Anesth Analg 2004;98:1705-1711
© 2004 International Anesthesia Research Society
doi: 10.1213/01.ANE.0000113548.27457.A3
ANESTHETIC PHARMACOLOGY
A Specific Alteration in the Electroretinogram of Drosophila melanogaster Is Induced by Halothane and Other Volatile General Anesthetics
Shantadurga Rajaram, PhD, and
Howard A. Nash, MD, PhD
Laboratory of Molecular Biology, National Institute of Mental Health, Bethesda, Maryland
Address correspondence and reprint requests to Dr. Howard A. Nash, Bldg. 36, Room 1B08, Laboratory of Molecular Biology, NIMH, Bethesda, MD 20892-4034. Address e-mail to howard.nash{at}mail.nih.gov
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Abstract
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In higher organisms, physiological investigations have provided a valuable complement to assays of anesthetic effects on whole-animal behavior. However, although complex motor programs of Drosophila melanogaster have been used to identify genes that influence anesthesia, electrophysiological studies of anesthetic effects in this invertebrate have been limited. Here we show that the electroretinogram (ERG), the extracellular recording of light-evoked mass potentials from the surface of the eye, reveals a distinct effect of halothane, enflurane, isoflurane, and desflurane. Behaviorally relevant concentrations of these volatile anesthetics severely reduced the transient component of the ERG at lights-off. Other prominent ERG components, such as the photoreceptor potential and the lights-on transient, were not consistently affected by these drugs. Surprisingly, for most anesthetics, a diminished off-transient was obtained only with short light pulses. An identical effect was observed in the absence of anesthetic by depressing the function of Shaker potassium channels. The possibility that halothane acts in the visual circuit by closing potassium channels was examined with a simple genetic test; the results were consistent with the hypothesis but fell short of providing definitive support. Nevertheless, our studies establish the ERG as a useful tool both for examining the influence of volatile anesthetics on a simple circuit and for identifying genes that contribute to anesthetic sensitivity.
IMPLICATIONS: Electroretinography (ERG) provides a useful monitor of anesthetic effects on the fruit fly. The effects of volatile anesthetics on the ERG are recapitulated by inactivation of potassium channels.
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Introduction
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Current studies in model organisms such as the fruit fly are identifying genetic loci that influence the way intact organisms respond to general anesthetics (1). The assumption that anesthetic action in these lowly invertebrates is similar to that of animals with a backbone is supported by several observations. For example, despite a recent statement implying the contrary (2), the absolute concentrations of volatile anesthetics required to disrupt behavioral end-points in wild-type flies are comparable to those needed to produce anesthesia in vertebrates (3–5). Moreover, compounds such as decane that disobey the Meyer-Overton correlation in vertebrates have also been found to do so in Drosophila (3). Also, just as in vertebrates, in which elementary motor and sensory functions are often retained at anesthetic concentrations that disrupt higher-order processing, in Drosophila a simple motor circuit remains active at concentrations of volatile anesthetics that render the organism behaviorally inert (6). Taken together, these observations encourage us to believe that genes identified in this model organism will be of general interest for studies of anesthetic action.
One of the most attractive features of the model organisms is that they expedite a forward mutagenesis strategy, i.e., creation of large sets of random mutants that can be screened for a phenotype such as altered anesthetic responsiveness. The end-product of this strategy is often a mutation in a gene that previously had been unsuspected to influence anesthesia or even to act in the nervous system. The challenge then is to determine how the mutation produces its anesthetic phenotype. When the end-point used to identify the mutation is the disruption of a complex behavior in an intact animal, one must be concerned that the anesthesia phenotype is a very indirect consequence of an effect on motor strength and/or coordination of the organism. What is needed is an end-point that does not rely on coordinated motor performance. Moreover, to enhance the possibility of studying the relationship between gene action and anesthetic sensitivity, it would be best if the assay system for this end-point involved a circuit of limited complexity. In this work, we show that the electroretinogram (ERG) of Drosophila, the light-evoked current recorded extracellularly from the surface of the eye (7), provides one such assay system. We find that modest concentrations of halothane and several other anesthetics produce a specific change in the ERG, a severe diminution of the off-transient. That this end-point is subject to genetic modification is proven by our demonstration that, in the absence of anesthetics, the identical change is associated with null mutations of Shaker, a gene that encodes a voltage-gated potassium channel. By studying variants with altered gene expression, we address the question of whether anesthetics affect the ERG by direct action on Shaker potassium channels.
