Electroconvulsive Seizure Behavior in Drosophila: Analysis of the Physiological Repertoire Underlying a Stereotyped Action Pattern in Bang-Sensitive Mutants

Seizure repertoires evoked by mechanical and electroconvulsive shocks

On mechanical agitation, such as vortexing or tapping the culture vial on the bench, all bang-sensitive mutants (includingbas, bss, eas, kdn,sda, and tko; see Materials and Methods) showed a stereotyped sequence of initial muscle spasm (uncoordinated movements), paralysis (lack of motion and responsiveness), delayed spasm, and recovery (normal posture). The two spasms were manifested by collapse of the body, high-frequency wing flapping, leg extension, and fully curved abdomen (see Fig.2A for an example ofbss.) Furthermore, female flies often laid eggs after mechanical agitation (Fig. 2A, arrowhead). These spasms and paralysis were reproducible with a predictable time to onset of the delayed spasm or to recovery of normal posture inbas and bss flies, which was significantly lengthened in bas bss double-mutant flies (Fig.2B). This orderly behavioral sequence implies that it results from patterned activities of neural circuits rather than unassociated events in individual muscles. Significantly, wild-type flies (Fig. 2C) and other categories of behavioral mutants do not show such behavioral repertoire even after extreme mechanical stimulation, such as vortexing the culture vial for tens of seconds (see Materials and Methods).

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Fig. 2.

Mechanically induced seizure in the bang-sensitivebas, bss, and bas bssflies. A, Seizure repertoire in bssmutants. Mechanical shock (10 sec vortexing; see Materials and Methods) induced a stereotyped behavioral sequence of initial spasm, paralysis, delayed spasm, and recovery of normal posture in both male (top panels) and female (bottom panels) mutant flies. Seizing females often lay eggs (arrowheads). The initial spasm, which occurred during the first few seconds after vortexing, was not captured in videotaping. B, Duration of seizure induced by vortexing vials containing five or fewer flies. Onset time of delayed spasm (hatched bar) and time to recovery (open bar) are quantified on the basis of thenumbers of flies indicated. C, Seizure susceptibility in different genotypes as indicated by percentage of flies displaying the seizure repertoire after vortexing. In this and the following figures, error bars indicate SD. **p< 0.01; ***p < 0.001.

We found that electroconvulsive stimulation (Fig. 1B) (see Materials and Methods) delivered to the brain of a tethered bang-sensitive mutant fly reproducibly induced the repertoire of seizure behavior, which paralleled the bang-sensitive behavioral sequence in unrestrained mutant flies. As confirmed by video recording, the same sequence of initial spasm, paralysis, delayed spasm, and recovery was preserved in the tethered flies after electroconvulsive stimulation (Fig. 3A). Although mechanically induced seizure is a unique phenotype seen only in bang-sensitive mutants, we found that electroconvulsive seizure could be induced reproducibly in tethered wild-type flies and mutants of other categories at more extreme stimulus intensities (Lee and Wu, 1998, 2000). Thus, it is possible to analyze mutant effects on seizure susceptibility and expression with well defined protocols in tethered flies.

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Fig. 3.

Electroconvulsively induced seizure behavior and its physiological correlates in the DLM flight muscle of abss¹ fly. A, Electroconvulsive stimulation applied to a tethered fly induced a behavioral repertoire of initial spasm, paralysis, delayed spasm, and recovery to normal posture. B, The same electroconvulsive stimulation in A induced a physiological repertoire of initial discharge (ID), which occurred during initial spasm, response failure (F), occurring during paralysis, delayed discharge (DD), during delayed spasm, and response recovery (R), concurring with recovery of normal posture in a DLM. In this and the following figures, the filled triangle indicates onset of DD, and the open triangle indicates R (recovery of consecutive responses) in traces of physiological recording.

Physiological correlates of the seizure repertoire in indirect flight muscles

The physiological correlates of the behavioral repertoire could be systematically analyzed in the GF pathway in which the motor outputs of jump and flight muscles coordinate the escape reflex (Fig.1A) (see Materials and Methods). A physiological repertoire (Fig. 3B) could be detected in the DLMs that temporally correlated with the entire behavioral sequence after electroconvulsive stimulation. The corresponding sequence in the DLM repertoire was an initial discharge of spikes, response failure, delayed discharge, and response recovery. The failure and recovery of the GF pathway were monitored by recording the DLM response to 1 Hz test pulses (Figs. 1B, 3B). Recovery time or failure duration were defined as the interval between the electroconvulsive stimulus and the restoration of the response in DLM to five consecutive test pulses.

