Download Distinct Roles of CaMKII and PKA in Regulation of Firing Patterns

Distinct Roles of CaMKII and PKA in Regulation of Firing Patterns
and K⫹ Currents in Drosophila Neurons
WEI-DONG YAO AND CHUN-FANG WU
Department of Biological Sciences, University of Iowa, Iowa City, Iowa 52242
Received 11 April 2000; accepted in final form 19 December 2000
INTRODUCTION
The Ca2⫹/calmodulin-dependent protein kinase II (CaMKII)
and cAMP-dependent protein kinase A (PKA) cascades play
critical roles in learning and memory behaviors in both vertebrates (Byrne and Kandel 1996; Chen and Tonegawa 1997)
and invertebrates (Davis 1996; Tully et al. 1996). CaMKII and
PKA are necessary for spatial memory performance in mice
(Huang et al. 1995; Mayford et al. 1996; Silva et al. 1992a,b).
In Drosophila, inhibition of CaMKII by genetic transformation
Address reprint requests to C.-F. Wu (E-mail: [email protected]).
1384
alters both nonassociative and associative conditioning (Griffith et al. 1993) and disruptions of cAMP pathways by mutations that alter the dunce (a phosphodiesterase gene), rutabaga
(an adenylyl cyclase gene), or PKA gene impair learning and
memory in a variety of behavioral paradigms (Aceves-Pina and
Quinn 1979; Drain et al. 1991; Dudai et al. 1976; Duerr and
Quinn 1982; Goodwin et al. 1997; Heisenberg et al. 1985; Li
et al. 1995; Quinn et al. 1974; Siegel and Hall 1979; Spatz et
al. 1974; Wustmann et al. 1996).
The cellular basis of CaMKII- and PKA-mediated behavioral plasticity has been intensively studied. The major focus
has been modifications in synaptic strength and nerve terminal
sprouting (Bailey and Kandel 1993; Bliss and Collingridge
1993), especially in the context of hippocampal long-term
potentiation in mammals (Huang et al. 1995; Malenka et al.
1989; Malinow et al. 1989). In Drosophila, inhibition of
CaMKII and mutations of dnc and rut genes alter synaptic
transmission and nerve terminal arborization at larval neuromuscular junctions (Wang et al. 1994; Zhong and Wu 1991;
Zhong et al. 1992).
Modulation of intrinsic membrane properties of neurons can
profoundly change network dynamics and performance (Getting 1989; Harris-Warrick and Marder 1991) and may represent another important cellular mechanism for behavioral plasticity, one that has received less attention in learning and
memory studies. To more fully understand the cellular basis of
learning and memory, it will be important to determine the
roles of PKA and CaMKII in regulation of intrinsic membrane
properties in neurons. Although dnc and rut mutations have
been shown to affect central neuron firing patterns (Zhao and
Wu 1997), it is not known to what degree this effect is
mediated by PKA, a downstream component of the cAMP
pathway. It is also not known how the electrical activities of
central neurons are regulated by CaMKII or how this regulation compares with modulation by PKA. Some useful insights
into the cellular operations that subserve learning and memory
processes may be gained from such studies given the apparently similar deficiencies in synaptic and behavioral plasticity
caused by gene knockouts of these two kinases in mammalian
systems (Huang et al. 1995; Mayford et al. 1996; Silva et al.
1992a,b).
Inhibition of CaMKII causes limbic epilepsy in mice (Butler
et al. 1995) and enhanced nerve firing in Drosophila (Griffith
The costs of publication of this article were defrayed in part by the payment
of page charges. The article must therefore be hereby marked ‘‘advertisement’’
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
0022-3077/01 $5.00 Copyright © 2001 The American Physiological Society
www.jn.physiology.org
Downloaded from http://jn.physiology.org/ by 10.220.33.3 on August 3, 2017
Yao, Wei-Dong and Chun-Fang Wu. Distinct roles of CaMKII and
PKA in regulation of firing patterns and K⫹ currents in Drosophila
neurons. J Neurophysiol 85: 1384 –1394, 2001. The Ca2⫹/calmodulindependent protein kinase II (CaMKII) and the cAMP-dependent protein kinase A (PKA) cascades have been implicated in neural mechanisms underlying learning and memory as supported by mutational
analyses of the two enzymes in Drosophila. While there is mounting
evidence for their roles in synaptic plasticity, less attention has been
directed toward their regulation of neuronal membrane excitability
and spike information coding. Here we report genetic and pharmacological analyses of the roles of PKA and CaMKII in the firing patterns
and underlying K⫹ currents in cultured Drosophila central neurons.
