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1Division of Neurobiology, 2Graduate Program in Physiological Sciences, and 3Department of Physiology, University of Arizona, Tucson, Arizona
Submitted 13 April 2008; accepted in final form 5 June 2008
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ABSTRACT |
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1G. We used mutants of Dmca1A and Dmca1D as well as targeted expression of an RNAi transgene to Dmca1D to determine the genes responsible for the voltage-dependent Ca2+ current recorded from two identified motoneurons. Our results implicate Dmca1D as the major contributor to the voltage-dependent Ca2+ current recorded from the somatodendritic processes of motoneurons, whereas Dmca1A has previously been localized to the presynaptic terminal where it is essential for neurotransmitter release. Altered firing properties in cells from both Dmca1D and Dmca1A mutants indicate a role for both genes in shaping firing properties. |
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INTRODUCTION |
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), voltage-dependent Ca2+ channels segregate to somatodendritic locations where they associate cellular activity to localized Ca2+ influx (Christie et al. 1995; Magee and Johnston 1995). Ca2+ channels in the somatodendritic processes of motoneurons may amplify postsynaptic current (Schwindt and Crill 1980; Heckman and Lee 1999; Hyngstrom et al. 2008; Johnson et al. 2003; Lee and Heckman 2000; Seamans et al. 1997; Simon et al. 2003) or regulate action potential firing frequency through Ca2+-activated K+ channels (McManus 1991; Vergara et al. 1998). On a somewhat longer time scale, voltage-dependent Ca2+ influx may drive activity-dependent gene regulation (Catterall 2000; Hardingham et al. 1997), mediating, for example, adjustments in intrinsic excitability (Peng and Wu 2007). Thus determining the cellular mechanisms required for the appropriate localization of voltage-gated Ca2+ channels and the integrative consequences of Ca2+ channel activation is a necessary step in understanding how the activity of neural circuits is maintained at the level of the single cell. Techniques available in Drosophila, such as cell-specific genetic manipulation and the ability to record in situ from identified neurons, make it an ideal system to study the influence of voltage-gated Ca2+ influx on neuronal function within an intact system. Thus the role of a particular channel type in determining the activity pattern of an identified neuron can be addressed rigorously through targeted genetic manipulation. Unfortunately, voltage-gated Ca2+ currents have not been well defined in Drosophila neurons in vivo. Our goal in the present study, therefore was to verify that the somatodendritic processes of Drosophila motoneurons include these currents and to take advantage of genetic approaches to determine the genes responsible.
The Drosophila genome contains three genes with known homology to voltage-gated Ca2+ channel 1 subunits in vertebrates; Dmca1A, Dmca1D, and Dm1G (King 2007; Littleton and Ganetzky 2000; Smith et al. 1996; Zheng et al. 1995). Dmca1A, also known as cacophony (cac), shares sequence homology with vertebrate N, P, and Q-type channels and is expressed at the Drosophila neuromuscular junction where it contributes to the Ca2+ influx responsible for synaptic release (Kawasaki et al. 2000, 2002, 2004), synaptic growth (Rieckhof et al. 2003), and regulation of the neuromuscular junction (Xing et al. 2005). Additionally, in the Drosophila giant neuron culture system, derived from cytokinesis-arrested embryonic neuroblasts, cac contributes the major Ca2+ current and plays a role in the homeostatic regulation of the A-type K+ current (Peng and Wu 2007). While the role of cac has been well described at the neuromuscular junction, as well as in cell culture, the contribution of cac to voltage-dependent Ca2+ currents in the central processes of neurons in situ has not been determined. Dmca1D shares homology with vertebrate L-type channels (Zheng et al. 1995) and is responsible for the major dihydropyridine-sensitive current recorded from Drosophila larval muscle fibers (Ren et al. 1998). Whether Dmca1D plays a role in the CNS is not known. Dmca1A and Dmca1D appear to play nonredundant roles in Drosophila as null alleles of both genes are independently embryonic lethal (Eberl et al. 1998; Smith et al. 1996). Dm1G shares homology with vertebrate LVA T-type channel. While the function of this gene has not been characterized in Drosophila, a current with steady-state inactivation at membrane potentials of –30 mV has been identified in embryonic motoneurons aCC and RP-2 (Baines and Bate 1998) as well as in larval body wall muscle (Gielow et al. 1995; Ren et al. 1998). These currents are sensitive to amiloride, a known blocker of vertebrate T-type currents.
