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Laboratory of Molecular Biology, National Institute of Mental Health, National Institutes of Health MSC 4034, Bethesda, MD-20892, USA
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Abstract |
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(Received 1 April 2004;
accepted after revision 25 May 2004;
first published online 28 May 2004)
Correspondence D. J. Sandstrom: Laboratory of Molecular Biology, National Institute of Mental Health, National Institutes of Health MSC 4034, Bethesda, MD-20892, USA. Email: sandstrd{at}mail.nih.gov
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Introduction |
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Experimental and theoretical analyses have implicated many molecules as possible anaesthetic targets, but it has been difficult to determine the link between altered molecular function and altered behaviour (Sonner et al. 2003). One potentially fruitful approach is genetic analysis, either via forward genetic screens for mutants with altered sensitivity to anaesthetic, or a reverse genetic approach that selects candidate genes on the basis of their known function (Nash, 2002). In either case, it is possible in principle to validate, or invalidate, candidate anaesthetic targets by examining the correlations between behavioural and physiological alterations in mutants. For example, a single amino acid alteration of a GABAA receptor subunit strongly reduces the effects of intravenous anaesthetics (Jurd et al. 2003). However, the same mutation had a less dramatic effect on the response to volatile anaesthetics, implying the presence of additional targets for these compounds.
The Drosophila larva presents an opportunity to analyse anaesthetic function with a combination of behavioural, neurophysiological, and genetic approaches. The axons and synapses at the larval neuromuscular junction allow detailed analysis of the physiological events underlying excitation and synaptic transmission (Jan & Jan, 1976; Kurdyak et al. 1994). In addition, the simplicity of larval locomotion (Berrigan & Pepin, 1995) makes behavioural analysis more straightforward than that in animals with jointed legs and elaborate postural control (Strauss & Heisenberg, 1990).
In previous work with hatchling Drosophila larvae, the volatile anaesthetic halothane reversibly inhibited glutamate release from the nerve terminal without affecting postsynaptic glutamate receptor function (Nishikawa & Kidokoro, 1999). This effect was blocked by mutations in the ion channel encoded by the narrow abdomen locus, suggesting a possible mechanism for the altered behavioural response to anaesthetics in these mutants (Krishnan & Nash, 1990; Nash et al. 2002). However, the cellular alterations caused by halothane were not further described. Anaesthetics have been shown to reduce excitability and Ca2+ influx (e.g. Butterworth et al. 1989; Study, 1994), and there is evidence that anaesthetics may act on the vesicle fusion apparatus as well (van Swinderen et al. 1999). At present, it is not known which of these cellular properties is affected by volatile anaesthetics in Drosophila larvae. In addition, because the concentration of halothane required to immobilize larvae is at present unknown, the behavioural relevance of the halothane-induced inhibition of glutamate release is unclear.
Because isoflurane can affect many aspects of excitability and synaptic transmission, the goal of the present study was to identify the primary cellular site(s) of action of volatile anaesthetics in wandering third instar larvae. Knowledge of the cellular mechanism(s) of isoflurane's action will permit one to pursue the connection between molecular targets and the whole-animal effects. At the neuromuscular junction, isoflurane reduces neurotransmitter release reversibly, leaving glutamate receptor function unaltered, as shown previously for halothane. The present study demonstrates that the reduction of neurotransmitter release is indirect, in that decreased vesicle fusion is a secondary consequence of reduced excitability in the axon and synaptic terminal. This reduction of excitability causes a measurable decrease in the probability of vesicle release in response to an action potential. These effects occur at the same concentrations of isoflurane that inhibit larval mobility. Thus, behaviourally relevant concentrations of isoflurane cause complex physiological effects, at the interface between excitability and neurotransmitter release.
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Methods |
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Cultures were reared on standard cornmeal-molasses medium in 50 ml vials at 25°C. Larvae were reared on food containing 0.05% bromphenol blue (Sigma, St Louis, MO, USA) to facilitate selection of early wandering larvae (Maroni & Stamey, 1983). With the exception of male Oregon-R larvae used for the locomotor assay, all experiments were performed on male Canton-S larvae.
Electrophysiology
Dissections and recordings were carried out in HL3 saline (mM: 70 NaCl, 5 KCl, 20 MgCl2, 10 NaHCO3, 5 trehalose, 115 sucrose, 5 Hepes; pH 7.2; Stewart et al. 1994), with Ca2+ concentrations as described in the text. Dissections were performed in chilled Ca2+-free HL3 saline with 0.5 mM EGTA. Recordings were performed at room temperature (
23°C).