These characteristic behavioral and physiological patterns were observed in all tethered bang-sensitive mutants examined, includingbas, bss, eas, kdn,sda, and tko (Fig.4) (and data not shown). In this report, we focused on the classical bang-sensitive mutants, bas andbss, because they have been well characterized genetically (Ganetzky and Wu, 1982), physiologically (Jan and Jan, 1978; Engel and Wu, 1994; Pavlidis and Tanouye, 1995), and behaviorally (Burg, 1987). Interactions between the two mutations were revealed inbas¹ bss¹ double-mutant flies.

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Fig. 4.

The DLM physiological repertoire and induction threshold in wild-type and bang-sensitive mutants.A, Wild type (Wt),bss¹,bas¹ , andbas¹bss¹ all showed the same stereotyped sequence of initial discharge (ID), response failure (F), delayed discharge (DD), and recovery (R) in the DLM. Note response failures (indicated by stimulus artifacts of 1 Hz test pulses) before recovery (open triangles) of the DLM action potentials (see Results). B, Profiles of the physiological repertoires in the bang-sensitive mutants. ID, DD, and F for each genotype were characterized based on the number of flies indicated. Time periods of DLM response failure (F) and recovery of GF pathway transmission (R) are indicated. For this figure and Figure 6, statistics are pooled from different alleles for single mutants (bas:bas¹ ,bas² ; bss:bss¹ ,bss² ), and the double-mutant data are based on the bas¹bss¹ combination. C, Duration-voltage relationship of stimulus trains for electroconvulsive seizure induction. Increasing the 200 Hz pulse train duration lowered the adequate voltage for induction of the DLM repertoire. Data points represent threshold levels of 50% success. One fly contributed only one test result at each intensity. The ranges of sample sizes for data points of each genotype are indicated. At stimulus duration of ≤0.5 sec, seizure induction in wild-type flies was not attainable (C), and the voltage required for seizure induction correlated with the sensitivity to mechanical stimulation (compare with Fig. 2C).

Traces of muscle activities recorded intracellularly in the muscle DLMa (Fig. 1A) are shown in Figure 4Afor the different genotypes. (Tungsten electrode recordings in this and following figures produced apparently varying amplitudes of spikes. This was caused by electrode polarization, which is worsened during higher spike frequency and by larger stimulus artifacts.) Temporal parameters such as the onset time and duration of each event were used to characterize the genotype-specific profiles of ID, DD, and F in wild type, bas¹ ,bss¹ , andbas¹ bss¹ based on large samples of flies (Fig. 4B). The above parameters were highly reproducible among flies within each genotype as evidenced by the relatively small SDs in Figure 4B. As shown in Figure 4, A and B, after ID ended, there was a period of response failure to 1 Hz brain stimulation, which is shortest in wild type and longest inbas¹ bss¹ . DD occurred during response failure and lasted for tens of seconds, whereas ID subsided within a few seconds (Fig.4A,B). These observations indicate that DD generation does not rely on the functioning of the GF pathway and that DD and ID may involve separate neural circuits.

We further compared threshold levels for evoking a physiological repertoire for wild-type, bas, bss, andbas¹ bss¹ flies. Adequate 0.1 msec, 200 Hz stimuli were obtained by adjusting either voltage or duration of the stimulus train (Fig. 4C). Threshold was defined as the voltage-duration combinations at which 50% of examined flies displayed F and DD in the physiological repertoire. Thus the threshold curves indicate minimally effective stimulus conditions (Fig. 4C) and provide a basis for selecting stimulus voltages and train durations appropriate for each genotype in the experiments to follow. We found that the voltage level required for seizure induction with pulse trains of 0.5 sec or shorter (Fig. 4C) correlated well with sensitivity to mechanical agitation in free-moving flies of each genotype (Fig. 2C), i.e., mechanically induced seizure was most readily produced inbas bss but not attainable in wild-type flies. Presumably, electroconvulsion is more effective than mechanical shocks for seizure induction in all genotypes and can provide a more precise and convenient tool to quantify seizure susceptibility.

As compared with bss and bas¹bss¹ , the higher threshold forbas electroconvulsive seizure (Fig. 4C) was consistent with its lower percentage of paralysis induced mechanically in unrestrained flies (Fig. 2C). Furthermore, inbas¹ bss¹ flies, the enhanced susceptibility to electroconvulsion (Fig.4C) as well as the increased durations of the evoked repertoire (Fig. 4B) correlated well with its extreme sensitivity to mechanical stress and prolonged paralysis (Fig.2B). It should be noted that although the same sequence is seen in both electroconvulsively and mechanically induced seizure behavioral repertoires, the electroconvulsive response appeared to be less variable and occurred in a compressed time scale (approximately half) (compare Figs. 2B,4B).

