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The Journal of Neuroscience, March 15, 1998, 18(6):2254-2267
Genetic Dissection of Functional Contributions of Specific
Potassium Channel Subunits in Habituation of an Escape Circuit in
Drosophila
Jeff E.
Engel and
Chun-Fang
Wu
Department of Biological Sciences, University of Iowa, Iowa City,
Iowa 52242
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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 (Engel and Wu, 1996
). 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.
Key words:
habituation; learning and memory; giant fiber escape
response circuit; Drosophila; insects; invertebrates; K+; potassium channels; 
subunit; 
subunit; Shaker; Sh; slowpoke; slo; ether à go-go; eag; Hyperkinetic; Hk
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INTRODUCTION |
Experience-dependent neural
plasticity implies the modification of synaptic strength or neural
firing properties by activity. K+ channels are
likely to be crucial, both as regulators of neural activity and as
targets of modulation leading to lasting changes in synaptic efficacy
(Rudy, 1988; Alkon, 1990; Hille, 1992; Klein, 1995; Byrne and Kandel,
1996). However, roles of different channel subunits are difficult to
dissect using conventional physiological and pharmacological
approaches, in which effects are rarely limited to one channel type.
Therefore there is value to a genetic approach that allows identified
channels to be mutated in intact animals, linking behavioral
alterations to cellular or molecular defects.
Several genes identified by hyperexcitable phenotypes (i.e., leg
shaking) have been shown to encode K+ channel
subunits (Kaplan and Trout, 1969; Hall, 1982; Wu and Ganetzky, 1992) of
different families that contribute to a variety of
K+ channels in Drosophila and other
animals including vertebrates (Jan and Jan, 1990; Rehm and Tempel,
1991; Salkoff et al., 1992; Chouinard et al., 1995; Ganetzky et al.,
1995; Baro et al., 1997). Furthermore, mutations that modify ion
channels can affect behavioral plasticity, e.g., in courtship
(Sh, eag) (Cowan and Siegel, 1984; Griffith et
al., 1994) and classical olfactory conditioning (Sh) (Cowan
and Siegel, 1986). However, the functional contributions of these
subunits to channels in neural circuits are not well understood.
Three such genes encode (pore-forming) subunits of multimeric
K+ channels with different properties.
Shaker (Sh) channels are voltage-gated (Salkoff
and Wyman, 1981; Wu and Haugland, 1985; Iverson et al., 1988; Timpe et
al., 1988), whereas slowpoke (slo) channels are
activated by cytoplasmic Ca2+ (Komatsu et al., 1990;
Atkinson et al., 1991). Both could regulate neuronal excitability and
synaptic transmission (Jan et al., 1977; Tanouye et al., 1981; Ganetzky
and Wu, 1982; Elkins et al., 1986; Gho and Ganetzky, 1992). The
ether à go-go (eag) subunit seems to
coassemble into channels with other subunit types, and eag mutations affect several K+ currents, including
those mediated by Sh and slo channels (Zhong and
Wu, 1991b, 1993b; Chen et al., 1996). Sequence analysis and physiological results suggest that the eag subunit is a
target for channel modulation by phosphorylation and cyclic nucleotide binding (Warmke et al., 1991; Brüggemann et al., 1993; Zhong and
Wu, 1993b; Griffith et al., 1994). Hyperkinetic
(Hk) encodes a (auxiliary) subunit (Chouinard et al.,
1995) that associates with heterologously expressed Sh
channels to confer modulation (Rettig et al., 1994; Chouinard et al.,
1995; Rhodes et al., 1995), and Hk mutations alter the
amplitude and kinetics of Sh-type currents, especially in
near-threshold voltages, in Drosophila muscle (Wang and Wu,
1996) and cultured neurons (Yao and Wu, 1995).
Differences in the action and modulation of Sh,
eag, slo, and Hk subunits suggest that
they could play distinct functional roles in neural plasticity.
However, differential expression and alternative RNA splicing of these
genes in different excitable tissues (Schwarz et al., 1988, 1990;
Stocker et al., 1990; Tseng-Crank et al., 1991; Becker et al., 1995;
Mottes and Iverson, 1995), with the potential for heteromeric subunit
combinations (Haugland and Wu, 1990; Isacoff et al., 1990; McCormick et
al., 1990; Ruppersberg et al., 1990; Wu and Chen, 1995; Chen et al.,
1996), raise the question of how a diversity of K+
channel types regulates different circuit components. Such functional complexity could be dissected via the use of mutations to analyze plasticity in a well defined circuit.