Voltage clamp recordings of synaptic currents were performed on muscle 6 of abdominal segment 5 in wandering third instar larvae, as previously described (Stimson et al. 1998). Synaptic currents were monitored using two-electrode voltage clamp (Axoclamp 2B; Axon Instruments, Union City, CA, USA), holding Vm at –70 mV. Microelectrodes were fabricated from borosilicate capillary glass (Friedrich and Dimmmock, Millville, NJ, USA), pulled on a horizontal puller (Sutter P-87; Sutter Instruments, Novato, CA, USA). Excitatory junctional currents (EJCs) were evoked by stimulation of the cut end of the segmental nerve (SN) to segment A5 via a glass-tipped suction electrode. Stimuli and solution changes were controlled and data were acquired using pCLAMP 8.1 (Axon Instruments), and synaptic currents were analysed using MiniAnalysis software (Synaptosoft, Decatur, GA, USA). Because physiological parameters drifted slightly during experiments, comparisons between treatment groups were always made at the same time point in the experiment. Parameters for isoflurane-treated preparations returned to control levels after removal of the drug, indicating that isoflurane had no effect on the rate or magnitude of drift.
Miniature EJCs (mEJCs) were recorded in the presence of 1.0 µM TTX. Amplitude, area and frequency were calculated from the average of all mEJCs in a 100-s period. The decay time constant was measured by fitting a single exponential to an averaged mEJC from the relevant 100-s period of each preparation (66–167 events).
To record conduction velocity the preparation was dissected as above, but the ventral ganglion (minus brain lobes) was left to anchor the segmental nerves proximally. A stimulating electrode was placed on the branch of the ISN between muscles 2 and 3 of segment A5, to stimulate a small population of motor axons antidromically (Fig. 3C1). Two recording electrodes, connected to a differential AC amplifier (Model 1700; A-M Systems, Carlsborg, WA, USA), were placed proximally, one adjacent to muscle 6 in segment A5, the other at the entry of the SN to the CNS. All values are given as the time difference between the arrival of the first negative peak of the compound action potential at the distal recording site to that at the proximal site.
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Anaesthetic delivery
Isoflurane (Baxter Healthcare, Deerfield, IL, USA) was dissolved in HL3 saline by placing a quantity measured with a gas-tight syringe into a vial with a Teflon-lined cap. The vial was vortexed at top speed for 1 min, at which point the isoflurane was dissolved maximally as measured by gas chromatography. Isoflurane solutions were superfused from separatory funnels connected to a solenoid valve array (Neptune Research, Caldwell, NJ, USA). Funnels were plugged with PTFE stoppers (VWR) into which were inserted polycarbonate check valves (Small Parts Inc., Miami Lakes, FL, USA) that allowed air to enter as the funnels drained. Isoflurane concentrations remained stable for many hours when solutions were stored in this way.
Solutions were delivered to the chamber with a peristaltic pump (Harvard Apparatus, Holliston, MA, USA) via PTFE tubing at 0.7 ml min–1, and removed by vacuum. The unenclosed chamber was constructed of Plexiglas, with a glass bottom, with a working volume of approximately 50 µl. Solutions arrived within 30 s of valve switching, and complete equilibration occurred within 2 min. The concentration in the chamber at equilibrium was approximately 40% below that in the reservoirs, comparing favourably with that in similar recording arrangements (e.g. Yamakura et al. 1999).
All volatile general anaesthetics tested caused spontaneous contractions of larval body wall muscles, appearing as waves of contraction moving along the lengths of the muscles. This problem has been described by others working with Drosophila muscles (Ueda & Kidokoro, 1996). In low-to-moderate concentrations of anaesthetic ethers, such as isoflurane or enflurane, the phenomenon appeared as occasional contractions occurring in random muscles at irregular intervals. At high concentrations of isoflurane, and even in relatively small amounts of halothane, contractions occurred regularly enough to impede stable recordings. Contractions persist in Ca2+-free saline (Ueda & Kidokoro, 1996; author's unpublished observations), and in TTX, and are associated with neither spontaneous synaptic currents in the muscle nor action potentials in the nerve (author's unpublished observations). Thus, the evidence supports a myogenic origin, possibly via Ca2+ release from intracellular stores. Recordings were possible, if sometimes challenging, to 0.4 mM isoflurane.