Genetic perturbation of the catalytic subunit of PKA (DC0) did not
alter the action potential duration but disrupted the frequency coding
of spike-train responses to constant current injection in a subpopulation of neurons. In contrast, selective inhibition of CaMKII by the
expression of an inhibitory peptide in ala transformants prolonged the
spike duration but did not affect the spike frequency coding. Enhanced
membrane excitability, indicated by spontaneous bursts of spikes, was
observed in CaMKII-inhibited but not in PKA-diminished neurons. In
wild-type neurons, the spike train firing patterns were highly reproducible under consistent stimulus conditions. However, disruption of
either of these kinase pathways led to variable firing patterns in
response to identical current stimuli delivered at a low frequency.
Such variability in spike duration and frequency coding may impose
problems for precision in signal processing in these protein kinase
learning mutants. Pharmacological analyses of mutations that affect
specific K⫹ channel subunits demonstrated distinct effects of PKA
and CaMKII in modulation of the kinetics and amplitude of different
K⫹ currents. The results suggest that PKA modulates Shaker A-type
currents, whereas CaMKII modulates Shal-A type currents plus delayed rectifier Shab currents. Thus differential regulation of K⫹ channels may influence the signal handling capability of neurons. This
study provides support for the notion that, in addition to synaptic
mechanisms, modulations in spike activity patterns may represent an
important mechanism for learning and memory that should be explored more fully.
DIFFERENTIAL REGULATION OF EXCITABILITY BY PKA AND CAMKII
were maintained in humidified chambers at room temperature (20 –
25°C) for 2–5 days prior to recordings.
Standard whole cell patch-clamp recording has been described
previously (Saito and Wu 1991). Recording bath solutions (Jan and
Jan 1976) contained (in mM) 128 NaCl, 2 KCl, 4 MgCl2, 1.8 CaCl2,
and 35.5 sucrose, buffered with 5 HEPES at pH 7.1. Patch pipettes
were filled with solution containing (in mM) 144 KCl, 1 MgCl2, 0.5
CaCl2, and 5 EGTA, buffered with 10 HEPES, pH 7.1. K⫹ currents
were isolated by adding TTX (0.2 ␮M) and Cd2⫹ (0.2 mM) to the bath
solution. PKA inhibitor Rp-cAMPS and CaMKII inhibitors KN-62
and KN-93 were from Calbiochem (La Jolla, CA). Recordings were
obtained at room temperature from isolated neurons with a patch
clamp amplifier (Axopatch 1B, Axon Instruments, Foster City, CA).
Data acquisition and analysis were carried out using pCLAMP and
Axograph software (Axon Instruments).
Heat-shock protocol
Induction of transgene expression in ShD, ala1, and ala2 cultures
was accomplished by placing cell cultures in a 38.5°C incubator for 45
min. The heat-shock temperature and duration were established in
previous studies to be sufficient for induction of either peptide (Griffith et al. 1993; Wang et al. 1994; Zhao et al. 1995). Detectable
expression of the CaMKII inhibitory peptide in ala lines, but not ShD
channels, was significantly induced by the cellular stress imposed by
the current cell culture procedures. ala phenotypes with or without
heat shock did not differ, and thus results from heat-shock-treated and
non– heat-shocked neurons were pooled in all experiments.
RESULTS
METHODS
Fly stocks
Canton S (CS) was the wild-type control strain used in all experiments. ShM and eag1 were both from the collection of Dr. S. Benzer
at Cal Tech. ShM is a null allele of Sh (Zhao et al. 1995). Shab1 was
generated in Dr. S. Singh’s Lab at SUNY Buffalo. Three lines of ShD
transformants were generated by inserting a P-element vector containing a ShD cDNA fused to a hsp-70 promoter into the first, second,
or third chromosome in the ShM genetic background. No basal expression of ShD channels is observed at room temperature (Zhao et al.
