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Department of Biological Sciences, University of Iowa, Iowa City, Iowa
Submitted 22 September 2006; accepted in final form 24 October 2006
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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; Shepherd 2004). A variety of inward Na+ and Ca2+ currents and outward K+ currents take part in shaping neuronal action potentials and firing patterns (Hille 2001). In particular, molecular studies have revealed a much greater diversity of K+ channel subtypes than that of Na+ and Ca2+ channels (Coetzee et al. 1999; Jan and Jan 1997). Regulation of such molecular diversity facilitates the fine tuning of neuronal excitability and thus enriches spike patterning.
K+ currents have been classified according to their gating, kinetic, and pharmacological properties. In a variety of excitable cells, voltage-activated outward K+ currents are composed of transient, inactivating IA and sustained, noninactivating IK (Hille 2001). Drosophila Shaker (Sh) mutants, initially isolated on the basis of their abnormal shaking behavior (Kaplan and Trout 1969), have made possible the cloning of the first K+ channel gene (Kamb et al. 1987; Papazian et al. 1987; Pongs et al. 1988) and subsequent identification of three additional Drosophila K+ channel genes of the Sh family, Shab, Shaw, and Shal (Butler et al. 1989), and their vertebrate counterparts Kv 1, 2, 3, and 4 (Coetzee et al. 1999). In heterologous expression systems, Sh and Shal channels mediate IA-like, fast-inactivating currents, whereas both Shaw and Shab regulate IK-like, slowly inactivating currents (Iverson et al. 1988; Timpe et al. 1988; Wei et al. 1990). Drosophila point mutations of Sh and Shab have demonstrated the in vivo roles of IA and IK channels at different levels, from cellular physiology to behavior (Fox et al. 2005; Hegde et al. 1999; Jan et al. 1977; Salkoff and Wyman 1981; Tanouye et al. 1981; Ueda and Wu 2006; Wang et al. 2002), and can provide information about the regulation of neuronal excitability. Rich repertories of neuronal spike patterns have been described in several Drosophila semi-intact preparations (Choi et al. 2004; Fox et al. 2006; Ikeda and Kaplan 1970). Nevertheless, it is difficult to delineate the spike patterns generated by synaptic interactions from those reflecting intrinsic neuronal membrane excitability in these preparations. Dissociated cell culture systems provide isolated conditions without cell-cell contacts and controlled ionic environment to eliminate contributions from synaptic interactions.
The "giant" neuron culture system of Drosophila derived from cytokinesis-arrested embryonic neuroblasts displays differentiated morphological and molecular characteristics of different neuronal lineages (Wu et al. 1990). These enlarged cells facilitate Ca2+ imaging (Berke and Wu 2002; Berke et al. 2006) and electrophysiological recordings of Na+ and Ca2+ action potentials of different firing patterns (Saito and Wu 1991; Yao and Wu 1999, 2001; Zhao and Wu 1997). To explore the biophysical characteristics and functional roles of Sh and Shab channels, we performed current- and voltage-clamp recordings on the same cells in this culture system. The remaining IA in Sh and IK in Shab null mutants could provide opportunities to distinguish properties of Sh versus non-Sh IA channels (encoded by Shal and possibly other unidentified genes) as well as Shab versus non-Shab IK channels (encoded by Shaw and other candidate genes) (cf. Chen et al. 2000; Robertson et al. 1996). Our results demonstrate the profound effect of Shab mutations on spike firing patterns and the distinctions between Sh channels and their counterpart, such as Shal channels. We also took advantage of the GAL4-UAS system (Brand and Perrimon 1993) to demonstrate different profiles of K+ current kinetics and firing properties in cell-lineage defined neuronal subsets and their specific alterations by Sh and Shab mutations. Preliminary accounts of this work have appeared in abstract form (Peng and Wu 2004).
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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The wild-type (WT) strain was Canton S. Two Shab mutant alleles, Shab1 (z66, a hypomorph) (Hegde et al. 1999) and Shab3 (9g, a null) (Singh and Singh 1999), were used in this study. Two Sh null alleles, ShM and Sh133 (Haugland and Wu 1990; Tanouye and Ferrus 1985; Wu and Haugland 1985; Zhao et al. 1995), were from the original collection of Dr. S. Benzer of Cal Tech. For upstream activating sequence-green fluorescent protein (UAS-GFP) expression experiments, the GH146-Gal4 UAS-CD8-GFP/CyO line (Wong et al. 2002) was a gift from Dr. J. Wang (University of California, San Diego), and two separate 201Y-Gal4 and UAS-CD8-GFP lines from Dr. Y. Zhong (Cold Spring Harbor Lab) were used to produce recombined homozygous lines. All fly stocks were maintained at room temperature with standard fly medium.