The study of mechanisms of neural plasticity has been facilitated by
simple conditioning paradigms such as habituation (e.g., Thompson and
Spencer, 1966; Klein et al., 1980; Fitzgerald et al., 1990; Krasne and
Teshiba, 1995). However, only recently has the genetic analysis of
habituation been applied to an identified neural circuit in the fly in
which molecular and behavioral phenotypes can be linked to physiology
(Engel and Wu, 1996). The giant fiber escape pathway mediates a
visually evoked jump-and-flight escape response (Levine and Tracey,
1973; Tanouye and Wyman, 1980; Wyman et al., 1984). Different
intensities of electrical stimulation, passed between electrodes in the
eyes, can be used to activate afferent elements in the brain
(long-latency response) or to bypass them and trigger the thoracic
stage (short-latency response) (Fig. 1),
enabling the analysis of distinct circuit components with different
response properties (Elkins and Ganetzky, 1990; Trimarchi and
Schneiderman, 1993; Engel and Wu, 1996; Lin and Nash, 1996). We have
demonstrated that the long-latency response shows characteristic parameters of habituation (Thompson and Spencer, 1966) and have used
the memory mutations rut and dnc (Dudai, 1988;
Tully, 1991; Davis, 1996) to show that perturbing cAMP metabolism
affects habituation in this circuit (Engel and Wu, 1996), consistent
with their effects on behavioral plasticity (e.g., Corfas and Dudai,
1989). We report here that mutations of identified
K+ channel subunits induce specific and distinct
effects on habituation in the giant fiber circuit of the fly, to an
extent as extreme as rut and dnc.
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Figure 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 the
CGF, 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.
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MATERIALS AND METHODS |
Controls were Canton-Special (CS) flies (n = 41). Mutant stocks were as follows: for Shaker,
ShKS133 (n = 15),
y cho f ShrKO120 (n = 11), and Sh5 (n = 6) (Wu and Haugland, 1985); for slowpoke, slo st
(n = 17) (Elkins et al., 1986) and
slo98 (n = 2)
(Komatsu et al., 1990); for ether à go-go,
eag1 (n = 11) and
eag4pm (n = 5)
(Ganetzky and Wu, 1983); and for Hyperkinetic,
Hk1 (n = 5) and the
viable deletion HkIE18
(n = 6) (Wang and Wu, 1996). Combinations were
eag1
ShKS133 (n = 10) and
eag1
ShrKO120 (n = 9)
(Ganetzky and Wu, 1983) and Hk1 f
ShrKO120 (n = 5),
Hk1 f
Sh5 (n = 5), and
Hk1 g
eag1
ShKS133 (n = 5) (Yao and
Wu, 1995). Values of n refer to the primary data set
displayed in the Results (see Figs. 2, 3, 6, 7, Table 1); results for
mutant alleles of each gene did not differ and were pooled, except that
ShKS133 is contrasted with
ShrKO120 and
Sh5 (see Figs. 3,
6A, Table 1). Viable genetic markers of eye color [chocolate (cho), garnet
(g), and scarlet (st)],
body color [yellow (y)], and bristle
morphology [forked (f)] are
described by Lindsley and Zimm (1992). Ether-induced shaking behavior
was present in these stocks as reported previously, and in eag
Sh double mutants, the characteristic occurrence of flies with
wings held downward and an indented notum was also noted (Engel and Wu,
1992).
Physiological methods were described in Engel and Wu (1996). High- and
low-voltage pulses from electrodes in the eyes were used to trigger the
cervical giant fiber (CGF) or afferent inputs to the CGF in the brain
(Fig. 1). For the short-latency response, stimuli were ~2 V above
threshold; for the long-latency response, stimuli were set 0.2-0.4 V
below the threshold for shorter latency responses (Engel and Wu, 1996),
except that stimulus voltage was reduced further for some trials as
noted (see Figs. 5, 10). Responses were recorded from a
tergotrochanteral muscle (TTM) (leg extensor) and a fiber of the
contralateral dorsal longitudinal muscle (DLM) (wing depressor) (Fig.
1). DLM fiber a was most commonly recorded; previous work indicates
that ipsilateral DLM fibers respond and fail together in habituation of
the long-latency response (Engel and Wu, 1996).
For habituation trials, stimulation was given at 5 Hz, found previously
to induce habituation within tens to hundreds of stimuli in a variety
of genotypes (Engel and Wu, 1996), except for some trials as noted (see
Figs. 5, 10). Habituation was indicated by a criterion of five
consecutive failures of the DLM flight muscle response (Engel and Wu,
1996), and trials were terminated after five consecutive failures to
test recovery or after 1000 stimuli (200 sec) if criterion was not
obtained first. Air puffs (Engel and Wu, 1996) were given immediately
after five consecutive failures to test dishabituation. For statistical
analyses, data for stimuli-to-failure criterion (see Fig. 3) and
refractory periods (see Fig. 8) were log-transformed to improve
normality (Engel and Wu, 1996).