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Methods
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The techniques for general handling and maintenance of the fly stocks have been described previously (6). Except where noted otherwise, flies used for ERGs were 2- to 7-day-old females that were collected under a brief exposure to carbon dioxide and allowed at least an overnight recovery period. The wild-type stock used in this study was the Canton-S strain, originally obtained from J. Steven de Belle (University of Nevada, Las Vegas, NV). The ShKS133 allele was obtained from Dr. M. Tanouye (Caltech, Pasadena, CA) and was made congenic to the wild-type Canton-S strain by repeated back-crossing (8). Because this allele is an antimorph, for controlled reduction of gene expression we acquired from Dr. F. Tejedor (Universidad Miguel Hernandez, San Juan, Alicante, Spain) a strain T(1;Y)B55-T(1;Y)W32 that carried a Shaker deletion. Males of this strain were crossed to Canton-S females to generate deficiency heterozygote females, which have only 50% of the normal Shaker complement. Dr. C. Oh (University of California, Berkeley, CA) provided the Tp(1;3)JC153 strain; males were crossed to Canton-S females to generate male offspring with an extra copy of the Shaker locus. Details concerning all the alleles in these strains have been catalogued (9,10).
The procedure for the vaporization, delivery, and monitoring of volatile anesthetics has been reported previously (6,11), as has the protocol for the administration of 4-aminopyridine (4-AP) (8). The ERG was measured as the voltage difference between the fly eye and a reference point, typically the thorax. The technique for tethering a fly without recourse to anesthesia was essentially as described (6,8). Although this tethering maneuver prevented escape, the fly was not completely immobilized. Because glass electrodes could not withstand the residual head movement, recording was accomplished with electrolytically sharpened tungsten wire electrodes (A-M Systems Inc., Everett, WA) inserted into dabs of electrode cream (Signa Creme; Parker Laboratories, Inc., Fairfield, NJ) applied to the eye and the thorax. The electrodes fed into a differential amplifier whose output was split between a digital oscilloscope and an analog-digital converter (National Instruments, Austin, TX).
After electrode placement, the fly was enclosed by a Plexiglas box through which flowed a stream of humidified air. After 10 min of dark adaptation, a set of reference ERGs was recorded, and the airflow was replaced with a stream of anesthetic vapor. After an hour of exposure, a period more than sufficient to ensure complete equilibration of the fly with the anesthetic, a second set of ERGs was recorded. Each fly was tested at only one concentration of a given anesthetic. Recovery of the ERG was often monitored after an additional hour of plain humidified air.
The optical equipment was from Oriel Instruments (Stratford, CT). Light from a 150-W xenon arc lamp filtered with an orange broadband filter (peak 
= 600 nm) to an intensity of 3 mW/cm2 was delivered to the fly by means of a fiberoptic guide. An electronic shutter generated a train of 5 light pulses (either 3 s or 200 ms long) that stimulated the fly at 15-s intervals. Data (collected every 4 ms) from all five responses were combined (with Labview software; National Instruments) to yield one average ERG. To quantitate the combined short-pulse ERG, a baseline voltage was obtained by averaging the values from the 35 sample points immediately before lights-on. The amplitude of the on-transient was calculated as the difference between the highest voltage reached after lights-on and this baseline value. Similarly, the amplitude of the maintained potential was measured as the difference between the baseline value and the last single point before lights-off. The off-transient amplitude was calculated as the difference between this last voltage and the lowest voltage value reached after lights-off.