We used in situ whole cell patch-clamp techniques to record voltage-dependent Ca2+ currents from identified motoneurons aCC and RP-2 in third instar Drosophila larvae. Motoneurons aCC and RP-2 were chosen based on their accessibility for recording and known influence on muscle function. We further recorded isolated voltage-dependent Ca2+ currents from larvae carrying mutant alleles of Dmca1A and Dmca1D. In both aCC and RP-2, Dmca1D carried the major component of the voltage-dependent Ca2+ current recorded from the cell body. To support these findings, we drove the expression of Dmca1D RNAi specifically in aCC and RP-2 and found a significant reduction in somatically recorded voltage-dependent Ca2+ current. Whereas Dmca1D contributed the major voltage-sensitive current recorded at the cell body, mutations of both Dmca1A and Dmca1D, as well as RNAi knock-down of Dmca1D, had an influence on the firing properties of aCC and RP-2.
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METHODS |
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Wild-type strains used were Canton-s and w1118. The GAL4 line, RRA, in which a transgene containing a region of the even skipped (eve) promoter drives GAL4 expression in specific neurons (Fujioka et al. 2003), was used to drive expression of green fluorescent protein (GFP) and other transgenes in aCC and RP-2 motoneurons in each thoracic and abdominal hemisegment. A recombinant was made that included RRA-GAL4 and UAS-GFP on the same chromosome [w-; Sco/SM6a; RRA-Gal4, UAS-mCD8-GFP]. The two motoneurons were identifiable based on dendritic morphology and target innervation (Choi et al. 2004; Hoang and Chiba 2001) (Fig. 1). Due to a decrease in the level of eve expression by the end of third instar (Fujioka et al. 2003), there was a mosaic pattern of GFP labeling, such that both cells were not always visible in each hemisegment. The homozygous viable allele of Dmca1D, AR66 (Eberl et al. 1998) (obtained from Dr. D. Eberl, University of Iowa, Iowa City, IA), contains a point mutation causing a hypomorphic phenotype (Ren et al. 1998). To obtain the AR66W1118 line, AR66 was crossed into a white background with a GFP-tagged balancer [w/w; L(2) 35fa/Cyo P[w+,Act:GFP]; +/+] so that the wild-type strain w1118 could be used as a control to examine the contribution of Dmca1D to somatically recorded Ca2+ current. Additionally, AR66W1118 allowed for the selection of homozygous mutants by deselection for GFP in the gut of heterozygous animals. Dmca1A [cacophony (cac)] temperature-sensitive alleles cacts2, cacts3, and cacts5 (Kawasaki et al. 2000; Rieckof et al. 2003) and a hypomorphic, hemizygous viable allele, cacs (Smith et al. 1996) (obtained from Dr. C. F. Wu, University of Iowa, Iowa City, IA) were used to determine the influence of Dmca1A on somatically recorded Ca2+ current. The UAS-RNAi-51491 line (RNAi91), targeted to Dmca1D, was obtained from the Vienna Drosophila RNAi Center (VDRC). Note that it was important to use appropriate control lines for each genotype because current densities varied among genotypes.
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Preparation
All experiments were performed on wandering late third instar Drosophila larvae. Larvae were placed on ice for 
2–3 h prior to dissection. Larvae were pinned dorsal side up and bathed in Ca2+-free A solution (which contained, in mM, 118 NaCl, 2 NaOH, 2 KCl, 4 MgCl2, 25 sucrose, 5 trehalose, and 5 HEPES, pH 7.1–7.2, 295 mosM) (Jan and Jan 1976). The CNS, segmental nerves, and body wall muscles were left intact. The preparation was visualized using an upright fixed stage Olympus microscope. To access motoneurons in the ventral nerve cord, Protease 14 (2 mg/ml extracellular solution- Sigma-Aldrich, St. Louis, MO) was focally applied to the ganglionic sheath by applying positive pressure to a recording electrode with the tip broken to a diameter of 10 µM (adapted from Choi et al. 2004). Treatment of the sheath was performed with constant laminar superfusion, and the debris was removed by applying negative pressure to the electrode. In experiments requiring temperature regulation of the bathing solution, a Warner TC-324B in-line bath heater was added to the perfusion system. Motoneurons RP-2 and aCC in thoracic and anterior abdominal segments were targeted in all experiments.