For each experiment, isoflurane concentrations were determined by gas chromatographic headspace analysis (Maiorino et al. 1979; Shimadzu GC-9 A; Shimadzu Scientific Instruments, Columbia, MD, USA). Details of chromatograph operation have been previously described (Allada & Nash, 1993). To measure concentration in the chamber, 200 µl of perfusate was placed into a 6 ml headspace vial and an aluminium seal with a PTFE septum crimped on. Sample vials were heated to 60°C and 250 µl of headspace gas was injected into the gas chromatograph with a Hamilton syringe. Three replicate injections were performed for each sample, with interreplicate variation being less than 5% in most cases. The concentration of isoflurane in the recording chamber was back-calculated (Allott et al. 1973; Franks & Lieb, 1993). Because the chamber concentration of volatile substances such as isoflurane could not be precisely controlled, and was determined at the end of each experiment, the values of anaesthetic concentration were binned as follows: 0.1 mM= 0.08–0.13 mM; 0.15 mM= 0.13–0.17 mM; 0.2 mM= 0.17–0.23 mM; 0.25 mM= 0.23–0.27 mM; 0.4 mM= 0.37–0.43 mM.
Larval locomotion assay
Pairs of actively wandering larvae were placed on 100 mm Petri dishes lined with 2% agarose gel, in an acrylic chamber fitted for gas flow. A stream of humidified air, mixed with isoflurane as previously described (Campbell & Nash, 1998), flowed through the chamber at 5 l min–1. Each larva was used for a single concentration. After equilibrating for 30 min, larvae were moved to the centre of the dishes and then allowed to recover from the physical manipulation for 30 s. Larvae were transilluminated from below with a low-wattage light box (VWR), and 1 min of activity was recorded at 2 frames s–1 as an AVI file (Panasonic WV-BP330 camera and 18–108 mm macro zoom lens, Noldus Information Technologies, Sterling, VA, USA; MiroVideo DC-30 plus Video Capture Card, Pinnacle Systems, Elk Grove, CA, USA). AVI files were converted to TIFF series in Adobe Premiere 5.1 for analysis in ImageJ, a JAVA-based image analysis program (http://rsb.info.nih.gov/ij/). Tracks were analysed with the MultiTracker plugin (written for ImageJ by Jeffrey Kuhn, University of Texas, Austin, USA). To prepare the data for curve fitting, they were averaged and normalized to the maximum average distance. Movement of the mouthhooks produced artifactual centroid displacement, even at the highest isoflurane concentrations, requiring residual movement to be subtracted. Larvae recovered and developed into normal adults at all concentrations of isoflurane.
Statistics
Data are presented as the mean ±S.E.M. Tests for significant difference between groups of normalized data were performed using one-way ANOVA, followed by Tukey's honestly significant difference test, when appropriate (SPSS Inc., Chicago, IL, USA). Some datasets contained significant heteroskedacity by the Levene test, and the results were analysed with the non-parametric Kruskal–Wallis test, with post hoc Mann-Whitney U tests. Differences were considered significant at P < 0.05. Concentration–response data were fitted in Origin 5.0 (OriginLab Corp., Northampton, MA, USA), weighting the data instrumentally with the standard errors.
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RESULTS |
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In control preparations, superfused continuously with saline containing 1.0 mM Ca2+ and stimulated every 10 s, initial EJC amplitude was 40.0 ± 5.2 nA and remained relatively steady over the course of a 25 min experiment (Fig. 1A). Isoflurane produced two readily apparent changes. The first was a decrease in EJC amplitude (Fig. 1A and B; P < 0.001, one-way ANOVA). The second was an increase in latency between nerve shock and the onset of the EJC (Fig. 1B; see also Fig. 3A and B). Both of these effects reversed after isoflurane was removed.