For a given genotype and/or treatment, we typically collected ERGs from a group of 8–15 flies. For the data of Figure 2, anesthetic effects were evaluated by comparing separate groups of treated flies with a group that had been exposed only to air. Because nonequality of the variances for different treatment groups precluded evaluation of confidence limits for potency, a nonparametric analysis of data was performed. For each anesthetic, significant differences were judged by ranking all the amplitude measurements of an ERG component together with the no-anesthetic controls that had been done in parallel. The mean rank of values for each concentration tested was then compared with the mean rank for the air-only control values. Finally, the magnitude of the resulting difference in mean ranks was compared with a theoretical value, calculated to ensure that the false-positive rate for claiming significant decreases in the size of the ERG component within the entire data set for the particular anesthetic was no larger than 0.05 (12). For the data of Figure 3, pairwise rank sum tests were performed.
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Figure 2. Quantitation of anesthetic effects on the electroretinogram (ERG). Each entry represents ERG amplitudes from separate groups of wild-type flies, presented as a box encompassing the central 50% of the data points, i.e., those ±25% around the median value (shown by a horizontal line). Vertical error bars encompass other data points that lie 1.5 box heights above and below the box limits, and the remaining data points (outliers) are shown individually as open circles. For each anesthetic concentration, the entries whose values are significantly different from those of air-only flies are marked with an asterisk. To obviate seasonal or other long-term fluctuations, these controls were performed within the same time frame as each set of anesthetic tests. The positions of the ERG used to measure the amplitudes of each component and the statistical analysis of the data are described in Methods.
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Figure 3. Manipulation of Shaker channels. A, Representative electroretinograms (ERGs) from a ShakerKS133 fly; responses were elicited in the absence of anesthetic by a 3-s light pulse (left) or a 0.2-s light pulse (right). B, The effects of 4-aminopyridine (4-AP) on short-pulse off-transient amplitude. The values obtained after feeding sucrose alone (diagonal hatching) or sucrose plus 1.5 mM 4-AP (cross-hatching) are presented in box plots (as in Fig. 1). *For wild-type flies, the off-transient after 4-AP feeding was significantly lower (P < 0.05) than after feeding sucrose. For Shaker deficiency and duplication lines (whose genotype and construction is detailed in Methods), the amplitudes after sucrose feeding (not shown) were not significantly different from the comparable values for wild-type flies.  After 4-AP feeding, the off-transient of deficiency heterozygotes was significantly lower (P < 0.01) than that of similarly fed wild-type flies. C, Halothane sensitivity of Shaker deficiency and duplication flies. The box plots present the amplitude of the short-pulse off-transient after an hour of exposure to halothane relative to that measured before exposure. The genotype and the halothane concentration are given below each box. *Halothane induced a significantly (P < 0.01) larger reduction in off-transient amplitude in deficiency heterozygotes than in wild-type flies.
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Figure 1. Effect of halothane on the electroretinogram (ERG). Representative traces elicited in wild-type flies by a 3-s light pulse (A) or a 0.2-s light pulse (B). The shaded gray area in each of the traces indicates the period of the light stimulus. Each row presents traces taken from different individuals after a 1-h exposure to humidified air with the indicated concentration of halothane; the last row shows the recovery of the ERG of the fly exposed to 0.70%.
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Results
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The traditional Drosophila ERG protocol calls for a light pulse that is kept on for a few seconds (7). Light prompts cation-selective channels in the fly’s photoreceptors (PRs) to open, and the consequent depolarization alters the synaptic output of PRs to second-order neurons of the underlying optic lobe. Extracellular currents generated by the changing transmembrane fluxes in the PRs and their synaptic partners produce potential differences between the eye and thorax. The resulting ERG (Fig. 1A) displays four features: an upward spike at the start of the light pulse (the on-transient), a sharp potential drop that is then largely sustained for the duration of the light pulse (the maintained potential), a downward spike at the termination of the light pulse, (the off-transient), and, finally, a biphasic decay of the maintained potential. A large body of evidence has established that the maintained potential reflects currents generated by processes intrinsic to the PRs, whereas both the on- and the off-transients reflect currents generated by synaptic transmission from the PRs to the underlying neurons of the optic lobe (7).