Electrophysiology
Motoneurons aCC and RP-2 were identified though the expression of GFP under the control of RRA-GAL4 or, in wild-type or mutant strains that did not carry the driver and the UAS-GFP transgene, through intracellular dye fills using Rhodamine Dextran 3000 added to the intracellular recording solution. Recordings were obtained from abdominal segments and from thoracic segments T2 and T3. The extracellular recording solution contained (in mM) 118 NaCl, 2 NaOH, 2 KCl, 4 MgCl2, 1.8 CaCl2, 25 sucrose, 5 trehalose, and 5 HEPES (Jan and Jan 1976). The pH was adjusted to 7.1–7.2 and the osmolarity to 295 mmol/kg. Because the CNS was partially desheathed in our preparations, it was deemed appropriate to approximate intraganglionic Ca2+ concentrations as opposed to higher Ca2+ levels that are used in salines that are designed to mimic Ca2+ concentrations in the hemolymph. To facilitate measurements through voltage-dependent Ca2+ channels and to reduce Ca2+ activated K+ currents, 1.8 mM Ba2+ was exchanged for 1.8 mM Ca2+ in select experiments. The intracellular solution contained (in mM) 144 KCl, 1 MgCl2, 0.5 CaCl2, 5 EGTA, and 10 HEPES (Peng and Wu 2007). The pH was adjusted to 7.1–7.2 and the osmolarity to 290 mmol/kg. To isolate voltage-dependent Ca2+ currents, KCl was replaced with CsCl in the intracellular recording solution and 1 µM tetrodotoxin (TTX), 50 mM TEA, and 1.5 mM 4-aminopyridine (4-AP) were added to the recording solution (all ion channel blockers were purchased from Simga-Aldrich). A small outward current remained under these conditions. Thin-walled borosilicate electrodes were pulled on a PP-83 (Narishigie) to a resistance of 2.5–5 M
and fire polished using an MF-35 microforge (Narishigie). Whole cell patch clamp was performed in situ using an Axopatch 1D amplifier (Axon Instruments). Clampex software (Molecular Devices- pClamp 10.1) was used to generate voltage and current commands and for data acquisition. Motoneurons with resting membrane potential less that –50 mV or seals <1 G were not used for experiments. For current-clamp experiments, resting membrane potentials were brought to –60 mV through current injection to the cell body. Spike and afterhyperpolarizing potential (AHP) amplitudes were measured (see Fig. 7) from traces in which depolarizing current injection brought the membrane potential to –30 to –20 mV.
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and was not well-corrected. The largest currents injected in current-clamp experiments (100 pA) would therefore have caused a voltage error of 2.8 mV. The largest Ca2+/Ba2+ currents measured in voltage-clamp experiments (350 pA) would have caused a series resistance error of 10 mV. In most cells, the maximal current was less (see Figs. 3–6). Current amplitude was normalized to whole cell capacitance, and values were reported as current density in voltage versus current experiments. Whole cell capacitance was determined from the charging transient following a 20-mV hyperpolarizing voltage command and used to calculate current density.
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Nifedipine (Sigma) was dissolved in 95% ETOH and added to the extracellular solution for a final concentration of 10 µM (final concentration of ETOH was <0.01%). PLTX-II (Alomone Labs) was added to the extracellular solution for a final concentration of 40–300 nM. Bay K 8644 (Alomone Labs) was used at a concentration of 1 µM.
Statistical analysis
Statistical analyses were performed using a standard t-test with Excel software (Microsoft). Standard error values are reported. Significance was assumed when P 
0.05.