1995).
DC0X4, a cold-sensitive allele of the DC0 gene that encodes the
major catalytic subunit (RI) of PKA, is lethal at 18°C but viable at
25°C (Li et al. 1995). The transgenic CaMKII inhibitor strains ala1
and ala2 were generated with P-element insertions into the first and
second chromosome, respectively (Griffith et al. 1993). Expression of
the inhibitory peptide is under control of the heat shock promoter
hsp-70. The ala transformants show a low basal level of expression of
the inhibitory peptide even without heat-shock treatment (Griffith et
al. 1993). All fly stocks were maintained on standard Drosophila
media at 20 –22°C except for DC0X4, which was kept at 25°C. All
strains used were homozygous for the corresponding mutations.
Cell culture and electrophysiology
The procedure for culturing Drosophila “giant” neurons has been
described previously (Saito and Wu 1991; Wu et al. 1990). Briefly,
embryos at the gastrulation stage were gently dissociated in modified
Schneider medium (GIBCO, Grand Island, NJ) containing 200 ng/ml
Insulin (Sigma, St. Louis, MO), 20% fetal bovine serum (FBS), 50
␮g/ml streptomycin, and 50 U/ml penicillin. Cells were washed in the
above medium and resuspended in medium containing 2 ␮g/mg
Cytochalasin B (Sigma), and then plated on glass coverslips. Cultures
Alterations of excitability and firing patterns in ala
and DC0 neurons
Whole cell current-clamp experiments were performed on
the soma of isolated giant neurons to minimize complications
introduced by synaptic connectivity. On injection of depolarizing currents, three types of voltage responses were observed
in both wild-type and mutant neurons: all-or-none, graded, and
nonregenerative (Saito and Wu 1991). Among these, the allor-none type action potentials have been best characterized
(Zhao and Wu 1997) and will be emphasized here. In wild-type
cells with all-or-none action potentials, firing patterns can be
functionally classified into adaptive, tonic, and delayed categories based on their instantaneous spike frequency and first
spike latency (Fig. 1), following the criteria described previously (Zhao and Wu 1997). Specifically, the delay to first
spike, and the intervals between the first two spikes (ffirst) and
the last two spikes (flast) in the spike train were used to
distinguish three different firing patterns. “Delayed” showed a
delayed onset (⬎100 ms) of the first spike. “Adaptive” displayed decreasing firing frequency within a spike train and
normally conformed to the criteria of latency ⬍100 ms and
flast/ffirst ⬍ 0.7. “Tonic” showed firing patterns with latency
⬍100 ms but flast/ffirst ⬎ 0.7, hence the firing frequency in these
neurons was relatively constant.
It should be noted that such categorization was based on
spike trains during the first run of a current-clamp protocol
immediately after a whole cell configuration was established.
This stimulation protocol consisted of a series of 400-ms
current steps ranging from ⫺30 to 300 pA. The spike train in
response to a current pulse at the strength of 40 pA above
threshold was taken for categorizing the firing patterns. Firing
Downloaded from http://jn.physiology.org/ by 10.220.33.3 on August 3, 2017
et al. 1994), suggesting a possible role for CaMKII in the regulation of ion channels. In Drosophila, voltage-dependent K⫹
currents are altered by dnc and rut (Delgado et al. 1998; Zhao
and Wu 1997; Zhong and Wu 1993), mutations that affect
cAMP metabolism (Byers et al. 1981; Levin et al. 1992;
Livingston et al. 1984), raising the possibility that K⫹ channels
may be downstream targets of PKA regulation. Correspondingly, central neurons of these memory mutants show aberrant
spike frequency coding (Zhao and Wu 1997). Furthermore K⫹
channel mutants display abnormal habituation in a defined
neural circuit, similar to altered habituation found in dnc and
rut flies (Engel and Wu 1996, 1998). These findings suggest a
close link among K⫹ channel activity, neuronal spike patterns,
and behavioral plasticity.