Halothane disrupts coordinated motor performance in Drosophila, with 50% effective concentration values that range (depending on the end-point) from 0.15% to 0.41% (3,4). The traditional 3-s ERG shows little or no alteration by halothane at these concentrations (Fig. 1A). However, halothane potently induces a reversible depression in the off-transient of a short-pulse ERG. The traces shown in Fig. 1B are representative of a larger sample of wild-type flies; this is quantified in Figure 2. In contrast to the off-transient amplitude, which is progressively decreased starting at small concentrations of halothane, there is no significant trend in the values for the maintained potential and the on-transient. Diminution of the off-transient of ERGs elicited by 0.2-s light pulses is also seen with other volatile anesthetics, again at concentrations known to induce defects in behavior. For example, enflurane depresses motor performance in flies with 50% effective concentration values that range from 0.23% to 0.51% (3,4); the off-transient is significantly depressed at 0.15% enflurane and almost disappears at 0.43% (Fig. 2). Although tested less fully, the potency of isoflurane and desflurane in depressing the off-transient (Fig. 2) is also commensurate with behavioral potency (3,4). Like halothane, the three other anesthetics have no significant effect on the maintained potential, and only enflurane has even a modest effect on the on-transient. Most of these anesthetics also share with halothane a much reduced potency for influencing the off-transient elicited by a 3-s light pulse (unpublished data).
The Shaker gene encodes a prototypical voltage-gated potassium-selective ion channel that plays an important role in a variety of neural processes. As shown by the example in Figure 3A, when elicited by a 3-s light pulse, the ERG of a ShKS133 mutant looks normal. However, with a 0.2-s pulse of light, although the on-transient and maintained potential are unremarkable, the off-transient is severely diminished. The trace shown in Figure 3A is representative of a larger sample. The median value for the short-pulse off-transient amplitude of ShKS133 flies (n = 53) is 0.3 mV, much smaller than the corresponding value for wild-type flies tested in air but similar to that of wild-type flies exposed to 0.45% halothane (Fig. 2). By genetic, molecular, and physiological criteria, the KS133 mutation is a severe loss-of-function allele of the Shaker locus (8). We tested several other strong alleles of this locus and found (data not shown) that they all produced ERGs that resembled that of ShKS133.
Feeding the channel blocker 4-AP to adult wild-type flies has been shown to recapitulate, at least partially, the effects of Shaker mutations (8). Such a feeding protocol produces little or no effect on the ERG induced by a 3-s light pulse. However, feeding 4-AP to wild-type flies results in a short-pulse ERG with a diminished off-transient (Fig. 3B); a rank sum test indicated that, compared with sucrose, feeding 4-AP resulted in significantly lower amplitudes. Because the channel blocker was given only after completion of neurogenesis, we conclude that Shaker cannot play a purely developmental role in the ERG. Instead, at least in part, Shaker ion channel function is needed for proper operation of the visual circuitry.
To explore whether halothane and 4-AP alter the ERG by the same mechanism, we examined the effects of these drugs on variants with altered Shaker gene dosage. Because Shaker channels are thought to be the principal target of 4-AP, reducing the gene dosage (which should decrease gene expression by 50%) should sensitize the ERG to this drug. This prediction was borne out. Although in the absence of drug, deficiency heterozygotes had ERGs of normal amplitude, after 4-AP feeding they had smaller off-transients (Fig. 3B); a rank sum test indicated that deficiency flies were significantly more sensitive to the drug than wild-type flies. To see whether reducing the gene dosage of Shaker had a comparable effect on anesthesia, we exposed wild-type female flies and deficiency heterozygotes to an intermediate concentration of halothane. In this case, the effect on the off-transient was calculated as the ratio of amplitudes measured before and after a 1-h exposure of each fly to anesthetic. As seen in Figure 3C, removing one copy of the Shaker gene made flies more sensitive to the effects of halothane. Less informative than tests with reduced gene dosage were those that used an extra copy of the Shaker gene. Compared with wild-type flies, duplication heterozygotes (in which gene expression should be increased by at least 50%) were not significantly more resistant (P < 0.27) to the effects of feeding 4-AP (Fig. 3B). Similarly, when we exposed wild-type flies or duplication heterozygotes to a moderate concentration of halothane, there was no difference in the effect on the off-transient (Fig. 3C).