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RESULTS |
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Our first goal was to determine whether extracellular Ca2+ plays a role in shaping the firing properties of two identified Drosophila larval motoneurons; MN1-1b (aCC) and MNISN-Is (RP-2) (Fig. 1). These cells were chosen based on their accessibility to recordings and because they have been well characterized in previous experiments (Baines and Bate 1998; Choi et al. 2004; Rohrbough and Broadie 2002). Whole cell current-clamp experiments were performed in situ on motoneurons aCC and RP-2.
In extracellular solution containing 1.8 mM Ca2+, the resting membrane potentials of aCC and RP-2 were –58 ± 1.38 and –66 ± 1.71 mV, respectively, and the input resistances were 699 ± 85.4 and 930 ± 91.0 M, respectively (RRA-GAL4 line: aCC n = 10, RP-2 n = 9). Removing Ca2+ from the extracellular recording solution caused a reduction in the AHP between spikes and a decrease in the interspike interval (Fig. 2, A and B). The resting membrane potential and input resistance values were not altered in either aCC (–60 ± 2.77 mV, 533 ± 50.8 M; n = 5) or RP-2 (–66 ± 2.95 mV, 910 ± 94.1 M; n = 5) by the removal of Ca2+ from the recording solution. Additionally, the action potential threshold was reduced by 10 mV, and there was a significant increase in action potential firing frequency in response to somatic current injection in both aCC and RP-2 (Fig. 2, A and C). Similar results were obtained when recordings were first generated in the absence of Ca2+, and subsequently 1.8 mM Ca2+ was added to the recording solution. Motoneurons aCC and RP-2 responded in a similar manner to the removal of Ca2+ from the recording solution (Fig. 2C). To summarize, removing Ca2+ from the recording solution caused a significant reduction in the spike AHP, a significant reduction in the interspike interval, a decrease in action potential threshold, and an increase in action potential firing frequency in response to somatic current injection. These observations suggest that aCC and RP-2 motoneurons express voltage-sensitive Ca2+ currents that influence the intrinsic firing properties of these cells. However, the removal of Ca2+ from the extracellular recording solution may also have influenced the function of additional voltage-dependent channels by altering membrane charge shielding effects, and the reduction in synaptic activity with reduced Ca2+ may have contributed to the observed alterations in firing properties.
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Whole cell voltage-clamp experiments were performed in situ to identify voltage-dependent Ca2+ currents within the central processes of aCC and RP-2 motoneurons in third instar larvae. Ca2+ currents were isolated by the addition of 1 µM TTX, 50 mM TEA, and 1.5 mM 4-AP to the extracellular recording solution and by replacing intracellular K+ with Cs+. With 1.8 mM Ca2+ in the extracellular recording solution, a depolarizing voltage command to –10 mV, from a holding potential of –70 mV, elicited an inward current, which underwent rapid reduction over the initial 20 ms of a 200-ms voltage command (Fig. 3A, i and ii). In a subset of cells, no inward current remained after 20 ms (Fig. 3Ai). In another subset, however, a reduced inward current persisted throughout the 200-ms voltage command (Fig. 3Aii).
Replacing 1.8 mM Ca2+ with 1.8 mM Ba2+, known to have a higher conductance than Ca2+ through voltage-gated Ca2+ channels (Byerly and Leung 1988), caused a substantial increase in current amplitude and reduced current decrement (Fig. 3A, i and ii). The latter may reflect Ca2+-dependent inactivation of Ca2+ channels. The addition of 500 µM Cd2+, a general blocker of voltage-gated Ca2+ currents, completely eliminated all inward current (Fig. 3Ai). The voltage-dependent Ca2+ current first became prominent at membrane potentials between –40 and –30 mV in both aCC and RP-2, and peak currents were elicited at approximately –10 mV (Fig. 3D). Motoneuron aCC displayed a significantly larger current density than RP-2 (Fig. 3, C and D). RP-2 displayed a larger variability in peak current amplitude than aCC, but the variability was reduced when currents were normalized to cell size (Fig. 3C). Similar differences in current density between aCC and RP-2 were observed in all wild-type strains including w1118 and Canton-S and the RRA-GAL4 line. There was relatively little steady state inactivation of the Ba2+ current. In both aCC and RP-2, long depolarizing prepulses (1 s) caused partial reduction in current during a test pulse to –10 mV (Fig. 3E).