Single embryo "giant" neuron culture
The "giant" neuron culture system derived from dissociated gastrula neuroblasts has been previously described (Saito and Wu 1991; Yao and Wu 1999, 2001; Yao et al. 2000; Zhao and Wu 1997). Briefly, the interior content of stage 7–8 embryos was sucked out with a glass micropipette and then dispersed in culture medium on an uncoated coverslip. The culture medium contains 80% Drosophila Schneider medium and 20% fetal bovine serum (GIBCO, Invitrogen, Carlsbad, CA) with the addition of 200 ng/ml insulin, 50 µg/ml streptomycin, and 50 U/ml penicillin. Drugs without specific labeling were from Sigma (St. Louis, MO). To generate multi-nucleated "giant" neurons from neuroblasts, cytochalasin B (CCB, 2 µg/ml) was added on the first day to arrest cytokinesis (Wu et al. 1990). CCB was removed by replacing the culture medium with CCB-free medium 24 h after plating. Actin filaments were restored within one day, as indicated by pattern stained by phalloidin in filopodia and lamellipodia (Berke et al. 2006). In Gal4-UAS GFP expression experiments, fluorescence was first detected in subsets of neurons between 12–24 h after plating. The intensity peaked after 
2–3 days.
Electrophysiology
Whole cell patch-clamp recording of cultured "giant" neurons has been previously described (Saito and Wu 1991; Yao and Wu 1999, 2001; Zhao and Wu 1997). Recording electrodes were prepared from 75-µl glass micropipettes (VWR Scientific, Chicago, IL). The tip opening of the electrodes had a diameter of 1 µm and an input resistance of 3–5 M
in bath solution. The bath solution contained (in mM) 128 NaCl, 2 KCl, 4 MgCl2, 1.8 CaCl2, and 35.5 sucrose, buffered at pH 7.1 with 5 mM HEPES. The solution for filling patch pipettes contained (in mM) 144 KCl, 1 MgCl2, 0.5 CaCl2, and 5 EGTA, buffered at pH 7.1 with 10 mM HEPES. Recordings were performed on the soma (diameters ranging from 13 to 18 µm) from 2- to 4-day-old cultures by using a patch-clamp amplifier (Axopatch 1B, Axon Instruments, Foster City, CA). The seal resistance was usually >5 G and junction potentials were nulled before establishing the whole cell configuration. A PC computer, an A/D-D/A converter and pClamp software (version 5.5.1, Axon Instruments) were used to generate the current- and voltage-clamp commands and for data acquisition. During current-clamp experiments, membrane potential was maintained at or near the resting level (around –60 mV) by applying polarizing current. Experiments were carried out at room temperature.
Several biophysical parameters in Table 2 were determined as previously described (Yao an Wu 1999). Briefly, currents were activated by step depolarization from a holding potential of –80 mV at 20-mV increments up to +60 mV. Boltzman fit was performed (reversal potential = –80 mV) to determine the half-activation voltage (V1/2). The recovery from inactivation protocol involved 500-ms twin pulses to +20 mV with varying interpulse intervals. Steady-state (st-st) inactivation was determined with a 500-ms preconditioning pulse (to –20 or 0 mV) followed by a +60-mV test pulse 5 ms afterward.
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Cultured "giant" neurons could generate nonregenerative and regenerative membrane potentials on current injection. The regenerative responses included graded oscillations with single or multiple peaks as well as all-or-none action potentials with clear thresholds (Fig. 1) (Saito and Wu 1991; Zhao and Wu 1997). With a fixed duration (400 ms) and current injection intensity (2–3 times the threshold level), all-or-none action potential firing patterns could be further divided into five categories (modified from Zhao and Wu 1997): single spike: only a single action potential was generated at different levels above threshold; delayed: the onset of spike activity showed a significant delay (>100 ms); tonic: action potential in the spike train occurred at regular intervals with onset latency <100 ms and flast/ffirst >0.7, where f represents instantaneous spike frequency in Hz; adaptive: spike frequency decreased over time with latency <100 ms and flast/ffirst <0.7; and damping: the spike amplitude diminished overtime.