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RESULTS |
Habituation in single mutants
We demonstrated previously six of the parameters of habituation of
Thompson and Spencer (1966) for the long-latency giant fiber response,
including frequency dependence, spontaneous recovery, faster
rehabituation, habituation beyond zero response, dishabituation by air
puff or light flash, and habituation of dishabituation (Engel and Wu,
1996). A seventh parameter, stronger habituation with weaker
stimulation, is described below. To characterize habituation in several
mutants of four gene loci, this work focused on the most essential
parameter, loss of the response, at a standard stimulus strength and
frequency (see Materials and Methods). Recovery and dishabituation were
also examined to demonstrate reversibility. Long-latency giant fiber
responses to electrical stimulation and habituation of the response
were obtained in flies of every genotype.
Mutations affecting different channel subunits led to profound
differences in the rate of habituation (Fig.
2, Table 1)
and the time required to reach a criterion level of habituation (i.e., five consecutive failures; Fig. 3).
Different mutant alleles of each gene produced consistent results,
except that two alleles of Sh that modify channel function
differed from an allele that eliminates function (see below).
Therefore, alleles have been combined in figures and tables unless
indicated otherwise. Similarities between mutant alleles of a gene
locus indicate the phenotypes are attributable to those loci rather
than unidentified variability in other parts of the genome.
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Figure 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 on
lines 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).
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Figure 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 and
eag 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 in
parentheses. SH133,
ShKS133;
Sh120,
ShrKO120; see Materials and Methods
for other mutant alleles.
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Sh and slo encode subunits with homologous
structure, with the major distinction that Sh subunits are
voltage-gated whereas slo subunits require cytosolic
Ca2+ for activation (Wu and Ganetzky, 1992). Two
slo mutations, slo1 and
slo98, markedly reduced the rate of
habituation (Fig. 2, Table 1) and the time to reach five-failure
criterion (Fig. 3), so that many slo mutant flies did not
achieve five consecutive failures within the trial length limit of 1000 stimuli. slo1 and
slo98 may be amorphic (null),
because both alleles eliminate a Ca2+-activated
K+ current in muscle (Elkins et al., 1986; Komatsu
et al., 1990). In contrast, Sh mutants habituated more
rapidly than did controls (Fig. 2, Table 1), but there were interesting
differences between alleles. Sh5
and ShrKO120, which express
functional but altered subunits, led to earlier habituation (Fig. 3,
Table 1). The Sh5 mutation, in an
exon common to all Sh transcripts (Gautam and Tanouye, 1990;
Lichtinghagen et al., 1990), alters channel-gating kinetics and voltage
sensitivity (Salkoff and Wyman, 1981; Wu and Haugland, 1985); the
ShrKO120 lesion site is not known
but may affect specific splicing variants because its phenotype is far
more extreme in neurons than in muscle (Wu and Haugland, 1985; Wu and
Ganetzky, 1992). ShKS133, a
missense mutation that affects the pore-forming region of all
transcripts (Lichtinghagen et al., 1990), is an antimorph (Haugland and
Wu, 1990) that eliminates a transient K+ current,
IA, in muscle (Salkoff and Wyman, 1981; Wu
and Haugland, 1985). Surprisingly, its effects on habituation were less
extreme than were those of Sh5 and
ShrKO120 (Fig. 3, Table 1).
Mutations of the two subunits implicated in channel modulation,
eag and Hk, had even stronger effects on
habituation than did Sh and slo, respectively.
eag1 and
eag4pm increased the rate of
habituation (Fig. 2, Table 1) and reduced the number of stimuli to
attain five consecutive failures (Fig. 3).
Hk1 and the amorphic deletion
HkIE18 both dramatically reduced
the rate of habituation (Fig. 2, Table 1) and delayed attainment of
five consecutive failures (Fig. 3), so that the majority of
Hk mutants did not attain this criterion within 1000 stimuli. It should be noted that habituation was assessed according to
DLM muscle responses (Engel and Wu, 1996), but in many Hk
flies, the TTM muscle continued to respond after the DLM pathway failed
(an otherwise rare phenomenon, described below). In this respect, the
phenotype of Hk mutants could be even more extreme than is
indicated by these plots (Figs. 2, 3).
Dishabituation, spontaneous recovery, and failure patterns
It is important to consider whether the conditioning induced in
these mutants is habituation as defined previously in this system
(Engel and Wu, 1996). As defined previously, habituation can be
localized to a portion of the circuit in the head (Fig. 1) because DLM
and TTM muscle responses failed in synchrony (an exception is described
below), and the pathway in the thorax can respond at frequencies well
above the 5 Hz used to induce habituation (Engel and Wu, 1992; see Fig.
8B for comparison). Response latencies were only
slightly altered in mutants (see below), and there were no abrupt
changes in latency during habituation that could have indicated a novel
activity-dependent circuit response.
Moreover, two critical characteristics of habituation, spontaneous
recovery and dishabituation induced by a novel stimulus (air puff),
were observed in flies of all genotypes. Figure
4 shows spontaneous recovery after 30 sec
of rest after habituation by 5 Hz stimuli. eag,
Sh, and slo mutants recovered at least as fully
as did controls. Hk flies are not represented in Figure 4
because they did not readily reach the five-failure habituation criterion at 5 Hz, but Hk animals did recover from
habituation when tested with increased stimulus frequencies in order to
attain habituation (see below).