To determine whether the voltage-dependent Ca2+ currents recorded somatically from aCC and RP-2 were sensitive to traditional pharmacological modifiers of voltage-dependent Ca2+ channels, we applied nifedipine, Plectreurys toxin-II (PLTX-II), and Bay K 8644 to the recording solution. Nifedipine has been shown to block the voltage-dependent Ca2+ current carried by Dmca1D in larval Drosophila muscle (Gielow et al. 1995; Ren et al. 1998) but did not block the Ca2+ current in aCC or RP-2, whether the holding potential was set at –70 or –40 mV. Similarly, Bay K 8644 enhances tail current amplitude of the Dmca1D-dependent Ca2+ current in larval muscle (Gielow et al. 1995) but did not have an effect on currents recorded in aCC and RP-2. PLTX-II blocks synaptic transmission at the Drosophila neuromuscular junction where cac channels support synaptic release (Branton et al. 1987; Kawasaki et al. 2000, 2004), suggesting that cac is PLTX-II sensitive. PLTX-II did not reduce the voltage-dependent current recorded somatically in aCC and RP-2.
Mutant alleles of cacophony (Dmca1A) did not reduce the voltage-dependent Ba2+ current recorded in either aCC or RP-2
To determine which genes contribute to the voltage-dependent Ca2+ current recorded from the cell bodies of aCC and RP-2, mutant alleles of Dmca1A (cac) and Dmca1D were examined. cac mutants have been well characterized at the larval neuromuscular junction and include temperature-sensitive alleles cacts2, cacts3, and cacts5 (Kawasaki et al. 2002; Rieckof et al. 2003) and a hypomorphic, hemizygous viable allele cacs (Peng and Wu 2007; Smith et al. 1998). Whole cell voltage-clamp recordings were generated in situ from cacs third instar larvae using 1.8 mM Ba2+ as the charge carrier. In hemizygous cacs mutant males, the peak current density generated was not significantly different from those generated in Canton-S control (Fig. 4A). Furthermore, the kinetics and voltage sensitivity of the current were similar in both cacs and control (Fig. 4B).
To confirm the results generated from the cacs mutants, recordings were obtained from cacts2 temperature-sensitive mutants. At permissive temperatures (<26°C) cacts2 mutant channels conduct Ca2+/Ba2+ current similar to wild-type channels; however, at restrictive temperatures (>32°C), cacts2 mutant channels no longer conduct Ca2+/Ba2+ current (Kawasaki et al. 2002). In hemizygous male cacts2 larvae, we recorded voltage-dependent Ba2+ currents from the same aCC and RP-2 motoneurons at permissive and restrictive temperatures. Threshold for current activation, peak Ba2+ current density, and reversal potential were indistinguishable between recordings generated at permissive versus restrictive temperatures in both aCC and RP-2 (Fig. 4C). Further, these values were not significantly different between permissive and restrictive temperatures in two additional temperature-sensitive cac alleles, cacts3 and cacts5 (data not shown). Current activation and inactivation kinetics were also similar in recordings generated at permissive and restrictive temperatures (Fig. 4D). Despite the lack of effect on currents recorded from the cell body, neuromuscular transmission was abolished reversibly at restrictive temperatures (data not shown), consistent with earlier reports and the known role of cac in synaptic release (Kawasaki et al. 2000, 2002, 2004). In summary, cac mutants cacs and cacts2 did not significantly reduce the voltage-dependent Ba2+ current recorded at the cell body of aCC and RP-2 motoneurons even though the cacophony protein is known to be localized in the axon terminals of these cells.
Hypomorphic mutation of Dmca1D (AR66) significantly reduced voltage-dependent Ba2+ current recorded in both aCC and RP-2
The contribution of Dmca1D to the voltage-dependent Ca2+ current in Drosophila neurons has not been determined. We used a homozygous viable mutant allele of Dmca1D, AR66, to determine the role played by Dmca1D in generating the somatic voltage-dependent Ca2+ current recorded in aCC and RP-2. AR66 is a hypomorphic mutation of Dmca1D that reduces the ability of the channel to carry current (Ren et al. 1998). Viability was reduced in AR66 homozygous mutants as evident by the reduced number of larva reaching the wandering third instar stage. Nevertheless, in third instar larvae that could crawl from the food, both aCC and RP-2 displayed healthy membrane potentials, were of normal size (as determined by whole cell capacitance), and maintained the ability to generate action potentials in response to current injection. Additionally, dye filling or GFP labeling of the cells did not indicate gross changes in cell morphology (Fig. 1), although we did not perform a quantitative analysis of dendritic branching complexity for this study.