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; Yao and Wu 1999; Zhao and Wu; 1997). Voltage-clamp measurements were performed on the soma with step depolarization from a holding potential of –80 m, at 20-mV increments up to +60 mV. Under these conditions, four types of total outward current responses (Types 1–4, T1-4, Fig. 4A) were distinguished based on their kinetics and ratios of IS/IP, where IP represented the peak current and IS the sustained component measured at the end of pulses. T1 and T2 currents were characterized by a larger inactivating component (IS/IP <0.5), with a single (
1)- and double (1 and 2)-exponential decay kinetics, respectively. In contrast, T3 and T4 neurons exhibited a larger sustained component (IS/IP >0.5) with a double (1 and 2)- or single (2)-exponential decay kinetics, respectively. Currents were normalized to membrane capacitance for current density estimates in pA/pF.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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To gain a general overview of the excitability patterns in Drosophila neurons, we performed current-clamp experiments on a large sample of individual, isolated neurons in single-embryo cultures. We observed both nonspiking membrane potential changes and spike activities, resembling a variety of excitability patterns previously described in different species, including Drosophila (Choi et al. 2004; Kuppers-Munther et al. 2004; Mee et al. 2004; O'Dowd 1995; Renden and Broadie 2003; Schmidt et al. 2000) and other invertebrates and vertebrates (Harris-Warrick and Marder 1991; Shepherd 2004).
During 400-ms step current injection, nonspiking neurons were unable to generate all-or-none action potentials (57% nonspiking vs. 43% spiking neurons) but still displayed different waveforms, including nonregenerative potentials and graded regenerative potentials with single- or multiple-peak responses (Fig. 1A). Similar nonspiking neurons have been described in insect nervous systems for a specific mode of signal processing (Burrows 1996). Among spiking neurons, the all-or-none repetitive firing patterns can be further divided into delayed, tonic, adapting, damping, or single-spike categories, based on spike onset time, and adaptation in spike frequency and amplitude (see Fig. 1 and METHODS for criteria). We found that each neuron retained the characteristics of a particular firing pattern in response to varying stimulus intensities, allowing a consistent, functional classification of neuronal subpopulations (Fig. 1, C and D). The proportions of neurons in each subcategory of nonspiking and spiking neurons were in general agreement with previously published results in the same preparation (cf. Saito and Wu 1991; Zhao and Wu 1997; with the additional categories of single-spike and damping firing patterns). Among the mono-, bi-, and multipolar cells examined in this culture system (cf. Saito and Wu 1991), we found no strict correlations between membrane excitability patterns with these morphological types (data not shown).
Distinct alterations of membrane excitability in Sh and Shab mutant neurons
The elimination of specific K+ currents by Sh and Shab mutations led to contrasting consequences in the regulation of neuronal excitability. Effects of Sh mutations have been documented in a number of in vivo preparations (Salkoff and Wyman 1981; Tanouye et al. 1981; Ueda and Wu 2006; Wu and Haugland 1985). In the "giant" neuron culture system, removal of Sh channels by a null mutation (ShM) resulted in quantifiable modifications in neuronal excitability patterns. Sh cultures contained a lower proportion of spiking neurons on current injection compared with WT cultures (Fig. 2C, WT vs. Sh, 43 vs. 30%, P < 0.05, 
2 test), but the proportions of the four multiple spike firing patterns remained unaltered (Fig. 2, A and C). It was also evident that ShM caused distinct modifications of action potential properties, including broadened action potentials during delayed and tonic firing and a slower firing rate in neurons of the damping category (Fig. 3 and Table 1). Although Sh IA plays a role in shaping the delayed firing pattern, neurons in Sh cultures showed only a slight, statistically insignificant shortening of spike onset time [means ± SE (n): WT, 193 ± 17 ms (11); Sh, 157 ± 22 ms (6)]. However, Sh neurons with delayed firing patterns had a higher voltage threshold for action potential initiation (Table 1).
