consistent with the participation of a novel region of the channel in voltage gating.
8065019
Molecular basis of altered excitability in Shaker mutants of Drosophila melanogaster.
Mutations in the Shaker (Sh) locus of Drosophila melanogaster have differing effects on action potential duration and repolarization in neurons as well as on A‐type K+ channels (IA) in muscle. The molecular basis of three exemplary Sh alleles (ShKS133, ShE62 and Sh5) has been identified. They are point mutations in the Sh transcription unit expressing aberrant voltage‐gated A‐type K+ channels. Replicas of each mutation have been introduced by in vitro mutagenesis into Sh cDNA. The expression of in vitro transcribed mutant Sh cRNA in Xenopus laevis oocytes reproduced the specific phenotypic traits of each Sh allele. The lack of IA in ShKS133 is due to a missense mutation within a sequence motif occurring in all hitherto characterized voltage‐gated K+ channel forming proteins. The reduction of IA in ShE62 is due to a mutation in an AG acceptor site. The intervening sequence between exons 19 and 20 is not spliced in ShE62 RNA. As a consequence, ShE62 flies do not contain the full complement of Sh K+ forming proteins. Finally, the Sh5 mutation leads to an altered voltage dependence of K+ channel activation and inactivation as well as to an accelerated rate of recovery from inactivation. This is due to a missense mutation altering the amino acid sequence of the proposed transmembrane segment S5 of the Sh K+ channels. Segment S5 is located adjacently to the presumed voltage sensor of voltage‐gated ion channels. The results explain the altered properties of excitable cells in Sh mutants and provide a general model for the possible role of A‐type K+ channels in modulating action potential profiles.
11151616
Altered synaptic transmission in Drosophila hyperkinetic mutants.
Synaptic transmission in Drosophila can be altered by mutations in specific genes. For example, mutations in the Shaker (Sh) gene, which encodes the rapidly inactivating A-type potassium channel, cause repetitive nerve firing and prolonged transmitter release at the neuromuscular junction. Here we show that mutations in the Hyperkinetic (Hk) gene also affect the properties of synaptic transmission at the neuromuscular junction. In particular, we find that whereas single or low frequency nerve stimulation evokes a wild type postsynaptic response, at higher frequencies of nerve stimulation, each stimulus results in repetitive nerve firing and increased postsynaptic response, which is similar to that observed in Sh mutants. Various experiments suggest that this increased postsynaptic response results from prolonged depolarization of the nerve terminal, leading to increased transmitter release at the neuromuscular junction. The similarity in phenotypes between Sh and Hk mutants, along with the observation that Sh is epistatic to Hk in its effects on synaptic transmission, suggest that Hk acts on synaptic transmission by an effect on A-type potassium channels.
23018011
The Passover locus in Drosophila melanogaster: complex complementation and different effects on the giant fiber neural pathway.
Drosophila melanogaster bearing the Passover mutation fail to jump in response to a light-off stimulus. Pas also disrupts some of the synapses between the neurons of the giant fiber system which mediate this escape behavior. We have mapped Pas to the 19E subdivision of the polytene X chromosome. Our genetic analyses reveal that deletions of either of two nonoverlapping regions fail to fully complement Pas. Heterozygotes of Pas with chromosomal deletions in the vicinity of polytene band 19E3 exhibit the full set of neuronal defects shown by Pas homozygotes. Alleles of the R-9-29 complementation group, which maps to band 19E3, exhibit a complex pattern of complementation with Pas. Heterozygotes combining the lethal R-9-29 alleles with Pas are all viable, some complement the neuronal defects of Pas, but most exhibit these defects. The viable shaking-B2 mutation also fails to complement Pas, the R-9-29 alleles or the 19E3 deficiencies. The R-9-29 locus may contain two functional domains, one required for viability the other for normal neuronal phenotype, trans-Heterozygotes bearing mutant alleles or a deficiency of the first region (19E3) together with deficiencies of the second region (19E5-6) also exhibit some of the neuronal defects shown by the Passover mutant. Deficiencies which delete the entire 19E3 to 19E6 interval do not produce this phenotype when heterozygous with a normal X chromosome. Thus normal function requires a cis-interaction between the two regions. These findings raise the possibility that the gene mutated by Pas is split or separated from a cis-activator by at least one other gene.
44992041
Activity-dependent changes in the intrinsic properties of cultured neurons.