The Drosophila “giant” neuron culture system has facilitated
patch-clamp characterization of neuronal spike activity and the
underlying ionic currents (Renger et al. 1999; Saito and Wu
1991; Wu et al. 1990; Yao and Wu 1999a,b; Zhao et al. 1995).
Two transgenic fly strains, ala1 and ala2, carry a heat-shock
inducible mini-gene for a specific peptide inhibitor of CaMKII
(Griffith et al. 1993). DC0X4 is an EMS-induced mutation that
inactivates a catalytic subunit of PKA, causing dramatically
reduced PKA activity (Li et al. 1995). These Drosophila
strains, together with several mutants and transformants of K⫹
channel subunits, allowed us to study how PKA and CaMKII
regulate neuronal firing patterns and how such effects may be
mediated by modulations of identified K⫹ channels.
1385
1386
W.-D. YAO AND C.-F. WU
patterns classified this way represented a condition closer to the
“native” excitability states of the neuron.
Because both CaMKII and PKA cascades are implicated in
mechanisms subserving learning and memory, we sought to
discern how the two systems modulate distinct aspects of
neuronal firing patterns. ala1 and ala2 neurons, in which
CaMKII activity is inhibited by a specific inhibitory peptide,
typically displayed prolonged action potential waveforms
(Figs. 1 and 2) and abnormal spontaneous activity (Fig. 3) with
the following characteristics. First, the spike duration, which
was determined as the width at the inflection point during an
action potential take-off in a spike train, was substantially
prolonged in ala neurons (Fig. 2, Table 1). Action potential
prolongation was observed in spike trains of all categories in
current injection experiments (Table 1). Second, during sustained step current injection, all-or-none spikes in the tonic and
adaptive categories in ala neurons exhibited a progressive
decrease in spike size (along with an increase in spike duration
over the period of stimulation), in contrast to the well-maintained spike shape in wild-type cells (Fig. 1). This might reflect
an inefficient repolarization mechanism. Third, a subpopulation of ala neurons fired highly irregular spike pattern in
response to current injection that could not be classified into
the three categories (Fig. 1). Fourth, long-lasting spontaneous
bursting activity with sporadic occurrence and varying fre-
FIG. 2. Action potential prolongation in ala neurons. Action potentials
were evoked by step current injections of 400 ms at an intensity 40 pA above
threshold. The duration of action potentials was determined as the width at the
inflection (- - -) during an action potential take-off in a spike train.
X4
FIG. 3. Abnormal spontaneous activity in ala, but not DC0 , neurons. A:
examples showing spontaneous sporadic bursts of action potentials in ala1 and
ala2, but not in wild-type (WT) and DC0X4, neurons. Membrane potentials
were allowed to free-run during recordings. B: the percentage of cells displaying sporadic firing but not persistent rhythmic firing was significantly increased
in ala cultures. In contrast, DC0X4 cultures did not show increases in either
type of spontaneous activity. * P ⬍ 0.01, 1-sample binomial tests of mutants
vs. WT.
quency was observed in 25–30% of ala neurons compared with
only 5% in wild type (Fig. 3), although the resting membrane
potentials were similar (data not shown). The action potentials
within spontaneous spike trains were also prolonged (data not
shown). This phenotype suggests that CaMKII plays a significant role in maintaining the quiescent state of neurons. Persistent rhythmic firing lasting for minutes (data not shown) (cf.