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Figure 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 symbols
and 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 on
lines 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; and
eag Sh = 14.
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Figure 5 shows examples of dishabituation
in each of the mutant types. To demonstrate dishabituation in slowly
habituating Hk and slo mutants, we could use a
higher stimulation frequency or a lower stimulus voltage to induce
habituation (Fig. 5, see Fig. 10). The latter approach takes advantage
of a negative relationship between stimulus intensity and the strength
of habituation (Thompson and Spencer, 1966), a parameter of habituation
that was not reported previously in this system (Engel and Wu,
1996).
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Figure 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 and
HkIE18 mutants shown here at
frequencies above the standard 5 Hz, and for
slo1, we also reduced the stimulus
voltage below normal at the start of the trial and reduced this voltage
further later. A segment of the
slo1 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.
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The mutant genotypes may be distinguished further by examining
the clustering of response failures. Again, eag and
Hk produced the most pronounced deviations from controls.
Figure 6A plots the
cumulative frequency of switches between responses and failures. The
slope of a curve indicates both the frequency of failures and the degree of clustering (longer strings of successes or failures lead to fewer switches). The curve for eag was similar to
that for ShrKO120 and
Sh5 during the first 100 stimuli
but was steeper than that for
ShKS133, reflecting a greater
number of failure strings. After approximately stimulus 90, the
eag curve flattened abruptly because of the termination of
faster-habituating trials at five-failure criterion (Fig.
6A, inset).
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Figure 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 stimulus
i [i.e., (number of switches occurring at
i)/(number of flies still being tested at
i)], 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 the line 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).
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The pattern of failures also differed between slo and
Hk mutants (Fig. 6A), even though both
showed diminished habituation (Figs. 2, 3). Failure onset was delayed
in slo mutants; the earliest failure (in 19 trials) occurred
at the 16th stimulus (Fig. 6A). After failure onset,
response-and-failure switching was as frequent as in controls until
approximately stimulus 175, when the rate diminished indicating that
the remaining subset of slo animals (i.e., trials not yet
terminated; Fig. 6A, inset) switched less frequently. In contrast, failure onset was much later in Hk
trials, no earlier than stimulus 62 (in 11 trials), and the frequency of switching (slope in Fig. 6A) never
attained the level seen in controls.
Figure 6B shows the frequency of strings of
consecutive failures in the first 100 stimuli, grouped by string
length. The first bar in each histogram in Figure
6B [no failures (nf)] indicates the proportion of trials without a single failure in 100 stimuli. The
last bar (5+) indicates the proportion of trials in which a
string of five or more failures occurred (equal to or more than five
because trials were terminated after five consecutive failures). The
most notable result in Figure 6B is that the rankings
for those two categories are consistent with the rates of habituation indicated in Figures 2 and 3; for nf, Hk > slo > controls > Sh > eag, and for 5+, Hk < slo < controls Sh < eag. Distribution patterns of one to four failure strings were less markedly different between genotypes. Slower habituation in slo is attributable
to shorter failure strings, because the average number of failures in
100 stimuli was similar to that in controls and Sh (Fig.
6B, failures/trial). In contrast,
Hk failed much less than did slo because of late
failure onset (compare Fig. 6A). eag
mutants showed the greatest failure frequency, even though the true
frequency is underestimated because most eag animals
habituated in under 100 stimuli (Fig. 6A,
inset).
Habituation in double mutants
Of the channel subunit genes studied, eag mutants
showed the most extreme increase in strength of habituation, whereas
Hk mutants showed the most extreme decrease (Figs. 2, 3, 6,
Table 1). There is strong physiological evidence (reviewed in the
introductory remarks) that both eag and Hk
subunits can interact with Sh channels. Therefore, double-
and triple-mutant combinations were examined to determine how these
interactions contribute to habituation. eag and
Sh mutations generally interact to produce phenotypes more
extreme than that produced by either mutation alone for characters including motoneuron excitability, terminal branching and synaptic transmission at neuromuscular junctions, leg shaking, novel wing position, and defective flight (Ganetzky and Wu, 1983; Budnik et al.,
1990; Engel and Wu, 1992). This is consistent with the finding that in
muscle, eag contributes to several K+
currents besides the Sh IA
(Zhong and Wu, 1993b; Wu and Chen, 1995). Therefore, it was surprising
that rates of habituation in eag Sh double mutants were no
more extreme than the rates in either Sh or eag
alone (Figs. 2, 3, Table 1). Failures began early in eag Sh
double mutants (Fig. 7A), as
in eag alone, but isolated failures (string length = 1)
were common (Fig. 7B). Spontaneous recovery from habituation
in eag Sh double mutants is shown in Figure 4.