In contrast, elimination of Shab current distorted the neuronal excitability patterns in a qualitative manner. There was a significant increase in the proportion of nonspiking cells, and the categories defining spike activities in WT and Sh could no longer describe the major spike patterns in Shab cultures (Fig. 2C). Most spiking neurons in Shab cultures displayed damping firing patterns (80%, Fig. 2C). In addition, we found a progressive damping of membrane regenerative activity during prolonged current injection in Shab cultures, which appeared as an abnormal decline in the membrane repolarization level, coupled with dwindling spike amplitude during repetitive firing (Fig. 2B). A declining membrane repolarization level between the initial and final regenerative events was frequently >20 mV among both nonspiking and spiking Shab neurons (top two traces in Fig. 2B; C, 
). These observations suggest that Shab currents play a major role in maintaining neuronal repolarization during repetitive firing or during nonregenerative activities over a time course of hundreds of milliseconds. It is worth noting that cells displaying such a repolarization decline were also found in a smaller population of WT and Sh neurons (Fig. 2C, ), suggesting that low levels of Shab expression also occur in a small subset of normal neurons. However, repetitive spikes in Shab neurons were characterized by an abnormally high firing rate and dwindling amplitude, which were not observed in either WT or Sh cultures (Fig. 3C and Table 1).
Voltage-activated outward K+ currents
The variety of firing patterns found in individual WT neurons and the extent of their disruptions in Sh and Shab cultures suggest the possibility of differential expression of K+ channels. Cultured "giant" neurons facilitated space-clamp control for analyzing the components of the total voltage-activated K+ currents that can be isolated in the presence of TTX and Cd2+ (Saito and Wu 1991; Yao and Wu 1999, 2001). A comprehensive voltage-clamp study could reveal the K+ current composition and kinetic profile in each neuronal subpopulation.
WT neurons displayed voltage-activated K+ currents with different degrees of inactivation in response to step depolarization (950 ms). The total K+ currents expressed by individual neurons could be classified into four types (T1-4, Fig. 4A) according to inactivation kinetics and the ratio of sustained/peak currents (IS/IP, see METHODS or Fig. 4 for criteria). In WT cultures, T3 neurons represented the largest population (40.3%), whereas T1 neurons represented the smallest (11.5%, Fig. 4B). In terms of current density (Fig. 4C), a larger IP and a smaller Is were found in both T1 and T2 neurons, yielding a lower IS/IP ratio. For current decay kinetics (Fig. 4D), the sole decay component in T1 neurons (1, 100–300 ms) was much slower than the fast component in T2 and T3 (1, <100 ms) but faster than their slow component 2. By contrast, T4 neurons were characterized with a single slow decay component 2 (>400 ms).
Distinct kinetics of voltage-activated K+ currents in Sh and Shab cultures
The properties of Sh- and Shab-mediated K+ currents could be revealed by comparing recordings on WT and null mutant cultures. In general, ShM cultures exhibited a great reduction in the peak transient current, IP, along with a substantial increase in the T3 subpopulation (Fig. 5, A and C, P < 0.05). In contrast, Shab3 neurons displayed a severely reduced sustained component, IS, and consequently T1 and T2 neurons became dominant in Shab cultures (Fig. 5, B and C, P < 0.01).
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Figure 6 summarizes a detailed analysis of current density and kinetics in T1–T4 neurons in WT and mutant cultures. The overabundance of T1 cells in Shab cultures was presumably due to a conversion from Shab-affected neurons, primarily T4 and T3, on removal of a sustained component. This new T1 subpopulation displayed distinct properties as reflected in an emergence of cells with an abnormally short decay constant 1 and a lowered IS density, compared with WT T1 neurons (Fig. 6, B and C, WT vs. Shab, IS in pA/pF, 12 ± 2 vs. 8 ± 1, P < 0.01; 1 in ms, 246 ± 11 vs. 167 ± 4 ms, P < 0.01). Similar results have been obtained from another independently isolated allele, Shab1 (data not shown).
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1 (Fig. 6C, note the log scale, 45 ± 1 vs. 56 ± 2 ms, P < 0.01), resembling the characteristics of 1 in WT T1 neurons.
In addition, differences in biophysical properties between Sh- and non-Sh IA (Table 2) provided explanations for distinct action potential and firing pattern phenotypes of Sh neurons (Table 1). In voltage-clamp recordings, T1–T3 Sh neurons had a more positive half-activation voltage (10 mV shift), and T1 Sh neurons inactivated at a more positive potential (–36 vs. –50 mV for half-inactivation) in a steady-state inactivation protocol (Table 2). Furthermore, the inactivating components in T2 and T3 Sh neurons showed a slower recovery from inactivation than WT neurons in a paired-pulse protocol (107–122 vs. 74–84 ms for half recovery, Table 2). Importantly, no significant changes were observed in T4 neurons between Sh and WT cultures, supporting the idea that the current in T4 neurons consists of mostly Shab currents.