Yao and Wu 1999a) has been observed in a small population of
cultured neurons. Such cells in wild-type cultures presumably
correspond to endogenous pace-making cells in the normal
TABLE
1. Firing properties of wild-type and mutant neurons
WT (44)
Adaptive
Tonic
Delayed
ala1 (25)
Adaptive
Tonic
Delayed
ala2 (28)
Adaptive
Tonic
Delayed
DC0 (11)
Adaptive
Tonic
Delayed
Incidence,
%
Mean Frequency,
Hz
Amplitude,
mV
Duration,
ms
18
68
14
19.0 ⫾ 1.7
16.5 ⫾ 1.5
18.3 ⫾ 3.4
31.7 ⫾ 4.7
49.9 ⫾ 1.9
31.6 ⫾ 5.1
3.9 ⫾ 0.4
4.0 ⫾ 0.4
4.2 ⫾ 0.4
20
64
16
21.6 ⫾ 1.9
18.0 ⫾ 2.2
13.2 ⫾ 1.5
39.6 ⫾ 2.6
42.7 ⫾ 3.3*
39.5 ⫾ 3.8
5.7 ⫾ 0.6*
6.0 ⫾ 1.1*
5.4 ⫾ 0.7
18
60
22
13.3 ⫾ 2.3*
24.5 ⫾ 2.0*
15.0 ⫾ 2.6
39.2 ⫾ 5.8
41.4 ⫾ 4.2*
45.8 ⫾ 10.0
6.0 ⫾ 0.8*
5.8 ⫾ 0.9*
5.7 ⫾ 1.4
33
50
17
36.3 ⫾ 6.0*
28.2 ⫾ 4.1*
17.8
17.1 ⫾ 0.8*
43.9 ⫾ 4.7
16.5
3.7 ⫾ 0.1
3.8 ⫾ 0.2
3.5
Data are means ⫾ SE except for DC0 delayed type, which had few cells.
Total number of cells firing regular all-or-none action potentials categorized
for each genotype is indicated in parentheses. Mean frequency is determined
based on the number of spikes elicited by a 400-ms depolarization current step
at a strength of 40 pA above threshold; Amplitude is measured as the voltage
difference between the peak and the valley of the first action potential in a
spike train; Duration is measured at the inflexion point during take-off of the
first spike. * P ⬍ 0.05, Student’s t-tests.
Downloaded from http://jn.physiology.org/ by 10.220.33.3 on August 3, 2017
X4
FIG. 1. Differential alterations of firing patterns in ala and DC0
mutant
neurons. Action potentials were evoked by step current injections of 400 ms at
an intensity 40 pA above threshold. Neurons were categorized into adaptive,
tonic, delayed, and interrupted classes based on the 1st spike latency and
instantaneous firing frequency as described by Zhao and Wu (1997). Highly
irregular firing patterns that could not be classified into these 4 categories were
found in mutant neurons (“Abnormal”). Note the fluctuations in firing rate in
DC0X4 neurons and action potential prolongation in ala neurons which maintained the regularity in firing rate.
DIFFERENTIAL REGULATION OF EXCITABILITY BY PKA AND CAMKII
Altered stability of spike frequency coding in ala
and DC0 neurons
Responses to individual current pulses can reveal erratic
patterns of spike trains in mutant neurons, such as ala1, ala2,
and DC0X4. However, a single-pulse paradigm cannot determine the stability of the spike patterns generated by a given
FIG. 4. Instantaneous firing frequency in WT and mutants. Instantaneous
firing frequencies of successive spikes in a spike train in each neuron were
determined as reciprocals of interspike intervals. The data points for each
neuron are connected (—). Spike trains were evoked by step current injections
of 400 ms at an intensity 40 pA above threshold. Information for the 1st spike
(reciprocal of the latency) in each neuron was excluded for simplicity.
neuron. Such endogenous stability of firing patterns is important to ensure a reliable and consistent input-output relationship
during signal processing by the neuron. We examined this
problem by using a revised paradigm involving repetitive stimulation at a low frequency. When neurons were stimulated by
step current injections (80 –120 pA) at 0.5 Hz or lower to
minimize the interaction between successive pulses, wild-type
neurons displayed highly reproducible firing patterns in response to these identical repeated stimuli (Fig. 5A). The firing
pattern, in particular the number of spikes in each spike train,
varied to a much greater extent in ala and DC0X4 neurons. To
quantify the variation in the number of spikes evoked by a
fixed current pulse for each genotype, the coefficient of variance (CV ⫽ SD/Mean) was determined for 20 repeated stimuli
and presented in relation to the mean spike number for each
cell (Fig. 5B). A markedly increased CV was seen in a large
proportion of ala1, ala2, and DC0 X4 neurons compared with
wild type, indicating significant intrinsic instability in the mutant neurons. Although our frequency analysis of different
firing patterns indicates that mutant neurons do not fire fewer
action potentials in general (Table 1), the variability among
responses to identical stimuli was most evident for neurons that
generated fewer spikes (Fig. 5B). Thus both CaMKII and PKA
play crucial roles in maintaining the stability of neuronal firing
patterns, which may be important for the proper performance
of neural circuits during information processing.