Learning and memory arise through activity-dependent modifications of neural circuits. Although the activity dependence of synaptic efficacy has been studied extensively, less is known about how activity shapes the intrinsic electrical properties of neurons. Lobster stomatogastric ganglion neurons fire in bursts when receiving synaptic and modulatory input but fire tonically when pharmacologically isolated. Long-term isolation in culture changed their intrinsic activity from tonic firing to burst firing. Rhythmic stimulation reversed this transition through a mechanism that was mediated by a rise in intracellular calcium concentration. These data suggest that neurons regulate their conductances to maintain stable activity patterns and that the intrinsic properties of a neuron depend on its recent history of activation.
4347563
Action-potential propagation gated by an axonal IA-like K+ conductance in hippocampus
Integration of membrane-potential changes is traditionally reserved for neuronal somatodendritic compartments. Axons are typically considered to transmit reliably the result of this integration, the action potential, to nerve terminals. By recording from pairs of pyramidal cells in hippocampal slice cultures, we show here that the propagation of action potentials to nerve terminals is impaired if presynaptic action potentials are preceded by brief or tonic hyperpolarization. Action-potential propagation fails only when the presynaptic action potential is triggered within the first 15–20 ms of a depolarizing step from hyperpolarized potentials; action-potential propagation failures are blocked when presynaptic cells are impaled with electrodes containing 4-aminopyridine, indicating that a fast-inactivating, A-type K+ conductance is involved. Propagation failed between some, but not all, of the postsynaptic cells contacted by a single presynaptic cell, suggesting that the presynaptic action potentials failed at axonal branch points. We conclude that the physiological activation of an IA-like potassium conductance can locally block propagation of presynaptic action potentials in axons of the central nervous system. Thus axons do not always behave as simple electrical cables: their capacity to transmit action potentials is determined by a time-dependent integration of recent membrane-potential changes.
4349788
K+ channel regulation of signal propagation in dendrites of hippocampal pyramidal neurons
Pyramidal neurons receive tens of thousands of synaptic inputs on their dendrites. The dendrites dynamically alter the strengths of these synapses and coordinate them to produce an output in ways that are not well understood. Surprisingly, there turns out to be a very high density of transient A-type potassium ion channels in dendrites of hippocampal CA1 pyramidal neurons. These channels prevent initiation of an action potential in the dendrites, limit the back-propagation of action potentials into the dendrites, and reduce excitatory synaptic events. The channels act to prevent large, rapid dendritic depolarizations, thereby regulating orthograde and retrograde propagation of dendritic potentials.
29131133
"Latent" inhibitory connections become functional during activity-dependent plasticity.
Simultaneous pre- and postsynaptic recordings from identified glycinergic inhibitory interneurons and the Mauthner cell showed that 25% of the afferents produced no or extremely small postsynaptic responses. Morphological determination of the number of contacts made by these cells on the Mauthner cell revealed a connectivity similar to that of functional neurons which always produce clear inhibitory postsynaptic potentials, suggesting that most of the endings, made by weak interneurons are silent. Intraaxonal injection of 4-aminopyridine or Ca2+ greatly enhanced transmission at functional connections but did not modify those which were ineffective. However, after eighth nerve tetanic stimuli, transmission at the weak connections was unmasked or enhanced for prolonged periods and was twice as likely to be potentiated, with a 6-fold greater mean enhancement than the potent ones. This result provides additional support for long-term potentiation of inhibitory synapses. Furthermore, weakly functional junctions represent a "reserve" pool which can be critical for the expression of plasticity within a network, and, consequently, for setting the threshold of reflex activities such as the escape reaction mediated by the Mauthner cell.
23410356
Occult Drosophila calcium channels and twinning of calcium and voltage-activated potassium channels
In the membrane of the flight muscle cells of developing Drosophila a large calcium-sensitive potassium current, IKc, was found. It was present before the development of voltage-activated potassium channels and seems to be the first potassium current to develop in the membrane. Also present in these early cells were large numbers of occult (hidden) calcium channels, which remained inactive until the end of pupal development. These inactive calcium channels could be made to function by injecting adenosine triphosphate or ethyleneglycol tetraacetic acid into the early cells. IKc has kinetic properties resembling the later developing voltage-sensitive current IKv, and is distinct from the fast, transient calcium-dependent outward current IAc, which appears much later in development. IAc closely resembles the voltage-sensitive current IAv, also present in these cells. Thus, both of the voltage-sensitive potassium channel types, IAv and IKv, have similar calcium-sensitive counterparts, IAc and IKc, that are present in the same cells.