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Discussion
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The work described in this article establishes the ERG as a useful tool for anesthesia research in Drosophila. At concentrations that interfere with behavior in flies and vertebrates (3,5), we found a specific effect of volatile anesthetics—a diminution of the off-transient that follows a short pulse of light. This is in contrast to previous reports that an unspecified concentration of ether depressed both transients of Drosophila (13) and that graded concentrations of halothane caused parallel decreases in all ERG components of Musca (14). We suspect that the major experimental distinction is the duration of the light pulse, which also affects the sensitivity of the ERG to mutational effects (15,16).
Compared with our previous physiological assay for anesthesia (6,8), the principal advantage of the ERG is that much more is known about the cells and the circuitry. For example, the maintained potential is known to be the end result of a well studied cascade of signal transduction in PRs (17). Similarly, the on-transient depends on the release of histamine from the axon terminals of PRs and the subsequent opening of ligand-gated ion channels in their synaptic partners in the lamina of the optic lobe (7). Because these two ERG features persist at relatively large anesthetic concentrations (Fig. 2), we can conclude that volatile anesthetics do not severely depress many of the well identified components in the fly’s visual system, for example, G-proteins, phospholipase C, TRP channels, or ligand-gated ion channels.
The differential effects we observed on the two transients, taken together with earlier observations (15,18), make it clear that the off-transient is not generated merely by reversing the steps that produce the on-transient. Although the neural substrate for the off-transient is not certain, the well described anatomy and physiology of the optic lobe (19) place limits on possible schemes. The key fact behind such schemes is that the histamine receptors in cells postsynaptic to PRs are chloride channels whose opening causes hyperpolarization during the light pulse (17). There are two principal neuronal cell types that receive histaminergic input from PRs: large monopolar cells (LMCs) and amacrine cells; the latter are thought to be excitatory neurons with several outputs (Fig. 4). When the light pulse ends, PR target neurons will be relieved from hyperpolarization and thus could generate an overshoot similar to that described for the classic anode-break response. We propose that it is such excitatory currents in these neurons or in their downstream targets that produce the off-transient. Figure 4 thus provides a framework for enumerating the ways in which anesthetics might disrupt the functioning of the visual circuit. These include several possibilities that are well precedented in vertebrate anesthesia, such as prolongation of an inhibitory signal (like that from PRs) and reduction of an excitatory signal (like that in LMCs or amacrine cells). Tests of these possibilities are the subject of continuing investigation in our laboratory, as is the way in which longer light pulses alter the functional anatomy of the circuit.
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Figure 4. The principal elements of the visual circuit involved in the electroretinogram (ERG). This scheme depicts the major neuronal types and synaptic contacts found in the retina and the lamina, the first layer of the Drosophila optic lobe (19). The width of the arrows is approximately indicative of the number of the synaptic connections. R1–R6 denote the photoreceptors (PR), which have their light-gathering apparatus and cell bodies in the retina and send axons into the lamina. These make synaptic contact with two neuronal cell types: large monopolar cells (LMC) that provide output to deeper layers of the optic lobe (not shown) and amacrines (Am), neurons whose input and output is entirely contained within the lamina. The principal output of amacrines is directed to PR axons, to LMC dendrites (intermingled with PR connections), and to T1 cells, neurons with an impressive dendritic field in the lamina but whose cell bodies and output fields lie in a deeper layer (not shown). The inhibitory nature of the PR synapses is indicated by blunt arrowheads, whereas the presumptive excitatory nature of amacrine contacts is indicated by pointed arrowheads. Not shown are epithelial glia, which receive synaptic input from both PRs and amacrines (19), but whose role in the visual circuit is unknown. Within the context of this scheme, volatile anesthetics could cause failure of the lights-off response by interfering with (a) the cessation of the inhibitory signal in PRs, (b) the generation of an excitatory response in neuronal or glial targets of PRs, (c) the delivery of an excitatory signal to the receptive field of disinhibited amacrines, or (d) the response to excitation by amacrine target cells.