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There was no detectable alteration in the size, shape or frequency of miniature EJCs (mEJCs), generated by spontaneous release of single vesicles (Fig. 2A). Initial mEJC amplitude was 0.54 ± 0.01 nA, decreasing slightly over the course of the experiment. The amplitude distributions of mEJCs in preparations treated with 0, 0.2 and 0.4 mM isoflurane were indistinguishable from one another (Fig. 2B). Mean mEJC amplitude, normalized to the initial value for each preparation, was not significantly affected by isoflurane concentrations up to 0.4 mM (0 mM, 0.94 ± 0.03, n= 6; 0.2 mM, 0.96 ± 0.02, n= 5; 0.4 mM, 0.92 ± 0.04, n= 5; P > 0.5, one-way ANOVA). Normalized mEJC area was also unaffected (0 mM, 0.86 ± 0.05, n= 6; 0.2 mM, 0.85 ± 0.06, n= 5; 0.4 mM, 0.79 ± 0.05, n= 5; P > 0.5, one-way ANOVA). There was no qualitative effect of isoflurane on mEJC shape (e.g. Fig. 2B, insets), and 
decay for mEJCs, was not significantly altered (Fig. 2C; P > 0.5, one-way ANOVA), indicating that glutamate receptor kinetics were not affected detectably by isoflurane at this synapse.
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Isoflurane reduces axonal excitability
As noted above, isoflurane increased the latency between the nerve stimulus and the onset of the EJC (Figs 1B and 3A). Isoflurane increased EJC latency significantly, to 22% above that in untreated controls (Fig. 3A and B; P < 0.001, one-way ANOVA), reaching full effect over essentially the same time course as the reduction in EJC amplitude (compare Figs 1A and 3A). This delay could occur at one or more steps between stimulation of the nerve and onset of synaptic currents. In the axon, excitability could decrease, increasing the delay to spike initiation and/or decreasing conduction velocity. There could also be increased latency between action potential invasion of the synapse and neurotransmitter release.
To examine the effect of isoflurane on axonal excitability, conduction velocity was measured directly. A distal branch of the intersegmental nerve (ISN), which contains only four to five motor axons (Hoang & Chiba, 2001) was stimulated and the time required for the compound action potential to travel between two proximal recording electrodes was monitored (Fig. 3C1). The result was a concentration-dependent increase in conduction time in response to isoflurane. In control experiments, conduction time was relatively stable, increasing only 1.1% by the midpoint of the experiment (Fig. 3C2). In isoflurane concentrations of 0.1, 0.2 and 0.4 mM, conduction time increased by 6.0 ± 1.7, 11.7 ± 1.4 and 14.8 ± 2.4%, respectively. The increases in 0.2 and 0.4 mM isoflurane were significantly different from 0 and 0.1 mM (P < 0.05, Kruskal-Wallis). When fitted with a Hill equation the maximum isoflurane effect was a 15% reduction in conduction velocity, with EC50= 0.117 mM. At all isoflurane concentrations, the effect on compound action potential velocity was less than the effect on latency from nerve stimulus to EJC onset. For example, at 0.2 mM, latency from nerve shock to EJC onset was increased by 22%, whereas action potential latency in the nerve only increased by 11% (Fig. 3B and C2). Thus, other effects, such as latency to spike initiation or additional slowing in the fine terminal branches of the motoneurone, also contribute to the delay. Nonetheless, these results demonstrate that axonal excitability, as reflected by conduction velocity, is significantly reduced by isoflurane.
Reduced excitability reduces glutamate release
The reduction in neurotransmitter output in response to isoflurane could be an additional consequence of altered excitability, in that decreased presynaptic depolarization will reduce Ca2+ influx and neurotransmitter release. Alternatively, isoflurane may act directly on Ca2+ channels or the fusion machinery to inhibit glutamate release. These alternatives can be distinguished by depolarizing the terminal electrotonically with a suction electrode placed near the synapse, while blocking action potentials with TTX (Rivosecchi et al. 1994; Song et al. 2002). If the effect of isoflurane on the EJC is a result of reduced depolarization by the action potential, it should be bypassed by depolarizing the terminal artificially. Conversely, if isoflurane affects excitability and neurotransmitter release independently, the EJC effect should persist during electrotonic stimulation.
In one set of experiments, EJCs were elicited with the standard protocol, and 0.25 mM isoflurane was applied to reduce neurotransmitter release (Fig. 4A). Once EJC amplitude had stabilized at its new level, saline containing 0.25 mM isoflurane and 1.0 µM TTX was perfused onto the preparation. The stimulating electrode was moved close to the synapse and the stimulus was adjusted to generate an EJC electrotonically. This direct, electrotonic, depolarization of the terminal produced an EJC with an amplitude indistinguishable from the control (P > 0.95, one-way ANOVA). Thus, electrotonic stimulation appeared to reverse the effect of isoflurane.