Larvae homozygous for the AR66 mutation displayed a significant reduction in voltage-dependent Ba2+ or Ca2+ current density compared with wild-type control (w1118) in both aCC and RP-2 (Fig. 5, A and B). Further, larvae heterozygous for the mutation displayed a current phenotype intermediate to that of the homozygote and wild-type (Fig. 5C), consistent with recordings generated from body wall muscle of AR66 mutants (Ren et al. 1998). The voltage sensitivity of steady-state inactivation was similar for AR66 mutants and wild-type larvae (Fig. 5D). To summarize, the AR66 mutation of Dmca1D significantly reduced the ability of aCC and RP-2 to generate a Ba2+ or Ca2+ current, implicating Dmca1D as a major contributor to the voltage-dependent Ca2+ current recorded from the central processes of aCC and RP-2 in situ.
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The GAL4-UAS system available in Drosophila allows for the expression of RNAi transgenes in a cell-specific manner. The expression of Dmca1D RNAi was driven in aCC and RP-2 motoneurons to determine if it would reduce the Ba2+/Ca2+ current recorded somatically in these cells. Drosophila lines carrying UAS-RNAi-51491 (RNAi91), an RNAi to Dmca1D, were crossed to the RRA-GAL4 line, driving expression of RNAi91 in aCC and RP-2 motoneurons. The expression of RNAi91 in aCC and RP-2 did not affect the development or the viability of the animals, and both aCC and RP-2 maintained normal resting membrane potentials. The overall morphology of these neurons within the CNS was normal (Fig. 1).
In aCC and RP-2 motoneurons expressing RNAi91, isolated Ba2+/Ca2+ currents were significantly reduced compared with control (RNAi91 not crossed to RRA-GAL4) (Fig. 6A). This result further implicates Dmca1D as the major contributor to voltage-dependent Ca2+ current recorded somatically in aCC and RP-2 motoneurons. The range for current activation was similar between RNAi91 expressing cells and control.
Hypomorphic mutations of Dmca1D and Dmca1A, as well as RNAi91 expression, alter the firing properties of aCC and RP-2 in response to somatic current injection
To determine whether Dmca1D and Dmca1A (cac) play a role in shaping the firing properties of aCC and RP-2, somatic current injections were administered to AR66 and cacs hypomorphic mutants and to cells expressing the RNAi91 transgene. Action potential shape was variable in both hypomorphic mutations (Fig. 7, A, B, and D). The mean spike amplitude was reduced significantly in AR66 mutants as compared with motoneurons from the control line (AR66, 5.54 ± 1.3 mV; n = 5; w1118, 10.24 ± 1.2 mV; n = 5; P = 0.03). The AHP amplitude was not significantly different (AR66, 11.28 ± 1.9 mV; n = 5; w1118, 14.67 ± 1.7; n = 5; P = 0.22). Spike amplitude in the cacs line was not significantly different from the Canton-S control line (cacs, 13.89 ± 1.6 mV; n = 4; Canton-s, 11.22 ± 1.1 mV; n = 4; P = 0.2). Similarly, the AHP amplitude was not significantly different (cacs, 12.43 ± 2.0 mV; n = 4; Canton-s, 13.73 ± 1.0 mV; n = 4; P = 0.59). Spike frequency was increased in both AR66 and cacs mutants compared with their respective controls and there was a reduction in spike threshold in both mutants (Fig. 8). Unlike control lines (see Fig. 2), removing Ca2+ from the recording solution did not cause a significant increase in spike frequency in either AR66 or cacs mutants (Fig. 8).