Correlation of firing patterns and voltage-activated K+ currents
Distinct firing patterns are known to be characteristic of identified cell types in nervous systems (Koch 1999; Shepherd 2004). Drosophila "giant" neuron cultures provided enlarged, isolated cells that help to reduce cell rundown during prolonged patch clamping and to overcome complications introduced by extensive neuronal branching and synaptic connectivity in vivo. We were able to perform sequential current- and voltage-clamp recordings on the same cells and found that neurons with delayed firing could express total K+ currents with T1–T3 but not T4 kinetics (Fig. 7). Conversely, in neurons with T1 and T2 current kinetics, we encountered only delayed firing. Because T1 and T2 neurons predominantly expressed inactivating K+ currents, whereas T4 neurons generated little inactivating current, these results are consistent with the idea that a major function of inactivating K+ currents is to regulate the latency of spike activity onset.
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Properties of membrane excitability and corresponding K+ current kinetics
Our correlation studies further established that the extent of delay in spike initiation found in T1–T3 neurons is determined by the strength of transient current. The delay in spike onset was inversely proportional to the IS/IP ratio (Fig. 8). T1 and T2 neurons exhibited a smaller IS/IP ratio and tended to have longer delays in firing than did T3 neurons (Fig. 8A). Notably, the longest delay in spike initiation was found in T1 neurons (Fig. 8B), which had the slowest decay and the smallest IS/IP ratio (Figs. 4D and 6C).
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4-aminopyridine and quinidine effects on outward voltage-activated K+ currents and firing patterns
We used specific K+ channel blockers to contrast the acute pharmacological removal of IA and IK from the effects of Sh and Shab null mutations. It is well established that 4-aminopyridine (4-AP) at low concentration blocks transient IA in different preparations including Drosophila larval motor axons and cultured embryonic neurons (Ueda and Wu 2006; Wu et al. 1989; Zhao and Wu 1997). Quinidine selectively blocks a component of delayed IK in muscles at low concentrations (Singh and Singh 1999; Singh and Wu 1989, 1990) and enhances motor axon excitability and neuromuscular transmission in Drosophila larvae (Ueda and Wu 2006; Wu et al. 1989).
We extracted the 4-AP-sensitive component from the difference between voltage-clamp responses before and after 4-AP treatment (Fig. 10, A and B, top). The peak current (IP) density in 4-AP-treated neurons was analyzed for individual current kinetic categories (T1–T4, Fig. 10, A and B, bottom). As expected, the effect of 1 mM 4-AP treatment on WT cultures was most drastic in T1 and T2 neurons, which predominantly expressed transient currents (Fig. 10A). We found that the 4-AP effects also held true for Sh T1 and T2 neurons (Fig. 10B), indicating that 4-AP can suppress both Sh IA and the remaining non-Sh transient currents. In either WT or Sh cultures, most of the T4 neurons, which expressed little inactivating currents, showed negligible sensitivity to 4-AP (Fig. 10A), with only a small 4-AP-sensitive transient component occasionally found in some T4 neurons (see example traces in Fig. 10B). As shown in Fig. 10, 4-AP treatment decreased the peak currents in T1 and T2 neurons more severely than in T3 and T4 neurons. The remaining 4-AP-insensitive currents in the treated neurons displayed T3- and T4-like kinetics, consistent with the apparent population conversion observed in Sh cultures, in which removal of Sh currents led to an excess of T3 and T4 neurons with diminished current amplitude (Figs. 4 and 5).
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Current-clamp experiments in WT cultures showed that quinidine affected the tonic and adaptive spike activities but did not alter the damping firing pattern (Fig. 11C). In fact, most tonic and adaptive neurons examined in WT cultures were converted into the damping category, whereas the damping firing encountered in Shab cultures (cf. Fig. 2) remained unaltered after drug treatment. Thus the combined voltage- and current-clamp data support the idea that the quinidine-sensitive Shab current (Fig. 10, C and D) is required for maintaining membrane repolarization during repetitive firing (Figs. 2 and 11, C and D).