14387266
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.
28780428
Role of calcium‐activated potassium channels in transmitter release at the squid giant synapse.
1. Several compounds known to block Ca2+‐activated K+ channels were microinjected into squid 'giant' presynaptic terminals to test the hypothesis that these channels mediate Ca2+‐dependent neurotransmitter release. 2. Injection of tetrapentylammonium, nonyl‐triethylammonium and decamethonium all reversibly blocked transmission evoked by presynaptic action potentials. 3. All three of these compounds blocked presynaptic Ca2+ channels. The actions of tetrapentylammonium on presynaptic Ca2+ influx were examined in detail and found to be quantitatively sufficient to account for the ability of this compound to inhibit transmitter release. 4. Injection of Ba2+, another agent known to block Ca2+‐activated K+ channels, also reversibly blocked evoked transmitter release. Ba2+ simultaneously enhanced basal (asynchronous) transmitter release and thus may be decreasing evoked release by depleting transmitter quanta available for release. 5. None of these results provide any support for the hypothesis that Ca2+‐activated K+ channels mediate Ca2+‐dependent release of transmitter at the squid synapse. However, our results have identified a new class of compounds that block Ca2+ channels from their cytoplasmic surface.
15123651
Presynaptic calcium signals and transmitter release are modulated by calcium-activated potassium channels
The regulation of synaptic transmission by Ca(2+)-activated potassium (gKca) channels was investigated at the frog neuromuscular junction (nmj). Charybdotoxin (CTX), a blocker of certain types of gKca channels, induced a twofold increase of transmitter release. Similar results were obtained with purified natural toxin, synthetic toxin, and recombinant toxin. Apamin, a blocker of a different type of gKca channel, did not alter transmitter release. CTX was ineffective after intraterminal Ca2+ buffering was increased by application of the membrane-permeant Ca2+ buffer dimethyl-BAPTA-AM. By itself, the permeant buffer first caused a slight increase in transmitter release before release was eventually decreased. This increase of release did not occur when the buffer was applied in the presence of CTX or Ba2+, another gKca channel blocker. Stimulus-evoked entry of Ca2+ in nerve terminals, detected with the fluorescent Ca2+ indicator FLUO-3, was increased after blockade of gKca channels by CTX. CTX had no effect on the amount or the time course of synaptic depression. The results are consistent with the hypothesis that CTX-sensitive gKca channels normally narrow the presynaptic action potential and thus, by indirectly regulating Ca2+ entry, can serve as powerful modulators of evoked transmitter release. In order to affect presynaptic action potentials, the gKca channels must be located close to Ca2+ channels.
41397221
Calcium-dependent modulation of the facilitation of transmitter release at neuromuscular junctions of Drosophila

The relationship between external Ca and twin pulse facilitation of transmitter release has been investigated in neuromuscular junctions of Drosophila larvae using external electrodes to record excitatory junctional currents (ejcs). The degree of synaptic facilitation decreases in inverse relation to [Ca2+]0, even at levels of transmitter release that do not cause depletion of quanta. This reverse-dependency of facilitation to external calcium is abolished by the slow mutation which suppresses the transient Ca-dependent K current (ICF). A likely explanation for these observations is that the activation of ICF by residual Ca shortens the action potential duration resulting in a reduced release of transmitter to the test stimulus. This represents a negative feed-back mechanism of synaptic function which opposes synaptic facilitation.