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Figure 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.
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In contrast to eag and Sh, Hk and
Sh mutations generally do not interact to produce more
extreme phenotypes than produced by Sh alone for characters
including neuromuscular synaptic facilitation and aberrant neuronal
spiking (Stern and Ganetzky, 1989; Yao and Wu, 1995; W.-D. Yao and
C.-F. Wu, unpublished observations), and in both muscle and neurons,
Hk has only been shown to affect the Sh
IA current (Yao and Wu, 1995; Wang and Wu,
1996). Yet, habituation in Hk Sh double mutants was markedly
retarded, as in Hk mutants, rather than more rapid as in
Sh (Figs. 2, 3, Table 1). The strong effect of Hk
was apparent even in Hk1
eag1
ShKS133 triple mutants, which
habituated more slowly than did controls, unlike eag,
Sh, or eag Sh (Figs. 2, 3). In Hk Sh
and Hk eag Sh mutants, failure onset was delayed compared
with Sh or eag Sh (Fig. 7A), yet rates
of response-and-failure switching (Fig. 7A) were more normal
than in Hk alone.
Double- and triple-mutant results indicate that the modification of
Sh subunits alone has less impact on habituation than does
altering Hk or eag subunits in the presence of
functional Sh subunits. Unlike the previously studied
excitability phenotypes listed above, habituation is an
activity-dependent process. Apparently, Hk and
eag mutations affect conditioning processes more severely than does Sh, even though Sh affects
nonactivity-dependent parameters of the giant fiber response, including
the latency and refractory period as described below.
Resting properties: refractory period and latency
Refractory period and response latency are temporal
characteristics on a millisecond scale that might influence the process of habituation, a type of activity-dependent conditioning lasting seconds or longer. These processes could be mediated
by overlapping mechanisms at overlapping sites in the circuit. In fact,
we found that alterations in refractory period and response latency in K+ channel mutants do not account for their effects
on habituation. To analyze response properties at two stages of the
giant fiber pathway, low-voltage stimuli were used to trigger the
long-latency response that begins in labile-afferent pathways in the
brain where habituation is mediated (Engel and Wu, 1996), whereas
stronger stimuli were given to bypass the afferent stage (see Materials and Methods) and trigger a short-latency response in the thoracic portion of the circuit (Fig. 1).
Refractory periods of the long-latency response
tended to be shorter than normal in all K+ channel
mutants examined except in eag Sh double mutants (Fig. 8A), which were normal.
This indicates that habituation rate is not coupled to refractory
period in this system, because the two parameters are not altered in
the same pattern across genotypes. A similar lack of correlation was
shown with cAMP-metabolic mutations of dunce and
rutabaga (Engel and Wu, 1996). Short-latency response refractory periods, mediated by the nonlabile thoracic pathway, were
affected quite differently in the same mutants (Fig.
8B); they were not shortened in any
K+ channel mutants but were prolonged in eag
Sh (cf. Engel and Wu, 1992), Hk, and Hk Sh
mutants.
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Figure 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 above
bars; *p < 0.05;
**p < 0.01; ***p < 0.001. ctrl, Control.
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Response latencies were also affected differently in cephalic and
thoracic stages of the pathway (Fig. 9).
The long-latency response was delayed slightly in Sh and
eag Sh mutants. Sh, eag Sh, and
slo mutations induced small but statistically significant delays in short-latency TTM responses, whereas Hk and
eag mutations led to earlier short-latency DLM responses.
The small scale of these differences indicates that the giant fiber
pathway is still functional (cf. Baird et al., 1990) and weighs against
the recruitment of potential collateral pathways in slo and
Hk mutants (see below). Although latency increased gradually
during habituation (Engel and Wu, 1996), sudden increments were not
observed. The most notable feature of these results, however, is that
the pattern of nonactivity-dependent properties of nerve conduction and
synaptic transmission in the circuit, including marked effects on the
long-latency response refractory period, do not correlate with
habituation phenotypes. The lack of a simple relationship among
habituation, response latency, and refractory period in
K+ channel mutants implies that activity-dependent
plasticity could involve multiple cellular mechanisms at different
sites in the circuit, influenced by K+ currents,
that shape efficacy in axonal conduction and synaptic transmission.
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Figure 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-tailed
t 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.
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Shift of long-latency response failure site in
K+ channel mutants
Habituation of the long-latency response is characterized by
synchronous failures of DLM and TTM responses (Fig. 5) (Engel and Wu,
1996), indicating that failures occur in the brain (Fig. 1). However,
in some animals of certain genotypes, the DLM branch of the circuit
failed more readily than did the TTM branch. This pattern was seen most
often in flies with Hk mutations (Engel, 1995) or
Hk combinations (Fig. 10),
less frequently in slo (Fig. 5) and eag Sh (data
not shown) mutants, and rarely in other mutants or controls (even in
individual flies that habituated unusually slowly, approaching the
level of Hk or slo mutants). These independent DLM failures probably occur after the circuit has bifurcated in the
thorax (Fig. 1), and consistent with this, a similar pattern of DLM
failures coupled with TTM responses could be observed when short-latency responses were induced by increasing stimulus intensity (Engel, 1995).