General electroconvulsive activity in the nervous system during the DLM repertoire

The seizure behavioral repertoire described above involves different body parts, which may require concurrent activities in a large number of neural circuits. In addition to the physiological events detected in the DLM of the GF pathway, we examined possible correlations between the DLM repertoire with the general electrical activity and signals trafficking between the brain and thorax (Fig.5). Differential recording between electrodes positioned in one retina and the abdomen preferentially registered action potentials occurring near the electrodes or propagating between the head and thorax (Fig.5A,B). There were several distinct features in these recordings. First, a suppression of the general activity level was evident during the period of DLM response failure. Second, large spikes were detected during the DLM burst periods of ID and DD (simultaneous recording traces in Fig.5A,B). Third, some spike activities of smaller amplitudes (Fig. 5A,B,asterisk) usually preceded the larger spikes, but it is not known whether they were involved in the initiation of the DD motor pattern generation. Therefore, the DLM repertoire primarily reflects the general activity levels of the neuromuscular systems during electroconvulsive seizures. A closer inspection indicated, however, that there was no strict correspondence between the occurrence of these extracellularly recorded spikes with individual DLM spikes (expanded panels a and b for wild-type andc and d for bss¹ mutant flies). Furthermore, we recorded action potentials traveling between the head and thorax directly from the cervical connectives after brain stimulation. Although the identity and direction of traffic for individual spikes were not resolved, cervical connective recordings again showed activities temporally correlated with DLM activities (Fig.5C) without a strict one-to-one correspondence between spikes (expanded panel e). These observations demonstrate that general CNS activities with an overall temporal pattern underlie electroconvulsive seizure and that the effects of bang-sensitive mutations are pervasive throughout the CNS. Thus the DLM repertoire provides refined, quantitative indicators of mutational effects and a convenient window for probing CNS activity during seizure.

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Fig. 5.

General nerve activities evoked by electroconvulsive brain stimulation in wild-type andbss¹ flies. A,B, Correlation of DLM activities with differential recording between the electrodes positioned in the head and abdomen in wild-type (A) andbss¹ (B) flies. These paired recordings demonstrated activity suppression and bursting events in the CNS that corresponded to DLM response failure (F) and discharges (ID,DD). However, individual spikes in the DLM were not coincidental with those in head–abdomen recordings (segmentsa–d are expanded 20× for temporal comparison; a, b, for wild-type;c, d, forbss¹ flies). C, Signal trafficking between the head and thorax detected in the cervical connectives correlated with simultaneous DLM recording after an electroconvulsive stimulus in a wild-type fly (e, 20×).DLM, Muscle recording;h-a, differential recording between head and abdomen; cer, cervical connective recording.

Distinct stimulus–response relations for ID, F, and DD

The DLM seizure repertoire may be generated under the influence of multiple inputs from separable neural circuits, as suggested by the temporally ordered ID, F, and DD and by their differences in sensitivity to electroconvulsive stimuli and mutational modification. The DLM responses induced at various stimulus intensity levels (50–100 V, 2 sec duration) in a wild-type fly is shown in Figure6A. F and DD reached a plateau expression level above 70 V. In contrast, ID expression was suppressed beyond a suprathreshold level (100 V). The distinct nonmonotonic dependence of ID on stimulus intensity was true for all genotypes examined (Fig. 6) (and data not shown). Furthermore, distinctions in expression levels between ID and DD (or F) were paralleled by the probability of seizure induction at different stimulus strengths. By varying duration of the stimulus train (200 Hz) at a fixed voltage (50 V), we determined the induction probability as a function of stimulus strength and observed a general trend of increased percentage of flies displaying F and DD when the stimulus was lengthened (Fig. 6C,D). In contrast, prolonged stimulation appeared to exert an inhibitory effect on ID induction (Fig. 6B). Therefore, maximal plateau expression of F and DD provided robust and convenient indicators for mutant characterization. In contrast, the optimal ID expression required fine-tuning of stimulus intensity, which varied among flies. It is worth noting that a plateau expression of F and DD parallels the phenomenon in mammalian species that stimulus intensities beyond 1.2× the electroconvulsion threshold result in the same maximal seizure expression (Kupferberg, 2001).

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Fig. 6.

Electroconvulsion stimulus intensity-response relationship in wild-type and bang-sensitive mutant flies.A, Stimulus intensity-dependent expression of the DLM physiological repertoire in a wild-type fly. ID evoked by 200 Hz, 2 sec stimulus trains showed a nonlinear dependence on stimulus intensity, reaching a maximum ∼70 V but disappearing at 100 V. In contrast, F and DD were lengthened at increasing stimulus intensity, reaching a plateau above 70 V. B–D, Induction probabilities of ID, F, and DD at 50 V with varying stimulus train durations. Probabilities of induction were determined by the fraction of flies displaying ID, F, and DD. The range of the sample size is indicated, with each fly subjected to only a single trial at each stimulus duration. Maximal ID induction was shifted toward lower stimulus intensities in the mutant flies (B). Single-mutant bss and double-mutant bas¹bss¹ flies exhibited greatly enhanced sensitivity for F induction (C) but only mild alterations in DD induction probability (D). Note that induction curves for ID, F, and DD displayed different profiles within each genotype, suggesting involvement of distinct neural circuits.

