Genetic dissection of functional contributions of specific potassium channel subunits in habituation of an escape circuit in Drosophila

Abstract

Potassium channels have been implicated in central roles in activity-dependent neural plasticity. The giant fiber escape pathway of Drosophila has been established as a model for analyzing habituation and its modification by memory mutations in an identified circuit. Several genes in Drosophila encoding K+ channel subunits have been characterized, permitting examination of the contributions of specific channel subunits to simple conditioning in an identified circuit that is amenable to genetic analysis. Our results show that mutations altering each of four K+ channel subunits (Sh, slo, eag, and Hk) have distinct effects on habituation at least as strong as those of dunce and rutabaga, memory mutants with defective cAMP metabolism (). Habituation, spontaneous recovery, and dishabituation of the electrically stimulated long-latency giant fiber pathway response were shown in each mutant type. Mutations of Sh (voltage-gated) and slo (Ca2+-gated) subunits enhanced and slowed habituation, respectively. However, mutations of eag and Hk subunits, which confer K+-current modulation, had even more extreme phenotypes, again enhancing and slowing habituation, respectively. In double mutants, Sh mutations moderated the strong phenotypes of eag and Hk, suggesting that their modulatory functions are best expressed in the presence of intact Sh subunits. Nonactivity-dependent responses (refractory period and latency) at two stages of the circuit were altered only in some mutants and do not account for modifications of habituation. Furthermore, failures of the long-latency response during habituation, which normally occur in labile connections in the brain, could be induced in the thoracic circuit stage in Hk mutants. Our work indicates that different K+ channel subunits play distinct roles in activity-dependent neural plasticity and thus can be incorporated along with second messenger "memory" loci to enrich the genetic analysis of learning and memory.

Fig. 1.

Schematic of the giant fiber pathway, showing one half of the bilateral circuit. Visual and other afferent pathways (here represented as a single neuron, aff.) feed to theCGF, which descends to activate TTM(jump) and DLM (wing depressor) branches in the thorax. Low-voltage stimulation, passed between electrodes in the eyes, activates afferent pathways (aff.) to trigger a long-latency response that can habituate. Higher-voltage stimulation directly activates the CGF to evoke a nonhabituating short-latency response. Numbered sites (1, 2, 3) are referred to in the Results. DLMn 5 is shown, because recordings were usually from its target muscle fiber, DLM a. For anatomy, see King and Wyman (1980), Strausfeld and Bassemir (1983), and Ikeda and Koenig (1988); for functional connectivity, see references in Engel and Wu (1992, 1996) and Lin and Nash (1996). aff., Afferent pathway; CGF, cervical giant fiber; DLM, dorsal longitudinal muscle;DLMn, DLM motoneuron; PSI, peripherally synapsing interneuron; TTM, tergotrochanteral muscle;TTMn, TTM motoneuron.

Fig. 2.

Habituation of the long-latency response in control and mutant flies. A, B, Electrical stimuli were given at 5 Hz until five consecutive failures or 1000 stimuli. DLM muscle responses for different alleles of each genotype were combined (see Materials and Methods for alleles used) and smoothed (three-point running average) for the first 100 stimuli, with the final 100 stimuli averaged to a single point (901–1000).Line dashing and symbols set onlines are to aid in distinguishing curves. Sample sizes are indicated below [see Fig. 3 (with Sh alleles combined)]. ctrl, Control; H-S,Hk Sh; e-S, eag Sh;H-e-S, Hk eag Sh; alleles of each mutant type are combined (see also Figs. 4, 8, 9; Materials and Methods).

Fig. 3.

Number of stimuli (means ± SEM) to attain one to five consecutive failures in control and mutant flies (same trials shown in Fig. 2). A, B, Results for each genotype were log-transformed (see Materials and Methods) before computing statistics. Except for Sh andeag Sh, different alleles gave similar results and are combined (see also Figs. 6, 7, Table 1). Numbers of stimuli to first failure (i.e., consecutive failures = 1) and five failures were tested against controls using a two-tailed t test (*p < 0.05; **p < 0.01; ***p < 0.001). Sample sizes are indicated inparentheses. SH¹³³,Sh^KS133;Sh¹²⁰,Sh^rKO120; see Materials and Methods for other mutant alleles.

Fig. 4.