A sequence of sensitivity to drug treatment among the different firing patterns emerges from the preceding results, suggesting a hierarchy of complexity of K+ current interactions among neurons displaying different firing patterns. As a whole, the most frequently observed conversions are from the delayed to damping firing patterns after 4-AP treatment and from tonic to damping after quinidine treatment (Fig. 11). However, conversions between delayed and tonic firing patterns on drug treatment have not been observed. These observations suggest that the damping firing pattern requires weaker or fewer K+ current components, whereas participation of 4-AP- and quinidine-sensitive components is critical for generating the delayed and tonic patterns, respectively. We also occasionally encountered conversions from tonic to adaptive and adaptive to damping but not vice versa (Fig. 11). Additional mutations and pharmacological agents may be employed in future investigations to elucidate this apparent hierarchy of complexity of the K+ current interactions in different firing patterns.
K+ channel distribution in "cell lineage-defined" subpopulations of neurons
In view of the wide range of excitability patterns and the underlying currents described in the preceding text, we explored the possibility of distinct expression patterns of K+ channels in more restricted, identifiable neuronal subsets in the "giant" neuron culture. We utilized enhancer detection lines to drive expression of the GFP marker in different neuronal lineages. We chose to focus on two specific subsets of neurons, marked by the GH146 and 201Y Gal4 drivers. In larval and adult preparations, these two drivers label olfactory glomerular projection neurons and their postsynaptic target, the Kenyon cells in the mushroom bodies (Stocker et al. 1997; Yang et al. 1995).
We found that only relatively small subsets of neurons were GFP-positive (Fig. 12A) in GH146 (7.1 ± 3.5%, n = 10) and 201Y (9.0 ± 4.7%, n = 8) cultures. GH146 GFP-positive [GH146(+)] cells tended to be monopolar (>60%) neurons, whereas 201Y GFP-positive [201Y(+)] cells had no clear dominant morphological types. Voltage-clamp recordings demonstrated distinct profiles of K+ current kinetics in these two neuronal subpopulations. Abundant T3 cells were observed among GH146(+) neurons, whereas T2 cells dominated the 201Y(+) neuronal subsets (Fig. 12, B and C). The IP component in GH-146(+) cells exhibited a more positive half-activation voltage and slower recovery from inactivation than other neurons (Table 3), reminiscent to the properties of IP in Sh neurons (Table 2) and thus resembling non-Sh (presumably Shal) transient currents. In some 201Y(+) neurons, predominantly T2 (Fig. 12C), we observed unusually large IP components, far greater than that in GH146(+) neurons as well as the total population (see box plots in Fig. 12D). Notably, the unusually large IP was eliminated by the Sh mutation (Fig. 12D). In contrast, the Shab mutation preferentially reduced IS without affecting IP in 201Y(+) neurons (Fig. 12D).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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Elimination of a sustained K+ current component and deterioration of firing patterns in Shab neurons
In Drosophila muscles, it has been demonstrated that IA is eliminated by Sh mutations (Haugland and Wu 1990; Salkoff and Wyman 1981) and IK is affected by Shab mutations (Singh and Singh 1999). Mutations of ether a go-go (eag), another gene encoding a K+ channel subunit, can also reduce IA and IK in muscles (Zhong and Wu 1991). Several studies propose a different K+ channel expression pattern in neurons: IA channels are encoded by both Sh and Shal (Baker and Salkoff 1990; Choi et al. 2004; Gasque et al. 2005; Yao and Wu 2001), but major component of IK is produced by Shab channels (Tsunoda and Salkoff 1995). Consistently, in "giant" neuron cultures, Sh and Shab null mutations only reduced, but did not eliminate IA or IK (Fig. 5), indicating the presence of a substantial non-Sh component for IA and a minor non-Shab component for IK in neurons.
Our results provide a first description of the striking phenotype of Shab neurons. During sustained current injection, Shab neurons rely on other noninactivating K+ currents for action potential repolarization as the transient IA becomes progressively inactivated. The resultant abnormal damping spike pattern and unusual regenerative activities contrast the roles of Shab and non-Shab channels (including Shaw) (cf. Hodge et al. 2005). In most Shab neurons, spiking or nonspiking, we observed a novel "repolarization decline" phenotype that reflects a failure in maintaining a steady level of membrane repolarization (Figs. 2 and 9). Furthermore, the spiking activity in Shab cultures was restricted to the damping firing pattern and was coupled with T3 current kinetics (Figs. 2 and 9). Consistently, quinidine-treatments, which specifically remove Shab currents in WT neurons (Fig. 10), closely mimicked the "repolarization decline" phenotype and converted spiking patterns into damping firing (Fig. 11).