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Of all the cell types in the fly’s retina and optic lobe lamina, only PRs appear to express the Shaker gene (20). An appealing idea for the role of Shaker in the generation of the off-transient is that the channel is needed for prompt repolarization of PR axons at lights-off, thus ensuring that the inhibitory signal decays swiftly to create a synchronous population of disinhibited LMCs and amacrines. One problem with this view is that many Shaker channels are known to inactivate rapidly (i.e., within a few milliseconds) and so would not be expected to be open at the end of a 200-ms light pulse. However, when expressed in a heterologous system, at least one of the Drosophila splice isoforms (one known to be expressed in the retina) (21) enters a "slow mode" in which it behaves as if it cannot inactivate (22). We propose that this or a related isoform contributes to the speed at which PRs repolarize at lights-off. Consistent with this idea is the recent observation that Shaker channels are important not for the response of Drosophila PRs to steady light but for the response to light that is modulated at a frequency of 1–50 Hz (23). Indeed, our observation on the short-pulse off-transient may be the low-tech counterpart of that information capacity analysis.
Is it possible that Shaker mutations and volatile anesthetics produce an identical alteration in the ERG because anesthetics act by closing the Shaker channels of the visual system? Studies by others (24,25) have provided little support for the idea that Shaker channels are sensitive anesthetic targets. However, these studies involved heterologous expression; they may have examined an insensitive isoform or omitted a critical component. As a first step toward an answer in an intact organism, we undertook a correlational analysis between the level of gene expression and anesthetic potency. In Drosophila, there is generally a linear relationship between gene expression and copy number, so we began by examining flies heterozygous for a deficiency that removes the core of the Shaker locus. We reasoned that if Shaker channels are a direct drug target, then reducing channel density by decreasing the number of gene copies should increase the potency of drugs that inhibit channel function. Indeed, deficiency heterozygotes were more sensitive than wild-type flies both to 4-AP, a known inhibitor of Shaker channels, and to halothane. This result certainly provides support for the direct action hypothesis but is also consistent with models that invoke Shaker as an element that determines the safety factor of a circuit (8). For example, within the context of the circuit shown in Figure 4, halothane might act not on PRs but on LMCs or amacrine cells; if so, reducing levels of Shaker in PRs might sensitize the lights-off component of the ERG to halothane merely by providing a less effective signal to the downstream cells.
We had hoped that generating flies with an extra copy of Shaker would provide a complementary test of the direct action hypothesis because, if the channels are a direct drug target, enhancing function by increasing the number of gene copies should decrease potency. However, although the naive expectation was that expression would be increased by at least 50% (26), a Shaker duplication had little effect on 4-AP sensitivity. Either Drosophila PRs fail to increase Shaker expression in response to increased gene dosage or channel function is limited by the supply of an accessory subunit. In any case, the absence of a clear effect of the genetic duplication on sensitivity to a known channel inhibitor renders uninformative the failure of this manipulation to alter halothane potency. We are left without a strong reason to eliminate a specific Shaker isoform as an anesthetic target but also without a clear way to distinguish whether anesthetics alter the ERG by affecting another component in neurons of the visual circuit.
We conclude that this work provides a resource that had been missing from previous studies with anesthetics in model invertebrates—a circumscribed circuit with well identified components that is sensitive to biologically relevant concentrations of volatile anesthetics. Although the small size of the involved neurons precludes a straightforward dissection of anesthetic action at the cellular level in Drosophila, the ERG can be exploited to narrow the search for anesthesia genes that operate at or close to the target of anesthetics. For example, attention would be focused on a mutation that altered halothane sensitivity of locomotor behavior in the fly if it also caused enhancement or suppression of halothane’s effects on the ERG. The same criterion could also be applied to mutations in candidate genes, chosen because they affect components of the fly’s nervous system that are thought to be anesthetic targets. Although further work would be needed to evaluate the importance of the homologous genes in vertebrate anesthesia, compared with studies relying entirely on complex behavioral assays, an ERG screen should be primed to identify loci whose function is intrinsic to anesthetic action in the fly.
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Acknowledgments
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For deft construction of the anesthetic chamber and for implementation of data analysis and acquisition programs, we respectively thank David Ide and George Dold of the Research Services Branch, National Institute of Mental Health. For useful discussion, we thank Drs. I. Meinertzhagen, B. White and M. Stern, and for comments on this manuscript, we are indebted to Drs. C.-H. Lee, P. Morgan, D. Sandstrom, and M. Sedensky.
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References
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Accepted for publication December 3, 2003.
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Abstract
Introduction