Stereotyped action pattern of the physiological repertoire

The ability to induce the electroconvulsive seizure even in wild-type flies suggests a natural capacity of neural circuits inDrosophila to exhibit a fixed sequence of activity under extreme stimulus conditions. The seizure repertoire is reminiscent of the release of fixed-action patterns of certain reflexive behaviors (Reichert and Wine, 1982). Usually, such distinct behavioral repertoires proceed to the end once they are initiated, even if additional stimuli were introduced. This property was demonstrated in the Drosophila electroconvulsive repertoire by using a pair of stimuli to determine the extent to which the second stimulus modifies the repertoire in progress. It was observed that before the onset of DD (during GF response failure), the second brain stimulus exerted essentially no influence on the repertoire (Fig.7). The stimulus delivered after the onset of DD introduced a brief suppression of DD activities and delayed the recovery of the GF pathway response to 1 Hz test pulses. The extent of modification depended on genotypes, with the least influence in wild type and the greatest in the double mutant (Fig. 7). The lack of significant influence of the second stimulus was observed for a wide range of intensities, even for suprathreshold stimuli shown in Figure 7(note ID suppression by suprathreshold stimuli; compare Fig. 6).

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Fig. 7.

Stereotyped action patterns in the DLM physiological repertoire of wild-type,bas¹,bss¹ , andbas¹bss¹ flies. A DLM repertoire once initiated completed the sequence even when interrupted by a second stimulus. Two identical suprathreshold stimuli (200 Hz, 2 sec, except for bas¹bss¹ , which was 1 sec; note that ID was suppressed by the suprathreshold stimuli) were delivered at different ISIs. A second stimulus (open rectangle) delivered before the onset of DD produced no change in the DLM repertoire initiated by the first (filled rectangle), whereas deliveries after the onset of DD introduced a brief suppression of DD and a slight delay in response recovery (compare second and third traces with control in the top trace in each panel). Traces shown are sequential recordings from the same fly with at least 10 min rests between trials.

Refractoriness of the physiological repertoire

The mild effects of a second stimulus on the physiological repertoire initiated by the previous stimulus prompted an examination of the opposite situation, i.e., how a previous stimulus influences the effectiveness of the ensuing stimulus. The mechanically induced seizure behavior in bang-sensitive mutant flies exhibits a striking refractoriness (Ganetzky and Wu, 1982). For instance, in basand bss mutant alleles, spasms and paralysis could not be induced within a refractory period of tens of minutes after a previous seizure episode.

To test whether similar refractoriness also exists for the electroconvulsively induced physiological repertoire, paired stimuli interposed by intervals longer than those in Figure 7 (minutes vs tens of seconds) were delivered to the head (Fig.8) (note the suprathreshold suppression of ID in control trials; compare Fig. 6). The responses to the second stimuli were analyzed for indications of a refractory period during which F and DD were either modified (because of relative refractoriness) or missing (because of absolute refractoriness). As expected, parameters such as F duration and DD onset time were gradually restored to the characteristic values of each genotype at an increasing interstimulus interval (ISI). For each genotype, traces from the same fly are shown in Figure 8A, and pooled data from five flies are plotted for F duration and DD onset time in Figure8, B and C. The two stimuli could induce identical responses only if a prolonged ISI (e.g., 10 min) was allowed. During the relative refractory period, F duration was shortened, and DD onset occurred earlier with diminished discharge duration seen in Figure 8A. Conceptually, the records shown in Figure7 can be interpreted as the absolute refractory period because the second stimulus lacked any effect when it was delivered before the onset of DD (Fig. 7, middle traces of all genotypes).

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Fig. 8.

Refractoriness of the DLM repertoire in wild-type and bang-sensitive mutant flies. A, Refractoriness indicated by reduced effectiveness of the second electroconvulsive stimulus. F and DD in the responses to the second stimuli were either modified because of relative refractoriness or missing because of absolute refractoriness. In each paired-stimulation trial, the first stimulus (200 Hz, 2 sec; filled rectangle) was delivered to a fly rested for at least 10 min, and the second stimulus (open rectangle) followed at different ISIs. In the sequential pair-stimulus trials (at 1, 3, 5, or 10 min ISI, executed in a reversal order in the same fly), the top trace for each genotype (Wt, bas¹,bss¹ , andbas¹bss¹ ) represents control (ctrl) taken from one of the first responses in the pairs. Note the reappearance of ID in Wt andbas¹ traces during the relative refractory period, which was suppressed by the suprathreshold stimuli in control traces. B, C, Restoration of response during refractory periods as indicated by recovery of F duration (B) and DD onset time (C), determined in the number of flies indicated. At each ISI, recovery is normalized to control values with line segments connecting the average values for each genotype.