The mutational effects demonstrate that Shab IK is more important for signal processing at time scales of hundreds of milliseconds. Shab neurons are capable of generating normal action potentials in response to brief stimuli (Fig. 2), but prolonged current injection causes abnormal high-frequency, run-away spikes that degenerate into dwindling oscillations. WT neurons expressing both Shab and non-Shab sustained currents fire full-blown action potentials 25 Hz on average (Table 1). In contrast, T3 Shab neurons (IS/IP >0.5) that retain substantial non-Shab currents, including Shaw, generate high-frequency firing (up to 60 Hz) either immediately on depolarization or with a gradual development (Fig. 3 and Table 1). Interestingly, it has been shown that pharmacological blockade or mutations of Shaw-like Kv3 channels disable the high-frequency firing (often up to 1 kHz) found in certain neuronal types in the hippocampus, basal ganglia, and auditory nuclei (Lau et al. 2000; Rudy and McBain 2001). Because the exact dynamics of membrane repolarization can determine the timing of recovery of inward Na+ and Ca2+ currents from inactivation for resetting another cycle of spike activity, the balancing act between Shab and non-Shab IK currents can enrich spike frequency control during repetitive firing.
Interaction between IK and IA in repetitive firing
An interesting parallel of the striking Shab phenotype during repetitive firing is observed at the larval neuromuscular junction (Ueda and Wu 2006). With single nerve stimuli, Shab synaptic transmission appears normal. During high-frequency nerve stimulation, an explosive neuromuscular transmission (up to 10-fold gain, termed the "big bang" phenomenon) could suddenly occur when cumulative inactivation of IA reaches a critical level. The phenomenon can also be induced in WT by repetitive stimulation following quinidine treatment and by single nerve stimuli in quinidine-treated Sh preparations (Ueda and Wu 2006). The generation of a plateau membrane potential in the motor axon terminals, which are enriched with both Na+ and Ca2+ channels, has been proposed to account for this phenomenon when both IA and IK are weakened by mutations, drugs, or activity-dependent inactivation. These observations are reminiscent of the "repolarization decline" in Shab neurons and quinidine-treated WT neurons during prolonged current injection (Figs. 2 and 9). Taken together, an intricate interaction between slowly inactivating IK and fast inactivating IA is important during the dynamic process of repetitive firing for maintaining the cycles of membrane excitation and repolarization.
Notably, simple removal of Shab current by acute quinidine treatment on WT cultures converted firing patterns to damping rather than nonspiking activities (Fig. 11). However, in Shab cultures, a drastic increase of nonspiking neurons was observed (Fig. 2C), contrary to the expectation of increased excitability caused by reduced IK. A clue to this unexpected finding is provided by the observation that in some nonspiking Shab cells, regenerative oscillations could still be initiated when the transient IA was suppressed by 4-AP or a depolarizing prepulse (data not shown). Voltage-clamp measurements (Fig. 6A) yielded direct evidence for an increase in the inactivating IA in Shab cultures. The peak total current (IP), which represents the sum of peak IA and IK, remained undiminished despite the fact that the sustained current component (IS) was significantly decreased in Shab neurons (Figs. 5B and 6A). In contrast, pharmacological removal of Shab currents reduced both IS (35%, Fig. 10B) and IP (30%, data not shown). Our observations resemble the homeostatic regulation of ion channel previously reported in other preparations. When neuronal spike activity is manipulated, a homeostatic regulation can be initiated to adjust the relative abundance of ion channels and other proteins (Guan et al. 2005; Spitzer 1999; Turrigiano and Nelson 2004). Over-expression of Shal IA in lobster neurons triggers a compensatory increase of hyperpolarization-activated inward Ih (MacLean et al. 2003). Therefore a compensatory upregulation of the transient K+ current component in Shab cultures could account for the lower percentage of spiking cells (Fig. 2). Overexpression of transient IA could prevent spike initiation in the standard current-clamp protocols employed here. Additional types of compensatory mechanisms, such as decreased expression of inward Na+ or Ca2+ currents, might also occur in Shab neurons. However, our preliminary results indicate that Ca2+ current density in Shab mutant cultures remains unaltered compared with that in WT cells (data not shown), although potential modification of Na+ currents require further investigation.