27164687
Activity-dependent potentiation of recurrent inhibition: a mechanism for dynamic gain control in the siphon withdrawal reflex of Aplysia
The siphon withdrawal response (SWR) of Aplysia supports several forms of learning that are under both excitatory and inhibitory control. Here we examine the role of interneuronal processing on the regulation of siphon responses, with an emphasis on the role of inhibition. We focus on the recurrent circuit formed by the excitatory interneuron L29 and the inhibitory interneuron L30, and show that this circuit provides a mechanism for use-dependent regulation of excitatory input onto siphon motor neurons. We utilized a reduced preparation in which input to the SWR circuit was elicited by taps applied to the siphon; tap-evoked EPSPs were measured in LFS siphon motor neurons. We first show that L29 is an important source of excitatory input to LFS motor neurons: voltage-clamp inactivation of a single L29 (out of five) results in a significant reduction of tap-evoked EPSPs. Next, we demonstrate that direct intracellular activation of L29, surprisingly, produces transient inhibition of evoked input to motor neurons that lasts up to 40 sec. We then provide several lines of evidence that the mechanism of L29-induced inhibition is through the recruitment and potentiation of recurrent inhibition from L30: (1) L29 activation results in reduced tap-evoked responses of other (nonactivated) L29s; (2) direct activation of L30 mimics the inhibitory effects produced by L29 activation (LFS neurons receive no direct synaptic input from L30); and (3) the L30 IPSP is significantly potentiated as a result of its own activity, whether produced directly (by L30 activation) or indirectly (through L29 activation). This IPSP potentiation has the same time course as L29-induced inhibition of motor neuron responses. Thus activity-dependent potentiation of L30 transmission can inhibit motor neuron responses, in part through inactivation of the L29 interneuronal pool. Finally, we propose that L29-L30 interactions provide a mechanism for dynamic gain control in the SWR.
25524462
Gain control in a proprioceptive feedback loop as a prerequisite for working close to instability
In the artificially closed femur-tibia control system of stick insects oscillations were induced in 3 different ways: Increasing the phase-shift by introducing an electronic delay, afference sign reversal and coupling the tibia to an inert mass. In all 3 cases the oscillations stopped after some time. The gain of the open-loop system was significantly smaller after the oscillations. Afference sign reversal by surgically crossing of the receptor apodeme of the femoral chordotonal organ for 25–85 days does not lead to altered characteristics of the control loop. When sinusoidal passive movements are forced upon the intact femur-tibia joint the forces resisting these movements do not decrease with time. In contrast to direct stimulation of the femoral chordotonal organ, these passive movements also influence the contralateral leg. The experiments show that the gain-control system of the femur-tibia control loop of stick insects consists of at least two components: A sensitization system (with inputs from many kinds of stimuli indicating some kind of disturbance) increases the gain of all reflex loops. A specific habituation-like system decreases the gain with repetitive stimulation only of one control system.
28020069
Genetic manipulation of an abnormal jump response in Drosophila.
The hyperkinetic mutants, Hk(1) and Hk(2), jump and fall over when an object moves near them. This behavior, the kinetogenic response, has been measured by the experimenter moving his hand above a vial containing a single fly and scoring the number of positive responses in fifty trials. The response is higher in Hk(1) than in Hk(2) and has remained so over a period of several years and in different genetic backgrounds. The Hk(1)/Hk(2) heterozygote also responds to movements, establishing the allelism of the two mutants.-When Sh(5), a shaker mutant at another locus on the X chromosome, is introduced into the same chromosome as Hk(1) or Hk(2), the response is reduced in proportion to the number of Sh(5) mutant genes added.-When Hk(1) or Hk(2) is heterozygous with a deficiency for the hyperkinetic region of the X chromosome, the expression is more abnormal than the respective Hk(1) or Hk(2) homozygote. This shows that the mutant genes are producing an altered gene product, or less of the normal, since one mutant gene by itself has a more abnormal expression than two. A tentative explanation has been offered for the observed mutant behaviors.
8511331
A family of putative potassium channel genes in Drosophila.
Mutant flies in which the gene coding for the Shaker potassium channel is deleted still have potassium currents similar to those coded by the Shaker gene. This suggests the presence of a family of Shaker-like genes in Drosophila. By using a Shaker complementary DNA probe and low-stringency hybridization, three additional family members have now been isolated, Shab, Shaw, and Shal. The Shaker family genes are not clustered in the genome. The deduced proteins of Shab, Shaw, and Shal have high homology to the Shaker protein; the sequence identity of the integral membrane portions is greater than 50 percent. These genes are organized similarly to Shaker in that only a single homology domain containing six presumed membrane-spanning segments common to all voltage-gated ion channels is coded by each messenger RNA. Thus, potassium channel diversity could result from an extended gene family, as well as from alternate splicing of the Shaker primary transcript.