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Figure 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 Figure
5. Note that during the Hk1
ShrKO120 trial, stimulus frequency was
increased as indicated; in the Hk1
eag1
ShKS133 trial, stimulus voltage was
reduced to 3.2 V during the deleted segment and further reduced to 3.0 V at the point indicated.
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This pattern could indicate a shift in the site of failures from
normally labile synapses in the head (e.g., site 1 in Fig. 1) to electrical or cholinergic synapses of the DLM branch in the
thorax (sites 2 or 3). One plausible explanation
could be the recruitment of normally silent afferents in the brain
leading to stronger transmission of the signal to the giant fiber
(i.e., at site 1). Decreasing stimulus intensity or
interstimulus intervals typically promotes habituation (Thompson and
Spencer, 1966), and to extend the above scenario, extra afferent inputs
activated in mutants could fail under these stimulus conditions.
Indeed, after the two muscle responses had become decoupled, it was
sometimes possible to induce the "typical" pattern of synchronous
DLM and TTM failures either by decreasing stimulus intensity to just
above the long-latency response threshold
(slo1 in Fig. 5,
Hk1
eag1
ShKS133 in Fig. 10) or by increasing the
rate of stimulation (Hk1
rKO120 in Fig. 10).
Conversely, drastically reduced TTM failures in Hk, Hk
Sh, and Hk eag Sh mutants could indicate recruitment of
parallel inputs to the thoracic TTM branch. Although the exact site of
modification in the pathway awaits further investigation, the
long-latency TTM response is clearly strengthened by Hk
mutations even more than is the long-latency DLM response. The typical
pattern of synchronized muscle responses could still occur in these
mutants (Figs. 5, 10), which indicates that the normal site of
habituation in the brain is labile in these flies as well. The apparent
shift in the site of failures under certain stimulus conditions in
Hk mutants reveals that there is considerable plasticity for
circuit function that could be regulated by the Hk channel
subunit in normal flies.
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DISCUSSION |
This is the first use of Drosophila mutations to
examine the influence of identified subunits of K+
channels on habituation. The giant fiber pathway system provides a
means to link molecular components to cellular physiology within a
defined circuit that shows plasticity (Engel and Wu, 1996). Our results
establish that different K+ channels have distinct
functional consequences for habituation in the giant fiber response,
with effects as extreme as those of the "learning mutations"
rutabaga and dunce (Engel and Wu, 1996).
Involvement of four potassium channel subunits in habituation
Potassium currents regulate signal transmission throughout the
nervous system, affecting presynaptic neurotransmitter release, postsynaptic integration, and the shape and frequency coding of action
potentials (Rudy, 1988; Hille, 1992; Turrigiano et al., 1994). In each
of these functions, K+ channels have been implicated
in neural plasticity (Alkon, 1990; Klein, 1995; Byrne and Kandel, 1996;
Debanne et al., 1997; Hoffman et al., 1997). K+
channels could have a dual role, as regulators of activity-dependent modulation of neural processes leading to plasticity and as targets of
modulation. Besides a range of mechanisms at the level of single neurons, habituation may involve various forms of plasticity at the
circuit level: conduction of excitatory paths may be depressed, or
previously latent inhibitory inputs may be facilitated and recruited
(cf. Charpier et al., 1995; Krasne and Teshiba, 1995), and there is
often a component of sensitization that could itself involve
K+ currents (Groves and Thompson, 1970). Therefore,
it is essential to study habituation in a defined circuit in the intact
organism, to complement work done with model synapses in reduced
nervous systems. In this manner, analysis of mutants of identified
K+ channel subunits has produced results that would
have been difficult to predict based on other experimental
approaches.
The distinct effects of mutations of four K+ channel
subunits indicate differences in their physiological characteristics
and localization within neurons of the pathway. Habituation was
enhanced in Sh and eag mutants and reduced in
slo and Hk mutants. Although Sh and
slo subunits are essential for kinetically similar
K+ currents in muscle (Wei and Salkoff, 1986; Singh
and Wu, 1990; Wu and Ganetzky, 1992), their mutations had opposite
effects on habituation, perhaps reflecting differences in their
activation mechanisms. Sh-type voltage-activated channels
are most likely to regulate the shape and frequency coding of sodium
action potentials in axons and dendrites (Tanouye et al., 1981; Debanne
et al., 1997; Hoffman et al., 1997). In contrast, slo-type
Ca2+-activated K+ channels, which
can colocalize with presynaptic Ca2+ channels, would
be well suited for regulating the activity-dependent accumulation of
presynaptic Ca2+ (Augustine et al., 1988; Gho and
Ganetzky, 1992; Robitaille and Charlton, 1992; Mallart, 1993;
Warbington et al., 1996). Although Sh and slo
subunits are workhorses in the nervous system, each essential for a
specific repolarizing current in muscle and neurons, the most extreme
positive and negative changes in habituation rates were induced by
mutations of eag and Hk subunits that alter K+ current modulation rather than eliminate currents
(Zhong and Wu, 1991b, 1993b; Yao and Wu, 1995; Wang and Wu, 1996).