An alternative explanation is that electrotonic stimulation produced larger EJCs which gave the appareance of reversing the isoflurane effect. We believe that this is not the case for the following reasons. First, the amplitudes of electrotonically evoked EJCs were the same as those generated by spikes in normal saline at two external Ca2+ concentrations (Fig. 4C). Thus, the release machinery appears to operate normally and shows the same Ca2+ dependence as in action potential-mediated glutamate release. Furthermore, as demonstrated previously (Rivosecchi et al. 1994) the level of paired-pulse facilitation is identical for electrotonic and normal EJCs (Fig. 4D), demonstrating that intraterminal Ca2+ dynamics are largely normal and that neurotransmitter release is not saturated under these conditions.
In a separate set of experiments, electrotonically generated EJCs were compared in the presence and absence of isoflurane (Fig. 4E and F). In control preparations treated with 1 µM TTX, electrotonically elicited EJCs resembled the control EJCs generated by action potentials before TTX application (Fig. 4E1; n= 9). Electrotonically generated EJCs were narrower than in controls, possibly because 0.2 ms square pulses decay more rapidly than action potentials. EJCs evoked in the presence of TTX plus 0.2 mM isoflurane appeared identical to those evoked in TTX alone (Fig. 4E2; n= 9). Neither the amplitude (Fig. 4F; P > 0.50) nor the area (0 mM 0.93 ± 0.08; 0.2 mM, 0.85 ± 0.07, n= 9 in both groups; P > 0.12) of EJCs in preparations treated with TTX plus isoflurane differed from those treated with TTX alone. Thus, electrotonically evoked EJCs were insensitive to isoflurane, and the fundamental effect of isoflurane at this synapse is to decrease excitability, rather than directly inhibiting Ca2+ influx, vesicle fusion or mobilization.
Isoflurane reduces release probability
At a large, branching synapse, such as the neuromuscular junction, decreased excitability could inhibit neurotransmitter output via at least two mechanisms. The first, global reduction in action potential-induced depolarization, would reduce Ca2+ influx and decrease release probability. The second, reduction of depolarization to below the safety factor in the terminal branches of the axon, would result in effective silencing of active zones in boutons distal to the failing branch. Branch-point failure will reduce EJC amplitude, leaving release probability unaffected.
The magnitude of paired-pulse facilitation (PPF), the ratio of two closely spaced EJCs (EJC2/EJC1), is a reliable indicator of the relative probability of neurotransmitter release (Zucker & Regehr, 2002). As the release probability of EJC1 decreases, the magnitude of PPF increases. When pairs of EJCs were evoked every 30 s, with an interpulse interval of 25 ms in 0.63 mM external Ca2+, PPF is 1.56 ± 0.09 (Fig. 4D). As would be expected with a decrease in release probability, PPF increased with increasing concentrations of isoflurane (Fig. 5A). In saline-treated controls, PPF was 0.91 ± 0.06 of the initial value. In 0.2 mM isoflurane, PPF was 1.08 ± 0.06 (P= 0.14; one-way ANOVA), increasing significantly to 1.14 ± 0.07 above initial value in 0.3 mM isoflurane (P < 0.05; one-way ANOVA). Although modest, this is the magnitude of change in PPF that would be expected from a 20% decrease in EJC amplitude, based on the effects of manipulating Ca2+ concentration (Fig. 5B). These data support the hypothesis that isoflurane reduces neurotransmitter output primarily by reducing release probability.
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Larval immobilization by isoflurane
To provide behavioural context for the physiological experiments, a simple locomotor assay was used to determine the concentration of isoflurane that inhibits locomotion. In this assay, control larvae in humidified air behaved as previously described (Wang et al. 1997), with epochs of straight crawling punctuated by pauses and turns (Fig. 6, inset). There was no difference between the two wild-type strains tested, with untreated larvae travelling at an average rate of approximately 40 mm min–1 (Canton-S, 40 ± 3, n= 12; Oregon-R, 44 ± 4, n= 10). When isoflurane was added to the air circulating through the chamber, partial pressures below 0.2% had no detectable effect, and locomotion ceased at partial pressures above 0.8% (Fig. 6). The concentration at which locomotion was reduced by 50%, the EC50, was approximately 0.4% (Canton-S, 0.42 ± 0.02%; Oregon-R, 0.43 ± 0.02%). In Canton-S, the EC50 of 0.42 ± 0.02% converts to a tissue concentration of 0.21 ± 0.01 mM at equilibrium (Franks & Lieb, 1993; 95% conf. 0.017), which is almost identical with the EC50 of 0.17 mM for the reduction of EJC amplitude and area (Fig. 1C and D). Therefore, isoflurane's effects on synaptic transmission occur at approximately the same concentrations that affect larval mobility, consistent with the possibility that reduced excitability and neurotransmitter release are involved in behavioural anaesthesia.