30450098
Genetic analysis of Drosophila neurons: Shal, Shaw, and Shab encode most embryonic potassium currents
In this study, we perform the first genetic analysis of K+ currents in Drosophila embryonic neurons revealing the identity of the currents present. Unlike muscles, where the presence of Shaker is obvious, Shaker currents are not detectable in these neurons. In contrast, we show that Shal is as important in these neuronal cell bodies as Shaker is in muscles. Only three single-channel currents were found, all of which are genetically separable. Shal encodes a 4 pS transient channel. Whole-cell Shal currents have a wide variety of inactivation rates which, in contrast to a mechanism such as heteromultimer formation, is due to single Shal channels assuming different gating modes. Shaw encodes a 42 pS noninactivating channel distinctive for its extremely low voltage sensitivity; Shaw channels have a total equivalent gating charge of 0.90 e- charges, in sharp contrast to 7 e- reported for Shaker channels. An 11 pS slowly inactivating channel also present in these neurons may be encoded by the Shab gene. Thus, of four voltage- dependent K+ channel genes now cloned in Drosophila, all except Shaker appear to be expressed in the cell bodies of these neurons. It is only in Drosophila that a study such as this one can be done. Because Drosophila contains only a single member of each of the four subfamilies of K+ channel genes (unlike mammals), we can eliminate an entire subfamily with a mutation to a single gene. Here, we have examined the effect of eliminating each of three subfamilies. Such a task is presently impossible to accomplish in any mammalian system.
87393224
The Mammalian Startle Response
One of the ultimate goals of neuroscience is to determine how the brain directs and changes behavior in man. Given the enormous complexity of this endeavor, an increasing number of neuroscientists have focused on invertebrates and lower vertebrates in an effort to simplify the problem. Eventually, however, it will be necessary to analyze the cellular basis of behavior in a complex mammalian brain. To approach the problem at this level, it would be useful to study a relatively simple behavior that can be elicited in mammals and that is sensitive to a variety of experimental treatments.
26928166
A behavioral analysis of habituation and sensitization of shortening in the semi-intact leech
We have previously demonstrated that the shortening reflex of the leech Hirudo medicinalis displays habituation, dishabituation, and sensitization. In this paper we demonstrate that the shortening reflex of the semi-intact animal also displays these phenomena. In the first experiment we found that the magnitude of the touch-elicited shortening reflex decreased as a result of repeated stimulations of the skin every 2 min. The second experiment examined the change in this reflex as a function of the interstimulus interval (ISI). The reflex failed to decrease when the ISI was 10 sec, but decreased significantly when the ISI was either 45 or 360 sec. Finally, in a third experiment we found that the presentation of noxious stimuli prior to habituation training prevented habituation. Thus, as we have observed previously in the intact animal, the semi-intact animal displays habituation, dishabituation, and sensitization.
1345183
Habituation of the ultrasound-induced acoustic startle response in flying crickets.
The ultrasound-induced negative phonotactic response of tethered, flying Australian field crickets habituates to repeated stimuli. Using the magnitude of the metathoracic leg's swing during a series of ultrasonic stimuli as a measure of habituation, we show that: (1) the response declines exponentially; (2) the response recovers spontaneously; (3) repeated trials produce more rapid and stronger habituation; (4) successive stimuli presented more rapidly produce more rapid and stronger habituation; (5) a weaker stimulus intensity produces more rapid and stronger habituation; (6) the habituation shows stimulus generalization (i.e. the response is similar for different ultrasonic frequencies); (7) a novel stimulus produces dishabituation; and (8) the effect of the dishabituating stimulus habituates after repeated trials. These findings place habituation of cricket negative phonotaxis in the context described for habituation in mammals.
56744759
Habituation of the Landing Response of Drosophila
A device was developed to automatically record the landing response of Drosophila. Tethered Drosophilae fly in front of a viewing screen onto which moving pattern can be projected. The movement of the front legs during a landing response can be monitored by two sensitive light gates: one records the onset of the forward thrust, the other the completion of the response as defined by a full forward extension of the legs.
8769705
Factors affecting habituation and recovery from habituation in the nematode Caenorhabditis elegans.
In four experiments, the factors that affect the rate of habituation, the degree of habituation, and the rate of recovery from habituation in a simple reflex circuit in Caenorhabditis elegans were investigated. The results showed that habituation was more pronounced and faster, and that recovery from habituation was more rapid, with short interstimulus intervals (ISIs) than with longer ISIs. Rate of recovery differed in animals that had reached asymptotic response levels when compared with animals still in the descending portion of the habituation curve. Once animals reached asymptotic response levels, rate of recovery appeared to be determined by ISI and not by additional stimuli.