Because habituation represents a change over time, it is reasonable
that the most important subunits should be those that confer lasting modulation. The opposing direction of eag and Hk
mutant phenotypes indicates that plasticity may be regulated by
counterbalancing mechanisms (Groves and Thompson, 1970). No channel
mutations eliminated habituation, which is likely to be shaped by the
interaction of multiple channel types, not by a particular
"habituation channel." Similarly, mutations that eliminate enzymes
for cAMP metabolism alter habituation but do not prevent it (Engel and
Wu, 1996).
Habituation was enhanced by Sh alleles
(ShrKO120 and
Sh5) that alter current density,
voltage dependence, or kinetics but was not significantly affected by
the antimorphic ShKS133 that (in
muscle) eliminates Sh current (Wu and Haugland, 1985). This
unexpected result implies that Sh subunits influence
habituation but not as a direct result of their modulation because
habituation seemed closer to normal in the absence of functional
Sh channels than in the presence of the defective but
functional subunits in ShrKO120 and
Sh5 mutants.
The eag channel subunit seems to coassemble into at least
four K+ channel types in fly tissues (Zhong and Wu,
1991b, 1993b) and can interact with Sh subunits to form
channels when expressed in Xenopus oocytes (Chen et al.,
1996) and the two eag alleles studied alter the modulation
of several K+ currents in muscle (Zhong and Wu,
1993b; Wu and Chen, 1995). Along with evidence of its modification by
kinases and cyclic nucleotides (Warmke et al., 1991; Brüggemann
et al., 1993; Zhong and Wu, 1993b; Griffith et al., 1994), this
indicates that eag would be an ideal link for the regulation
of a broad group of K+ channels. If habituation
involves the modulation of channels via eag subunits, then a
change in the efficacy of modulation could account for accelerated
habituation in eag mutants.
The Hk subunit is known to associate with and modulate
Sh channels (Rettig et al., 1994; Chouinard et al., 1995;
Rhodes et al., 1995; Yao and Wu, 1995; Wang and Wu, 1996). The
importance of this auxiliary subunit in habituation is indicated by the
fact that Hk mutations produced the most extreme phenotype,
sometimes modifying the site of failures as well as slowing the rate of habituation. Habituation may serve to regulate sensitivity to external
or internal stimuli (Fischer and Carew, 1993; Bässler and Nothof,
1994), and it is notable that the visually induced jump response
(mediated by the giant fiber pathway) is hypersensitive in
Hk mutants (Kaplan and Trout, 1969; Levine, 1974). This
hypersensitivity is less extreme when Hk is combined with
Sh (Kaplan and Trout, 1974), consistent with our results in
multiple mutants (Figs. 2, 3). Likewise, abnormal spontaneous rhythmic
firing seen in cultured Hk neurons becomes less extreme in
Hk Sh double mutant neurons (Yao and Wu, 1995).
Mutations of Sh affected habituation less strongly than did
either eag or Hk. If eag and
Hk subunits modify Sh currents, as has been
proposed, then it seems that plasticity is disrupted by the
maladjustment of modulatory mechanisms (in eag or
Hk mutants) far more than by the alteration or elimination
of their target (Sh) channels. Hk subunits have
so far only been shown to associate with Sh channels; yet
even though ShKS133 eliminates
functional Sh channels (Chouinard et al., 1995; Wang and Wu,
1996), Hk1
eag1
ShKS133 differed markedly from
eag1
ShKS133, and Hk Sh differed
from Sh (Figs. 2, 3, 7, Table 1). The Hk subunit
may therefore prove to modulate other K+ channels
besides Sh, potentially including products of the genes sei, shal, shab, and shaw
(Butler et al., 1989; Tsunoda and Salkoff, 1995; Titus et al., 1996;
Wang et al., 1996) that were not examined here.
Characteristics of habituation for a given behavior can depend
critically on the interstimulus interval (ISI) (e.g., Groves and
Thompson, 1970; Davis, 1984; Boulis and Sahley, 1988), and it is
possible that K+ channels play different roles in
habituation at different ISIs. A broad range of ISIs have been used in
the study of behavioral habituation in diverse phyla: less than a
second (May and Hoy, 1991), seconds (Thompson and Spencer, 1966; Davis,
1984; Wittekind and Spatz, 1988; Corfas and Dudai, 1989), tens of
seconds to minutes (Pinsker et al., 1970; Long et al., 1989; Rankin and
Broster, 1992; Krasne and Teshiba, 1995; Weil and Weeks, 1996), or
hours (Brown et al., 1996). The visually induced jump response in
Drosophila habituates at ISIs of 1-10 sec (Engel and Wu,
1996). In the present experiments, an ISI of 0.2 sec (5 Hz) was used;
the kinetics of habituation are not expected to be identical because
visual stimulation may recruit only a subset of the cervical giant
fiber afferents that are activated by the electrical stimulation used
here (Fig. 1).