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There was a significant reduction in the AHP in the evoked spikes of RNAi91 expressing cells (RNAi91, 6.49 ± 0.4 mV; n = 6; control, 12.41 ± 1.6 mV; n = 5; P = 0.02), and a reduction, although not significant, in the amplitude of evoked spikes when compared with control (RNAi91, 3.42 ± 0.4 mV; n = 6; control, 6.71 ± 1.6 mV; n = 5; P = 0.1). The frequency of evoked spikes was not altered by RNAi91 expression (Fig. 8). In the absence of Ca2+ in the extracellular solution, motoneurons expressing RNAi91 displayed a high-frequency of spontaneous spike-like events (Fig. 7C), such that it was difficult to obtain frequency versus current relationships for the evoked spikes.
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DISCUSSION |
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1 subunit of voltage-dependent Ca2+ channels, and each may experience alternative splicing (Lipscombe et al. 2002) and posttranslational modifications. In addition, the voltage sensing and current conducting 1 subunit is usually found in association with auxiliary subunits (Dickman et al. 2008). These processes ensure that each cell is equipped with the assortment of voltage-dependent Ca2+ currents needed to function appropriately. The Drosophila genome contains three genes with homology to each class of voltage-dependent Ca2+ channel found in vertebrates (King 2007; Littleton and Ganetzky 2000; Smith et al. 1996; Zheng et al. 1995). While the Drosophila Ca2+ channel genes share sequence homology with their vertebrate counterparts, classification of currents by voltage threshold and pharmacology is not as clear-cut. In Drosophila, all three gene products generate whole cell currents that activate at membrane potentials approximating –40 mV (Gielow et al. 1995; Leung and Byerly 1991) and vertebrate Ca2+ channel blockers have varied effects (King 2007). Genetic tools available in Drosophila have been used to dissect voltage-dependent Ca2+ currents in larval muscle (Ren et al. 1998), at the neuromuscular junction (Kawasaki et al. 2000, 2002, 2004), and in the giant neuron culture system (Peng and Wu 2007).
To determine which of the genes encoding voltage-dependent Ca2+ channels was responsible for the current observed in aCC and RP-2, we examined mutant alleles of Dmca1A and Dmca1D. Whole cell voltage clamp revealed a significant reduction in current amplitude in the Dmca1D mutant AR66. In support of these findings, targeted expression of a Dmca1D RNAi transgene (UAS-RNAi-51491) in aCC and RP-2 significantly reduced the voltage-dependent Ca2+ current. Together, these results implicate Dmca1D as the major contributor to the voltage-dependent Ca2+ current recorded somatically in aCC and RP-2 motoneurons. In both experiments, however, a relatively small residual inward current remained. Because AR66 is a hypomorphic mutation, which reduces but does not abolish current (Eberl et al. 1998), and Dmca1D RNAi expression may not completely eliminate Dmca1D protein, it was not possible to determine whether the residual current was through Dmca1D-encoded channels or through channels encoded by Dmca1A or Dm1G. The residual current in AR66 mutants was relatively insensitive to voltage-dependent inactivation. This suggests that the residual current may not reflect Dm1G expression, as this putative T-type current displays rapid voltage-dependent inactivation (Gielow et al. 1995; Ren et al. 1998). As mentioned in the preceding text, the pharmacological tools used to classify vertebrate Ca2+ channels have varied effects in Drosophila (King 2007). Nifedipine was ineffective in our experiments even though it has been reported to block the current carried by Dmca1D in larval Drosophila muscle (Gielow et al. 1995; Ren et al. 1998). Different splice variants of Dmca1D may be expressed in neurons and muscle (Zheng et al. 1995). Alternatively, the alpha subunits may be associated with different auxiliary subunits or cytoskeletal proteins in the different cell types.
Differential distribution of voltage-dependent Ca2+ channels has been established in a variety of cell types including vertebrate hippocampal neurons and motoneurons (Cristie et al. 1995; Simon et al. 2003; Westenbroek et al. 1990). The results of the present study suggest that Dmca1A and Dmca1D are differentially distributed in Drosophila motoneurons. Although our recordings were from the cell body, Ca2+ channels that reside in dendritic processes probably contributed to the whole cell current. The presence of poorly clamped and delayed onset currents in some records (see METHODS) suggests that the Ca2+ channels are located, at least in part, in dendritic processes that are somewhat distal to the cell body (Heckman et al. 2008; Johnson et al. 2003). Dendritic Ca2+ currents have been revealed in other insect motoneurons using both voltage clamp and Ca2+ imaging (Duch and Levine 2002). Additional Ca2+ channels, such as those in presynaptic terminals, probably lie outside the range for adequate space clamp and did not contribute to the voltage-dependent current we recorded from the cell body. It is well established that cac is expressed in motoneurons and localizes to the neuromuscular junction (Kawasaki et al. 2000, 2002, 2004). Thus Drosophila motoneurons may serve as a favorable model for investigating the mechanisms that determine the distribution of distinct Ca2+ channel types.