45499151
Habituation and iterative enhancement of multiple components of the Tritonia swim response.
To understand the relationship between memory storage sites in the brain and learned changes in behavior, the learned behavior must be characterized. However, even simple types of learning may be quite complex. Repeated elicitation of the Tritonia swim produced multiple changes in the response. Several types of acquisition curves were observed in a single experiment depending on the response component measured. Habituation (response decrement) and iterative enhancement (response facilitation) occurred simultaneously in different swim components. The acquisition curve for one component could be modulated by stimulus strength. Because the Tritonia swim neural network is well studied, it may be possible to causally relate experience-dependent behavioral changes to the underlying memory trace in this marine mollusk.
39067456
A neuropeptide gene defined by the Drosophila memory mutant amnesiac.
Mutations in genes required for associative learning and memory in Drosophila exist, but isolation of the genes has been difficult because most are defined by a single, chemically induced allele. Here, a simplified genetic screen was used to identify candidate genes involved in learning and memory. Second site suppressors of the dunce (dnc) female sterility phenotype were isolated with the use of transposon mutagenesis. One suppressor mutation that was recovered mapped in the amnesiac (amn) gene. Cloning of the locus revealed that amn encodes a previously uncharacterized neuropeptide gene. Thus, with the cloning of amn, specific neuropeptides are implicated in the memory process.
3142373
Learning without Performance in PKC-Deficient Drosophila
The performance of a task is often assumed to be a prerequisite for the learning of many tasks, including the associative conditioning of courtship in the fruit fly, Drosophila melanogaster. Transgenic flies specifically inhibited for the enzyme protein kinase C dissociate the acquisition of learning and memory from performance of the task. They fail to show immediate suppression of courtship but nonetheless develop normal memory of it.
44908182
Patterned neural activity of a mutant Drosophila melanogaster.
Hyperkinetic (1P), a behavioral mutant of Drosophila melanogaster, shows rhythmic leg-shaking action derived from mutation of a single, sex-linked gene. The neural mechanism governing this abnormal motor function consists of rhythmic bursts of activity produced by motor neurons located within the thoracic ganglion. The mechanism of the pattern formation was independent both of sensory information and higher centers in the central nervous system but endogenous within the ganglion. Two types of neurons which appear to be involved in the pattern formation were found within six limited regions.
30373344
A simple chemosensory response in Drosophila and the isolation of acj mutants in which it is affected.
Although the Drosophila visual system has been described extensively, little is known about its olfactory system. A major reason for this discrepancy has been the lack of simple, reliable means of measuring response to airborne chemicals. This paper describes a jump response elicited by exposing Drosophila to chemical vapors. This behavior provides the basis for a single-fly chemosensory assay. The behavior exhibits dose dependence and chemical specificity: it is stimulated by exposure to ethyl acetate, benzaldehyde, and propionic acid but not ethanol. Animals can respond repeatedly at short intervals to ethyl acetate and propionic acid. The response relies on the third antennal segments. To illustrate the use of this behavior in genetic analysis of chemosensory response, nine acj mutants defective in response are isolated (acj = abnormal chemosensory jump), and their responses to two chemicals are characterized. All of the acj mutants are normal in giant fiber system physiology, and two exhibit defects in visual system physiology.
23156045
Development and maintenance of a simple reflex circuit in small-patch mosaics of Drosophila: effects of altered neuronal function and developmental arrest.