Several features about refractory period should be noted. First, it is known that minor disturbances such as rough handling of the culture vial without inducing any seizure-paralysis can still suppress the bang-sensitive behavior. Correspondingly, stimuli insufficient to produce a seizure response could still prevent the full expression of the physiological repertoire (data not shown) (cf. Lee, 2000). Second, within the relative refractory period, modified responses resembled those evoked by near-threshold stimuli (less than optimal expression), suggesting a temporarily increased threshold level. The typical near-threshold responses was evidenced in the 1′ and3′ traces for wild type and bas (Fig.8A), showing the reappearance of ID, earlier onset of DD, and shortened F and DD durations (compare Fig.6A). Third, the refractory period was clearly genotype dependent, with the longest in wild type and shortest inbss and bas bss. Furthermore, the time courses of regaining GF pathway function and DD pattern generation could be modified differentially by mutations. For example, a more gradual restoration of DD duration could be seen in wild-type,bas¹ , andbas¹ bss¹ flies in contrast to a rapid and abrupt restoration of full DD expression in bss¹ (Fig.8A).

Relations of ID and DD to other motor patterns in DLMs

In tethered flies, both sporadic irregular and sustained regular motor patterns were observed in DLMs under normal conditions (Fig.9A). Visual inspections indicated that flight activity of the tethered flies coincided with the regular spike activity (8–10 Hz) (Fig. 9A, trace F1), whereas twitching and cleaning motions often correlated with sporadic spike activities (trace S) in DLMs. These nonelectroconvulsion-associated activities have been encountered in all DLMa–f fibers. Significantly, sporadic spike activities were observed during the electroconvulsive repertoire (sometimes superimposing on the DD pattern), especially at near-threshold stimulus intensities (data not shown). In contrast, flight activities have never been seen during the electroconvulsive repertoire. Furthermore, after an electroconvulsive seizure, flight initiation (either spontaneous or air-puff induced) was suppressed during refractory period, and its reappearance coincided with the regained expression of DD. Therefore, a flight pattern generator could participate in driving the DD pattern, whereas the circuits responsible for sporadic activities appeared to function independently of DD generation.

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Fig. 9.

Effects of GABAergic blockade on spontaneous activity and electroconvulsive repertoire of DLMs. Wild-type flies were fed with 1 mm picrotoxin (ptx-fed) in food dye-colored medium. A, DLMs of control flies (fed with medium without drug) could express three distinct activities: sporadic spontaneous bursts (S) during the rest period, sustained flight activity (Fl), and the electroconvulsive repertoire (E).B, Picrotoxin-fed flies exhibited periodic discharges (S) in the DLM and failure of transmission in the GF pathway before experiencing an electroconvulsive shock (data not shown). The periodic bursts appeared to occlude electroconvulsive induction of ID and DD (compare traces E in A andB), suggesting that GABAergic blockade induces another seizure-like state. Suprathreshold stimuli (100 V, 200 Hz for 2 sec) were applied immediately before traces E in bothA and B. An arrow next to the open triangle indicates that no recovery from GF pathway failure (F) was observed throughout the recording.

In mammalian seizure studies, high-frequency electrical stimulus trains have been shown to evoke a stereotypic firing sequence in normal hippocampal slices, reminiscent of the electroconvulsive repertoire seen in Drosophila DLMs, whereas inhibition of GABAergic transmission induces sustained rhythmic bursts (Hablitz, 1984; Stasheff et al., 1985). We therefore examined the possibility that GABAergic blockade in Drosophila could lead to a new pattern of seizure activity. Indeed, a rhythmic bursting pattern distinct from flight and electroconvulsive discharges occurred spontaneously in wild-type flies fed with picrotoxin (2.0 mm), which blocks GABAergic transmission (Fig. 9B, trace S, pix-fed). Such conspicuous periodic bursting activity lasted for tens of minutes. In contrast, the sham control fly (fed with dye-colored food without drug) showed only spontaneous sporadic activity (Fig. 9A, trace S) or sustained flight patterns (trace Fl) in DLMs. Significantly, in picrotoxin-fed wild-type flies, the characteristic electroconvulsive repertoire was no longer attainable, presumably revealing a different seizure state in which GF failure persisted and induction of ID and DD were occluded (note sustained response failure and absence of ID and DD in Fig. 9B, trace E). In fact, GABAergic blockade caused transmission failure of the GF pathway before the application of electroconvulsion (data not shown). Importantly, picrotoxin feeding in wild-type flies also occluded all other motor patterns, including flight activities (Fig. 9B).