Alteration of action potential duration and firing frequency in Sh neurons
In contrast to Shab channels, Sh channels play a role in regulating rapid events within a millisecond time scale. We observed broadened action potentials in Sh neurons with delayed and tonic firing patterns (Fig. 3 and Table 1). This demonstrates a role of action potential repolarization for Sh channels, consistent with greatly prolonged action potentials previously documented in the cervical giant fiber of Sh mutants (Tanouye and Ferrus 1985).
A well-established function of transient K+ currents is pertinent to the control of spike initiation time during excitatory inputs (Hille 2001). Our mutational and pharmacological analyses confirm the important but overlapping roles of Sh and Shal IA channels in controlling spike initiation (Figs. 7 and 10) (cf. Choi et al. 2004; Zhao and Wu 1997). Neurons exhibiting delayed firing persisted in Sh cultures, supporting the idea that within only a small subset of neurons, IA channels are exclusively or predominantly encoded by Sh (Baker and Salkoff 1990). Furthermore, no disproportional reduction in any of the categories of firing patterns was observed in Sh cultures (Fig. 2), indicating that Sh and Shal IA may serve redundant roles in suppressing transient depolarization, and thus the action of Shal IA alone could retain the dynamic manifestation of the delayed and other firing patterns.
Delayed or tonic firing in WT and Sh cultures could be converted into damping patterns after 4-AP treatment (Fig. 11), suggesting that with total elimination of transient IA, spike repolarization deteriorates during repetitive firing (cf. Connor and Steven 1971). Nevertheless, such damping firing patterns were distinct from those observed in Shab or quinidine-treated WT neurons, in that the high-frequency oscillations typical of Shab neurons were never observed (Figs. 1, 2, 9, and 11). In summary, a clear rank of firing rate was observed in the damping patterns of the three genotypes: Shab > WT > Sh (Table 1).
The slower decay of transient IA in Sh cultures (Table 2) is in agreement with previous reports on pupal and larval cultures (Baker and Salkoff 1990; Gasque et al. 2005), indicating that the remaining Shal channels inactivate more slowly than Sh channels. This is also consistent with the conclusion based on Sh and Shal channel expression experiments in the frog oocyte (Wei et al. 1990). Our results also suggest that Shal channels follow a slower kinetics of recovery from inactivation (Table 2), something that awaits confirmation in other preparations. However, our steady-state inactivation measurements of IA in Sh cultures suggest that Shal channels inactivate at voltages more positive than that for Sh channels (Table 2), contrary to the preceding reports (Baker and Salkoff 1990; Gasque et al. 2005). The reason for the discrepancy is unknown, although expression patterns of the Sh and Shal splice variants as well as their potential association with auxiliary channel subunits could differ among the embryonic, larval and pupal cultures used in these studies. Splice variants of the Sh products are known to mediate K+ currents of varying degrees of inactivation when expressed heterologously in Xenopus oocytes (Iverson et al. 1988; Timpe et al. 1988). Similarly, subtypes of mammalian Kv1 channels also display different inactivation properties (Coetzee et al. 1999; Stumhmer et al. 19
100 ms; tonic, latency <100 ms and flast/ffirst >0.7 (instantaneous spike frequency f = 1/interspike interval); adaptive, latency <100 ms and flast/ffirst <0.7; damping, repetitive spikes with diminishing amplitudes. Current steps: –30 pA and 2–3 times the threshold level (usually 40 pA above). C and D: distribution of individual regenerative responses and firing patterns among neuronal populations. Numbers in the parentheses represent sample sizes.
. C: distributions of individual excitability categories for WT, ShM, and Shab3 cultures. 
0.5) at +60 mV with single (
and 
in the T1 cell, IP and IS. TTX was added to eliminate Na+ current and Cd2+ was used to reduce Ca2+ current as well as Ca2+-activated K+ current. B: distribution of individual current types. T3 neurons represented the largest subpopulation, whereas T1 neurons were the smallest. The sample size is shown in the parentheses. C: IP and IS at +60 mV were normalized to membrane capacitance to indicate current density for comparison. D: decay time constants 
) are indicated. See A for the sample size.
) 1 mM 4-AP treatment were superimposed (voltage steps from –80 to +60 mV). The 4-AP-sensitive components (right) were extracted from the difference between sequential recordings. The plots showed changes in IP (left for T1 and T2, middle for T3 and T4) and in kinetics categories (right) caused by 4-AP. T1–T4 current kinetics are categorized based on IS/IP ratios and inactivation kinetics (see METHODS and 