Recovery from habituation in control and mutant flies. A–C, After habituation to five-failure criterion (1st, filled symbols and solid lines), flies were allowed to recover for 30 sec and given a second stimulus bout (2nd, open symbolsand dashed lines). Two aspects of recovery to be noted are the response likelihood in the initial stimuli of the second bout and the rate of subsequent habituation. Hk mutants are not included in this paradigm because of the infrequency of five consecutive failures with 5 Hz stimulation. Results were combined and smoothed as described for Figure 2. Symbols set onlines are to aid in distinguishing curves. Trials are a subset of those shown in Figure 2. Sample sizes are control (ctrl) = 35; eag = 10;slo = 6; Sh = 25; andeag Sh = 14.

Fig. 5.

Dishabituation in control and mutant flies. Each example shows responses to the first 400 stimuli of a trial on a single fly, with dishabituating air puffs indicated (dots). Mutant alleles are specified. To induce habituation more readily, we stimulated the slo¹ andHk^IE18 mutants shown here at frequencies above the standard 5 Hz, and forslo¹, we also reduced the stimulus voltage below normal at the start of the trial and reduced this voltage further later. A segment of theslo¹ record has been deleted (100 skipped) to show the effect of the second voltage reduction. Note episodes of dissociation between DLM and TTM failures in this slo¹ mutant (see Results), indicated by short horizontal bars for single DLM failures or extended bars for groups of DLM failures separated by ≤ 15 stimuli. L-DLM, left DLM;R-TTM, right TTM.

Fig. 6.

Kinetic properties of habituation in control and mutant flies. A, Cumulative frequency of switching between responses and failures (i.e., response after failure or vice versa). Because individual trials were terminated with five consecutive failures, the number of flies still being tested diminished over time (inset shows the proportion remaining). Accordingly, the frequency of switches as shown is per fly per trial at each stimulusi [i.e., (number of switches occurring ati)/(number of flies still being tested ati)], and these weighted frequencies were accumulated for stimuli i = 1–1000. Note the different scaling of the x-axes for 0–100 versus 100–1000. Line dashing and symbols set on theline are to aid in distinguishing curves.B, Distribution of lengths of strings of consecutive failures in first 100 stimuli. String numbers were normalized for each genotype by dividing by the number of trials. Because trials were terminated after five consecutive failures, the five-failure category would also include any longer failure strings (5+). Thus, at most, one 5+ string could occur in a trial. Conversely, nf (no failures) indicates the proportion of trials without a single failure in the first 100 stimuli. The absolute frequencies of failures (up to trial termination if reached within the first 100 stimuli) are indicated (failures/trial). Sample sizes are indicated (same trials shown in Fig. 3).

Fig. 7.

Kinetic properties of habituation in control and mutant combination flies. A, Cumulative frequency of switching between responses and failures. B, Distribution of failure string lengths in the first 100 stimuli. For explanation, see Figure 6 legend; plots are scaled to match those in Figure 6.

Fig. 8.

Giant fiber response refractory periods in control and mutant flies (means ± SEM). Refractory periods were measured as described previously (Engel and Wu, 1992, 1996) and log-transformed for statistics (two-tailed t tests comparing mutants with control). A, Long-latency response. Refractory periods for the long-latency response are nearly always identical for DLM and TTM responses (Engel and Wu, 1996); differences seen here resulted from a few flies in which only DLM was recorded.B, Short-latency response. Refractory periods for the short-latency response are typically different for TTM and DLM pathways (cf. Engel and Wu, 1992, 1996). Sample sizes are indicated abovebars; *p < 0.05; **p < 0.01; ***p < 0.001.ctrl, Control.

Fig. 9.

Giant fiber response latencies in control and mutant flies (means ± SEM), measured as described previously (Engel and Wu, 1992, 1996) and compared using two-tailedt tests (mutants vs controls). A, Long-latency response. B, Short-latency response. Sample sizes are indicated above bars; *p< 0.05; **p < 0.01; ***p < 0.001. ctrl, Control.

Fig. 10.

Decoupling of DLM and TTM long-latency responses during habituation in Hk-combination mutants. Layout is as described in Figure 5; a segment of each record has been deleted (skipped) to show transitions between decoupling and coupling of responses. Episodes of dissociation between DLM and TTM failures are indicated by horizontal bars as in Figure5. Note that during the Hk¹Sh^rKO120 trial, stimulus frequency was increased as indicated; in theHk¹eag¹Sh^KS133 trial, stimulus voltage was reduced to 3.2 V during the deleted segment and further reduced to 3.0 V at the point indicated.