A combined genetic, anatomical, and behavioral approach has been undertaken to study the developmental and functional plasticity of identified bristle mechanosensory neurons in Drosophila. A stereotyped grooming reflex in decapitated flies enabled simple but reliable assessments of the functional output of individual bristle sensory cells to correlate with their axonal projections and terminal arbors revealed by the cobalt backfill technique. Construction of small-patch mosaics that contain only a single mutant bristle allowed functional perturbation of individual neurons within an otherwise normal environment. Mutations that affect nerve excitability and membrane recycling have been used to examine their effects on neuronal pathfinding, arborization, and the initiation and maintenance of functional connections. Previous studies (Burg and Wu, 1986, J. Neurosci. 6:2968-2976; 1989, Dev. Biol. 131:505-514) have demonstrated that para(ts)nap(ts) double-mutant sensory neurons, in which action potentials are unconditionally blocked by defects in sodium currents, and eag Sh double-mutant sensory cells, in which membrane excitability is increased by alterations in potassium currents, can establish and maintain central projections that are indistinguishable from their functionally normal counterparts. Mutations of the shi(ts) gene cause a temperature-sensitive, reversible block of the membrane recycling process, resulting in arrest of neuronal growth in culture (Kim and Wu, 1987, J. Neurosci. 7:3245-3255) and depletion of synaptic vesicles that leads to transmission blockade at established synapses (Ikeda, Ozawa, and Hagiwara, 1976, Nature 259:489-491; Koenig and Ikeda, 1983, J. Neurobiol. 14:411-419; 1989, J. Neurosci. 9:3844-3860). Prolonged heat treatments (up to 16% of total development time) of small-patch shi(ts) mosaics at different pupal stages did not prevent the establishment of central projections characteristic of the various sensory cell types. However, none of the shi(ts) sensory neurons heat-pulsed during the initial or the final 16% of pupal development were able to initiate the reflex behavior, although a proportion of those treated in other periods apparently established functional contacts with appropriate targets to support the characteristic cleaning reflex. The possibility exists that the membrane recycling process blocked in shi(ts) cells provides a crucial mechanism for cell-cell interactions taking place during initial differentiation and final synaptic stabilization, and possibly competition, in the developing sensory neuron. Heat treatments of adult shi(ts) mosaics blocked the reflex initiated by the mutant (but not the surrounding normal) bristles, as expected from the effect of synaptic block.(ABSTRACT TRUNCATED AT 400 WORDS)
7386702
Genetic feminization of brain structures and changed sexual orientation in male Drosophila
The neural basis of sexual orientation in Drosophila was studied by the production of males with regionally feminized brains. Such flies express the female form of the sex determination gene transformer in a limited number of neurons under the control of GAL4 enhancer trap inserts. This method facilitated the creation of lines with a stable pattern of feminization. In tests of sexual preferences, flies that were feminized in a portion of the antennal lobes or in a subset of the corpora pedunculata (mushroom bodies) courted both males and females. These two brain structures, both of which are involved in olfactory processing, may function in the recognition of sex-specific pheromones, in the control of sex-specific behaviors, or both.
19346168
The Drosophila brain revisited by enhancer detection.
The patterns of gene expression in the Drosophila brain were studied by using the lacZ reporter gene carried on an enhancer detector element. From the analysis of serial sections of the heads of 6000 enhancer detector lines, reporter gene expression in some lines was found to generally follow boundaries established by cell type or anatomy, revealing distinct patterns of lacZ expression restricted to the lamina, the medulla, mushroom bodies, antennal lobes, or other anatomical subdivisions. About 15% of the lines showed ubiquitous expression in most or all head tissues and 25% of the lines showed expression throughout the CNS. Another quarter of the lines showed widespread expression in the CNS, with large regions of the brain showing expression. This suggests that the majority of detected genes are expressed with little spatial specificity. The expression patterns produced by 12 different insertions at the rutabaga locus were found to be extremely similar in the brain and offer strong evidence that the enhancer detector elements generally report the activity of an adjacent gene. Only 15% of the lines were judged to have relatively specific expression in one brain region, including those with preferential or specific expression in the mushroom bodies, antennal lobes, lamina, medulla, etc. The cytological insertion sites for elements showing preferential mushroom body expression were found to be dispersed in the genome at approximately 50 different chromosomal regions. In addition to providing a broad picture of the transcriptional activity in the Drosophila brain, these enhancer detector lines offer access to interesting new genes and form a novel collection of lines in which identifiable brain cells are marked in a reproducible way.
19872000
Drosophila mitotic domain boundaries as cell fate boundaries.
Fate determination in Drosophila embryos is evidenced by the appearance of mitotic domains. To identify fate or fates of cells, individual cells in mitotic domains 2, 8, and 15 were marked and monitored through development. Comparison of the different fates indicated that domain boundaries are cell fate boundaries. Cells were marked by expression of GAL4-dependent transgenes after photoactivation of a caged GAL4VP16 analog that had its DNA binding activity inhibited with a photolabile blocking reagent. Caged GAL4VP16 was also used to induce gene expression in Xenopus embryos. Thus, photoactivated gene expression is a versatile tool for spatiotemporal control of gene expression.