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Discussion |
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Reduced excitability reduces transmitter release
The reduction of neurotransmitter release was the most robust effect on larval neurophysiology I observed. Based on several lines of evidence, this was an exclusively presynaptic effect, not involving alterations in the sensitivity or kinetics of postsynaptic glutamate receptors. First, there was no detectable change in the size or decay time of mEJCs. Second, electrotonic stimulation of the presynaptic nerve terminal reversed the isoflurane effect on EJC amplitude, an unlikely result if postsynaptic function were reduced by isoflurane. Finally, isoflurane produced a measurable increase in PPF, indicating reduced vesicle release probability.
The fundamental presynaptic effect of isoflurane was a modest reduction in excitability which was amplified into a larger reduction in glutamate release. Volatile anaesthetics have commonly been observed to reduce excitability, measured as spike threshold, conduction velocity, or both (reviewed in Elliott & Urban, 1995). Although these effects are often dismissed because of their relatively small magnitudes, they reflect events that can have a significant impact on neural function. Alterations in channel properties that reduce conduction velocity will also affect the size and shape of the spike, which can cause disproportionately larger alterations in presynaptic Ca2+ currents and neurotransmitter release (Augustine, 1990). Thus, even though isoflurane reduced conduction velocity by a maximum of about 15%, the associated alterations at the synapse decreased EJC amplitude by almost 30%. Indeed, at the giant synapse in the calyx of Held, it was shown that a very small reduction in spike amplitude induced by isoflurane caused a much larger reduction in neurotransmitter output, due to the non-linear relationships between spike area, Ca2+ influx and neurotransmitter release (Wu et al. 2004). The present study demonstrates that this effect is not limited to a particular developmental stage, brain region, or even phylum.
Larval sensitivity to isoflurane
In the locomotor assay, larvae were anaesthetized by isoflurane, with an EC50 of 0.42–0.43%atm, quite similar to the EC50 of 0.39% for adult flies (Campbell & Nash, 1994) and to the minimum alveolar concentration (MAC) of 0.6 % in mammals (calculated from a MAC of 0.3 mM; Franks & Lieb, 1996). The similar sensitivities of the stages and species is consistent with conservation of isoflurane targets between larvae and adult flies, and possibly mammals as well. Interestingly, the isoflurane EC50 values for Oregon-R and Canton-S larvae were almost identical, despite considerable genetic separation between the strains. Although this result is somewhat surprising, in that genetic background often has a strong impact on complex behaviours (Tully, 1996), it is possible that the relative simplicity of crawling behaviour renders it less sensitive to subtle molecular differences in the nervous system. Regardless, the apparent absence of strain differences will simplify subsequent genetic analysis, allowing behavioural screening without requiring elaborate steps to isogenize or outcross the mutant stocks.
Although Nishikawa & Kidokoro (1999) did not determine the halothane sensitivity of hatchling larval locomotion based on the EC50 values for adult flies (0.41%atm; Campbell & Nash, 1994) and wandering larvae (0.5%; author's unpublished observations), the concentration range they used (0.9–2.7%atm) may have been substantially higher than that required for immobilization. This may explain why they observed a halothane-induced reduction in mEJC frequency, while I recorded no such effect. Alternatively, this discrepancy could result from differences between the actions of halothane and isoflurane or developmental changes in synaptic function between first and third instar larvae.