Localization of DD pattern generation in the thorax

Individual DLM motor neurons and central motor pattern generators for flight have been localized in the thoracic ganglia (King and Wyman, 1980; Ikeda and Koenig, 1988). We examined whether neural circuits in the thorax are sufficient to generate the electroconvulsive repertoires in the DLMs. Figure 10 shows the pattern of DLM response to direct thorax stimulation in intact or decapitated flies, supporting the thoracic localization of the circuits involved in DD pattern generation. This was achieved by applying electroconvulsive stimuli across two electrodes positioned bilaterally in the anterior portion of the thorax, which effectively initiated the DD. Furthermore, similar results were obtained from both decapitated wild-type and bss flies to remove the influence from the head (Fig. 10A,B). Electroconvulsive stimuli delivered to the thorax also evoked ID (data not shown). In Figure 10, ID was suppressed by the suprathreshold stimuli used to initiate full-blown DD and F.

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Fig. 10.

Localization of DD pattern generation to the thorax and F induction along the GF pathway. A,B, Intact and decapitated (decap) preparations were examined for the effect of direct thorax (thx) electroconvulsion (second andthird traces) bypassing the brain, as compared with the standard brain (br) electroconvulsion (top traces) in wild-type (A) andbss¹ mutant (B) flies. Thorax electroconvulsive stimulation (200 Hz, 2 sec, suprathreshold) could induce characteristic DD discharges of the DLM repertoire with or without the head, suggesting the thoracic localization of DD pattern generator(s). Test pulses (1 Hz) applied to the thorax after thorax electroconvulsion detected no occurrence of F in DLMs, indicating direct activation of motor neurons by test pulses.C, Both brain and thorax electroconvulsion induced failure in DLM response to test pulses delivered to the brain (first and third traces), but not to the thorax (second trace), suggesting multiple sites along the GF–PSI pathway, but excluding the motor neurons, as the sites of transmission failure. Open triangles withbackward-directed arrows indicate the lapse of manual switching time before the onset of test pulses.

Note that after electroconvulsive stimulation applied to either the thorax (Fig. 10A,B, bottom two traces) or the brain (Fig. 10C, middle trace), DLM responses to 1 Hz test pulses to the thorax persisted (i.e., no failure), presumably because of direct activation of DLM motor neurons. This is in contrast to the standard brain electroconvulsive protocol (Figs. 3-9) in which test pulses were applied to the brain and the DLM response would disappear during GF pathway transmission failure. Moreover, test pulses delivered to the brain after thoracic electroconvulsive stimulation (Fig.10C, bottom trace) failed to initiate a DLM response, indistinguishable from brain electroconvulsive stimulation (Fig. 10C, compare top and bottom traces). Therefore, either type of electroconvulsive stimuli induced DLM response failure to brain test pulses but no failure to thorax test pulses that could activate DLM motoneurons directly. Thus, severe local shocks to either the brain or the thorax portion of the GF pathway can induce temporary transmission failure.

Temporal correlation among different motor outputs of the GF pathway

Figure 11, inset, shows the placement of the identified nerve and muscle cells innervated by the right cervical GF neuron (for details, see also Fig.1A and Materials and Methods). For clarity, the symmetrical elements associated with the left GF neuron are omitted. Isomorphic representations shown next to individual traces indicate the left- or right-side (filled or open symbols) locations of the neuronal somata (GF,PSI, mn) and muscle fibers (DLMa–DLMc, TTM) in the GF pathway.

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Fig. 11.

Electroconvulsive repertoires in different motor outputs of the GF pathway in wild-type andbas¹bss¹ double-mutant flies. Theinset shows a schematic diagram of one half of the bilaterally symmetrical GF pathway (see Materials and Methods). Cells with somata on the left (l) side are filled, and those on the right (r) are open. Isomorphic representations of the GF pathway shown next to individual traces indicate the left- or right-side locations of the neuronal somata and muscle fibers involved. A, Discharges in DLM pairs driven by the same versus different motor neurons. A pair of DLM fibers (lDLMa and lDLMb) that share input from the same motor neuron showed synchronized spike firing (left panel). In contrast, firing in lDLMa and lDLMc, which are driven by separate motor neurons, lacked synchronicity despite their similar temporal characteristics of ID, DD, and R (right panel). This indicates that motor neuron activity rather than muscle excitability determines the electroconvulsive spike discharge patterns. B,C, Electroconvulsive responses of bilateral muscle pairs in wild-type (B) and bas bssdouble-mutant (C) flies. Both ID and DD were missing in TTM responses, indicating specificity of the ID and DD pattern generators for DLMs. TTM recovery was faster than that of DLMs, demonstrating a delay imposed by the mutant PSI (comparelDLMa–rTTM in C). Note also the temporal coupling between TTM recovery and DLM DD onset (lDLMa–rTTM pairs), coinciding with the resumption of general CNS activities (compare Fig. 5). Left and right pairs of DLM fibers (lDLM andrDLM), although receiving input from two bilateral GF–PSI chains, showed similar temporal sequence of ID, DD, and R in both wild-type (B) and mutant (C) flies (simultaneous recordings, each pair from a different fly).