In contrast to our findings from in situ recordings, Dmca1A was found to be the major contributor to voltage-dependent Ca2+ currents in the Drosophila giant neuron culture system (Peng and Wu 2007). Three separate mutant alleles for Dmca1A (cacs, cacts2, and HC129) reduced Ca2+ current recorded from the cell body of these cell-division-arrested embryonic neuroblasts. This may reflect differences in Ca2+ channel gene expression among cell types, although it is not clear what neuronal types the giant neurons represent. Alternatively, there may be a shift in the contributions of different channel types during embryonic and postembryonic development, with cac providing the dominant contribution at embryonic stages. It is also likely that the contribution of Dmca1A to the Ca2+ current in axon terminals is more readily measurable from the cell body in the more compact cultured cells.
Whereas mutant alleles of cac did not significantly reduce whole cell voltage-dependent Ca2+ currents in our experiments, they did alter cellular firing properties. This may represent a role for distant cac-encoded Ca2+ channels in action potential initiation or shape, or a low-density population of this channel type within the dendritic membrane, that would not have been prominent in our voltage-clamp analysis or in images of fluorescently tagged channels (Kawasaki et al. 2004). The firing frequency was increased in cac motoneurons. This may reflect reduced activation of Ca2+-activated K+ channels. Alternatively, there may have been a compensatory developmental reduction in the expression of K+ channels in cac mutants (Peng and Wu 2007). It may be possible to test this possibility using cacts lines or by expressing an appropriate RNAi transgene in the motoneurons during a restricted temporal window (Nicholson et al. 2008).
The same hypotheses apply to the increased firing frequency that was observed in AR66 mutants and is consistent with the effect of removing external Ca2+ from the extracellular solution. Furthermore, the reduction in spike amplitude in AR66 mutants may reflect a contribution of somatic or dendritic Ca2+ channels in these regions. Although in most insect motoneurons the action potential is initiated where the axon leaves the dendritic region and is thought to invade the cell body passively (Gwilliam and Burrows 1980), somatodendriic Ca2+ channels can boost this depolarization (Duch and Levine 2000, 2002). The spontaneous spike-like events observed in motoneurons expressing RNAi91 may reflect spike initiation in a second, more distal, location. Initiation at this site may have been allowed by a reduction in K+ channel activation or expression.
An important next step will be to determine whether the Ca2+ current described in this study reflects the dendritic compartment of the motoneurons. Ca2+ imaging will help to resolve this question. Voltage-dependent Ca2+ channels localize to the dendrites of vertebrate motoneurons where they are proposed to increase the sensitivity to both excitatory and inhibitory inputs (Heckman 2003; Heckman et al. 2008; Hultborn et al. 2004; Hyngstrom et al. 2008; Lee and Heckman 2000). Furthermore, modulation of voltage-gated Ca2+ channels in dendrites may shape motoneuron recruitment patterns during locomotion (Heckman et al. 2008) or other types of patterned motor activity (Johnson et al. 2003). Whereas it is challenging to investigate the contribution of Ca2+ channels and their modulation to the activation patterns of specific neurons in the intact mammalian nervous system, these important mechanisms can be addressed readily in Drosophila. With the development of in situ whole cell recording techniques and the availability of cell-specific genetic manipulations that are possible in Drosophila, this system will allow rigorous examination of mechanisms contributing to motoneuron recruitment in diverse systems.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Address for reprint requests and other correspondence: R. Levine, Division of Neurobiology, University of Arizona, Tucson, AZ 85721 (E-mail: RBL{at}neurobio.arizona.edu)
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