Possible sites of action
The effects I measured in the periphery may be more pronounced at central axons and synapses, where circuitry is likely to be more sensitive to reduced excitability and concomitant inhibition of synaptic transmission. Central axons have smaller diameters, and, where they have been examined, central synapses have low and variable release probability (e.g. Dobrunz & Stevens, 1997). Thus, central transmission may fail at anaesthetic concentrations that inhibit neuromuscular function only modestly. Indeed, previous experiments have demonstrated that a central component of the visually evoked jump reflex fails at behaviourally relevant anaesthetic concentrations, while the peripheral component is not detectably altered (Lin & Nash, 1996; Campbell & Nash, 1998). The giant interneurone that mediates this reflex is also relatively insensitive to volatile anaesthetics, as shown by its high following frequency and unchanged latency when treated with halothane. This may reflect the high reliability of the axon, which is also relatively insensitive to reduction in Na+ channel function (Nelson & Wyman, 1990). My working hypothesis is that isoflurane immobilizes larvae by the reduction of excitability, and concomitant disruption of neurotransmitter release, in central neurones generating or modulating locomotor behaviour.
A large number of candidate anaesthetic target proteins have been identified by physiological assays and genetic screens (reviewed in Nash, 2002; Perouansky & Hemmings, 2003; Sonner et al. 2003). Some proteins, such as ion channels, are obvious candidates for mediating changes in axonal excitability. The voltage-gated Na+ channel, encoded by the paralytic (para) locus in Drosophila, is a strong candidate. Na+ channels are critical in determining spike size, threshold and conduction, and para mutants are hypersensitive to volatile anaesthetics (Gamo & Nakashima, 1991; Leibovitch et al. 1995). In addition, isoflurane has been shown to reduce glutamate release from synaptosomes by inhibiting Na+ channel activity (Lingamenini et al. 2001), an effect that resembles the inhibition of glutamate release reported here. Another attractive class of candidate targets comprises non-voltage-gated ‘leak’ or ‘background’ currents. Leak K+ conductances have been shown to be strongly enhanced by volatile anaesthetics, including isoflurane (Franks & Lieb, 1991; Shin & Winegar, 2003), and one family of leak channels, the 2P/KCNK potassium channels, has several members whose conductance is enhanced by isoflurane (Yost, 2003). A putative interaction site for volatile anaesthetics has been described for these proteins, which is conserved in the presumptive Drosophila orthologues (Talley & Bayliss, 2002).
The observation that isoflurane's effect saturates at a point at which conduction velocity is reduced by 15% and EJC amplitude is decreased 30% gives an important clue to the nature of the target. Whether isoflurane is inhibiting an excitatory channel, like Para, or activating an inhibitory one, like a KCNK family member, the maximal effect does not cause complete loss of propagation or neurotransmission. This provides an important criterion for identification of isoflurane target(s) in this system.
Experiments are ongoing to analyse the effects of mutations in channel genes, such as para, and narrow abdomen, on locomotion and excitability in the presence of anaesthetics. Preliminary results suggest that some mutants have parallel effects on the behavioural and physiological responses to isoflurane, while others affect neither (J. N. Walter and D. J. Sandstrom, unpublished observations). In addition, the larval behavioural assay can be used to screen for new mutations that affect isoflurane sensitivity. Such a screen would complement a candidate gene approach by generating a collection of mutants unbiased by preconceptions regarding site of action. Thus, isoflurane targets will be identified regardless of whether they are present in neurones, glia or other cell types.
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), but increases rapidly and reversibly upon treatment with 0.2 mM isoflurane (
). B, relative EJC latency at 700–800 s time point. Latency is increased significantly (P < 0.005) in treated (grey bar) versus control preparations (open bar). In this and subsequent figures, an asterisk indicates a statistically significant effect at P < 0.05. C, axonal conduction velocity. C1, schematic diagram of the preparation. The stimulating electrode is placed distally to stimulate a small number of motor axons, while two recording electrodes are placed along the nerve to record compound action potentials as they progress antidromically toward the CNS. C2, interelectrode delay at varying concentrations of isoflurane. Propagation time increases in a concentration-dependent manner, with 0.1, 0.2 and 0.4 mM isoflurane causing mean increases of 6.0, 11.7 and 14.8%, respectively. When the data are fit with a concentration–respose curve, the effect asymptotes at 15.0% delay, with EC50= 0.117 mM.
1.5 mM. C, isoflurane does not increase PPF in synapses stimulated electrotonically. In preparations not treated with isoflurane, PPF remains relatively steady, at 104 ± 6% control. PPF is not significantly different in 0.2 (light grey bar) or 0.3 mM isoflurane (dark grey bar) under these conditions.