We first examined the possibility that altered muscle excitability, rather than neuronal activity, is responsible for the characteristic DLM discharge pattern in each mutant. We took advantage of the fact that DLMa and DLMb are innervated by the same motor neuron (Fig. 11,inset) and compared their spike activities in simultaneous recordings. We found exact spike-to-spike correspondence between the firing patterns recorded in these two muscle fibers (Fig.11A, left panel), indicating that the electroconvulsive spike pattern is determined by input to the motor neuron. A contrasting result was obtained from simultaneous recording, on the same side, of DLMa and DLMc, which are innervated by separate motor neurons (Fig. 11, inset). In these two muscles, a spike-to-spike correspondence was no longer observed, despite a similar DD onset time (Fig. 11A, right panel), reflecting some degree of independence among motor neurons in expressing electroconvulsive activities. The above finding suggests that fine-tuning in DD patterning involves contributions from individual motor neurons and potentially inputs from different interneurons.

To reveal electroconvulsive discharge profiles specific to distinct functions, we examined the physiological repertoire exhibited in another GF pathway motor output, the jump muscle TTMs. Figure 11 shows traces of simultaneous recordings from TTM–DLM pairs in both wild-type (B) and bas¹bss¹ double-mutant (C) flies. The TTM motor neuron receives direct innervation via an electrical synapse from the GF on the same side (Fig. 11,inset). Bursting activity corresponding to either ID or DD was never observed in TTM after electroconvulsive stimuli applied to the brain (Fig. 11B,C) or thorax (data not shown). This is consistent with the idea that the ID and DD pattern generation involves circuits regulating only specific sets of motor neurons such as DLMns. However, the recovery of TTM from response failure concurred with the time of DD initiation in DLMs (Fig.11B,C, top pairs), which coincided with onset of general bursting activities in the CNS (Fig.5). Note the earlier response recovery for the TTM compared with that of DLMs, most evident in the double mutantsbas¹ bss¹ , although the two muscles share the same GF input. A possible explanation is that a partial recovery of GF is sufficient to drive the TTM motor neuron directly by electrical coupling but is not enough to induce DLM responses, which depend on successful activation of the interposed PSI chemical transmission (see also mosaic data below).

A comparison of bilateral DLM motor activities suggested that electroconvulsive discharges in muscles on the opposite sides involve recruitment of two separate motor pattern generators. Simultaneous recordings from right–left DLM–DLM pairs indicated that they were activated to display DD approximately, but not exactly, at the same time (Fig. 11B,C). Thus, bilaterally located pattern generators may be recruited in a coordinated manner to initiate DD activity in different muscles. However, as mentioned above (Fig. 11A), individual DLMns differ in the exact firing pattern even if their somata are located on the same side (see especiallybas¹ bss¹ double mutants in Fig. 11B, middle pair).

Genetic mosaic analysis of the physiological repertoire

Gynandromorph mosaics of Drosophila have been used to identify the anatomical loci in the nervous system associated with specific behavioral defects and dissect components in a behavioral repertoire (Ikeda and Kaplan, 1970; Hotta and Benzer, 1972, 1976; Burg, 1987). Gyandromorph mosaics consisting of heterozygous and hemizygous tissues (in our study, bas¹bss¹ /+ +//bas¹bss¹ /O) (Fig.12, inset) were generated by a loss of the unstable ring-X chromosome in a developing heterozygous female embryo. An earlier loss of the ring-X engenders a larger hemizygous mutant patch in the adult fly, identifiable by cuticular phenotypes of recessive markers plus male features of different body parts (see Materials and Methods). If the ring-X loss occurs during the first mitotic division, exact bilateral mosaicism can be found in some gynandromorph flies (Hotta and Benzer, 1972). Such bilateral mosaics are especially useful for distinguishing contributions between specific DLMs and their motor neurons to a particular mutant phenotype by taking advantage of the special organization of the GF pathway (compare Fig.12, inset). This has been elegantly demonstrated by Koenig and Ikeda (1983) to localize shibire mutant defects to the presynaptic motor neurons. Here we used a similar approach to distinguish the contributions of different circuit elements to the electroconvulsive seizure repertoire inbas¹ bss¹ double-mutant mosaics. In a perfectly bilateral mosaic, the genotypes of the GF, PSI, mn, and muscle cells can be assigned as indicated by the isomorphic circuits shown above individual traces in Figure12A. It can be seen that different groups of DLMs are innervated by different numbers of mutant neurons (open symbols) in the GF–PSI–DLMn chain of a bilateral mosaic.