Potassium channels and the mutational analysis of learning
and memory
The classical approach to the genetic analysis of learning and
memory sought mutations or transformants without obvious confounding behavioral or developmental defects. This led to genes involved with
various second messenger pathways, including rutabaga,
dunce, and amnesiac (cAMP cascade),
ala (CaM-kinase), and KCI and turnip (protein
kinase C) (Dudai, 1988; Choi et al., 1991; Tully, 1991; Griffith et
al., 1993; Feany and Quinn, 1995; Davis, 1996; Kane et al., 1997), but
tended to exclude ion channel mutations because these caused easily
detectable behavioral defects such as ether-induced leg shaking (Dudai,
1988). It seems unlikely that the changes in habituation performance
shown here in channel mutants are a spurious consequence of pleiotropic
defects elsewhere in the circuit, because the response attenuation in
mutants was localized to the afferent stage of the circuit where
habituation ordinarily occurs (see Results) and showed recovery and
habituation that appeared grossly normal.
In fact, concerns about pleiotropy also apply to classical learning
mutations such as dnc (Dudai, 1988; Qui and Davis, 1993). The distinction between "pure" learning mutations and mutations with secondary physiological, developmental, or behavioral effects is
now blurred. It has become clear that mutations of ion channels and
second messenger systems have overlapping effects (Dudai, 1988; Davis,
1996; Wu, 1996). Mutations affecting K+ channel
subunits alter activity-dependent conditioning of neural excitability
and firing (Ikeda and Kaplan, 1970; Tanouye et al., 1981; Ganetzky and
Wu, 1982; Saito and Wu, 1991, 1992) and synaptic efficacy (Jan et al.,
1977; Ganetzky and Wu, 1983; Mallart, 1993; Delgado et al., 1994;
Warbington et al., 1996). However, second messengers modulate ion
channels, and mutations of second messenger pathways cause
physiological effects resembling channel mutations. rut and
dnc mutations that alter cAMP metabolism have been shown to
alter muscle K+ currents (Zhong and Wu, 1993a),
excitability in cultured neurons (Zhao and Wu, 1997), and facilitation
at neuromuscular junctions (Zhong and Wu, 1991a). Branching of motor
neuron terminals is increased by eag, Sh, and
Hk mutations that increase neural activity (Budnik et al.,
1990), but cAMP metabolic mutations have identical effects. Indeed, the
most extreme terminal branching occurs in dnc eag and
dnc Sh double mutants and is suppressed by rut in triple mutants (Zhong et al., 1992). On the other hand, channel mutations can alter behavioral plasticity. Mutations of Sh
and eag reduce conditioning in courtship (Cowan and Siegel,
1984; Griffith et al., 1994) and alter habituation of a jump response to olfactory stimulation (McKenna et al., 1989; Tully and Koss, 1992)
(C.-F. Wu and T. Tully, unpublished observations), and Sh mutations alter classical olfactory conditioning (Cowan and Siegel, 1986).
We have shown here that mutations of four different
K+ channel subunits alter habituation of the giant
fiber response in gene-specific ways, with modulatory eag
and Hk subunits appearing particularly influential. In the
future, expression of normal or mutated genes may be concentrated in
particular regions of the nervous system using gynandromorph mosaics or
enhancer trap lines or temporally controlled with conditional promoters
(Burg et al., 1993; Ferveur et al., 1995; Zhao et al., 1995; Han et
al., 1996; Cambridge et al., 1997). The effects of such novel spatial
or temporal patterns of mutant gene expression can be contrasted with
the phenotypes of mutations expression patterns closer to normal, such
as those used in this study, to give indications of the localization of habituation and the colocalization and potential interaction of different molecular players. More broadly, these results open the
possibility to compare the effects of channel mutations and second
messenger mutations at cellular, circuit, and behavioral levels within
a single system.
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FOOTNOTES |
Received June 30, 1997; revised Jan. 5, 1998; accepted Jan. 6, 1998.
This work was supported by National Institutes of Health Grants NS18500
and NS26528 to C.-F.W. We thank Xianjin Xie for technical assistance
and Drs. D. Baro and R. Harris-Warrick for comments on this
manuscript.
Correspondence should be addressed to Dr. Jeff E. Engel, Section of
Neurobiology and Behavior, Cornell University, Seeley Mudd Hall,
Ithaca, NY 14853.
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Abstract
Introduction