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Fig. 12.

Mutational effects on the identified neurons and muscles of the GF pathway in bas¹bss¹ bilateral mosaic flies.A, The inset shows one GF pathway of the bilaterally symmetrical pair (compare Fig. 10, inset) with “control” nerve or muscle elements (filled) on the left(ipsi) side and “mutant” elements (open) on the right(contra) side. Also shown is a schematic bilateral mosaic with the phenotypically control (bas¹bss¹ /+ +) and mutant (bas¹bss¹ /O) tissue on theleft and right, respectively. The predicted genotypes of GF and PSI, and DLM, TTM, and their motor neurons (mn) are determined by whether the soma is located on the ipsilateral (i) control or contralateral (c) mutant side of the body, as shown in the relevant portion of the GF pathway for DLM and TTM inputs next to individual traces. Suprathreshold electroconvulsive stimuli (filled bars) were applied to compare modifications in the DLM physiological repertoire for DLMs on the mutant side [DLMa (trace 3,cDLMa: no mutant neuron in the pathway) vsDLMc (trace 4, cDLMc: one mutant neuron)] and on the control side [DLMc(trace 5, iDLMc: two mutant neurons) vsDLMa (trace 6, iDLMa: three mutant neurons)]. Note that the number of mutant neurons along the GF pathway correlates with DLM recovery time (open triangle). In contrast, bilateral TTMs showed similar recovery regardless of the number of mutant neurons (trace 1,iTTM: all normal cells; trace 2,cTTM: all mutant cells), suggesting bilateral coupling for the GF pathways. Note also the similar timing of DLM DD onset and TTM response recovery on both sides regardless of the number of mutant elements along the GF pathway (traces 1–6; compare Fig.10). B, C, Consistent results were obtained from four bas¹bss¹ bilateral mosaics. Separate symbols were used to distinguish data from different mosaic flies. F duration of DLM recovery lengthened with increased numbers of predicted mutant neurons in the GF pathway (B). In contrast, DD onset time was independent of the number of mutant neurons in the GF–PSI–mn chain (C).

Four bilateral bas¹bss¹ mosaics have been examined, demonstrating consistent results. Example traces obtained from one (Fig. 12A) illustrate how the repertoires of DLMa, DLMc, and TTM fibers ipsilateral (i) or contralateral (c) to the control side were influenced by mutant elements in the GF–PSI–DLMn or GF–TTMn chains (see corresponding isomorphic chains and inset in Fig. 12A).Traces 3–6 in Figure 12A demonstrate that the recovery time (open triangles) of the DLM responses to test stimuli applied to the brain was progressively lengthened as the number of presumptive mutant neurons in the GF pathway increased (from zero in trace 3 to three in trace 6). However, when the DLM motor neuron was mutant, the recovery time was delayed, whether the GF–PSI pair was wild type (traces 3and 4) or mutant (traces 5 and6). In contrast, DD onset time (filled triangles) was clearly less dependent on the number of mutant neurons in the GF pathway (traces 3–6), supporting the notion that the recruited circuits for DD generation in DLMs can operate independent of the GF pathway. Consistent with the results shown in Figure 11, there was no DD-like activity in TTMs (traces 1 and 2), and their recovery time coincided roughly with the onset time of DD in DLMs (traces 3–6). Moreover, TTM response recovery was not significantly influenced by the genotypes of the neuronal elements, even if both GF and TTM motor neurons were mutant (compare traces 1 and 2). Interestingly, the recovery time of the bilateral TTM pair in the double-mutant mosaic was intermediate between those seen in wild-type and bas bss double-mutant flies (compare Fig. 12,traces 1 and 2, with Fig.11B,C, top pairs), reflecting electrical coupling between the two GF neurons (Phelan et al., 1996; Sun and Wyman, 1996).

The above observations were true for all four bilateralbas¹ bss¹ gyandromorphs, as summarized for F duration (Fig.12B) and DD onset time (Fig. 12C) of TTM and DLMs with different numbers of mutant neurons in their GF pathway inputs. Note that in each bilateral mosaic, DD onset time was again intermediate between those seen in wild-type and bas bssdouble-mutant flies (compare Figs. 12C and4B). The close correspondence among the DD onset time of bilateral DLMs and the similar recovery time of the bilateral TTM pair again indicate an overall resumption of the CNS activity (Fig. 5), regardless of the locations on the mutant or control side. These results based on bas¹bss¹ bilateral mosaics were further supported by observations in 5 additional bilateraleas² bas¹ double-mutant gyandromorphs (data not shown) and 12bas¹ bss¹ mosaic flies with bilateral divisions in either head or thorax (Lee, 2000).