Downloaded from http://jn.physiology.org/ by 10.220.33.3 on August 3, 2017
CNS (Yao and Wu 1999a). The percentage of these putative
pace-making cells in ala (3–5%) was similar to that of wildtype culture (2%; Fig. 3). The incidence of adaptive, tonic, and
delayed firing as well as the spike amplitude and the mean
spike frequency in response to suprathreshold stimulation were
also summarized in Table 1.
Both abnormal spontaneous activity and aberrant spike frequency coding have been demonstrated in neurons with dnc
and rut mutations (Zhao and Wu 1997), both of which disrupt
cAMP metabolism and cause learning and memory deficiencies (Aceves-Pina et al. 1983; Byers et al. 1981; Dudai et al.
1976; Livingstone et al. 1984). Although the majority of the
cAMP effects in eukaryotes may be mediated through activation of PKA, intracellular cAMP has also been shown to
interact directly with ion channels in some cell types (Delgado
et al. 1991; Dhallan et al. 1990). Thus to establish the functional roles of PKA in mediating the phenotypes of the memory
mutants dnc and rut, it is necessary to employ mutants such as
DC0X4 in which direct perturbation of the kinase activity
occurs.
We found that very few DC0X4 neurons displayed spontaneous sporadic bursting activities with a percentage not statistically different from that of wild type (Fig. 3). This indicates
that the increased spontaneous activity in dnc and rut neurons
(Zhao and Wu 1997) may be mediated by mechanisms other
than PKA modulation. The resting membrane potential, distribution of firing patterns, and action potential duration are not
affected in DC0X4 (Table 1 and data not shown). The firing
frequency was significantly increased in adaptive and tonic
DC0X4 neurons. The action potential amplitude was significantly decreased in adaptive but slightly reduced in tonic
neurons in DC0X4. We noticed that the most remarkable mutant
phenotype of firing patterns in DC0X4, however, is the erratic
firing rate during current injection (Figs. 1 and 4). These
findings show that abnormality in firing frequency coding can
occur without altered action potential duration (Figs. 1 and 2).
With respect to this particular phenotype, the DC0X4 results
were in general consistent with the dnc and rut study (Zhao and
Wu 1997), suggesting that defective frequency coding caused
by abnormal cAMP levels may be mediated by PKA.
In contrast, frequency coding of all-or-none spike trains in
ala neurons in response to current injections appeared normal,
and the instantaneous firing rates in tonic and adaptive categories were indistinguishable between ala and wild type (Figs.
1 and 4). It is remarkable that the spike frequency coding
remained intact despite the fact that the action potential duration in some ala neurons is significantly prolonged compared
with wild-type neurons (Figs. 1, 2, and 4 and Table 1). These
results indicate that perturbations of CaMKII and PKA activities exert influences on distinct neuronal firing parameters.
CaMKII appears to play a major role in defining the width of
individual action potentials and maintaining their waveform
during sustained activity. PKA, on the other hand, is more
important to the regularity of spike frequency coding.
1387
1388
W.-D. YAO AND C.-F. WU
Altered K⫹ currents in ala and DC0 neurons
Voltage-activated K⫹ currents regulate membrane repolarization and hence excitability and firing patterns in neurons
(Hille 1992; Rudy 1988). The altered neuronal firing patterns
and aberrant spontaneous hyperexcitability observed in ala and
DC0X4 neurons suggested that CaMKII and PKA affect K⫹
current mechanisms. In the giant neuron culture system, various voltage-activated K⫹ currents and the effects of mutations
of identified K⫹ channel subunits have been well characterized
(Yao and Wu 1999a; Zhao et al. 1995). Abnormality in kinetics
and amplitude of different current components may be readily
determined. Some of the striking mutant phenotypes observed
in current clamp experiments might be correlated to changes in
K⫹ current properties that could be revealed in voltage-clamp
records.
Voltage-activated K⫹ currents were examined in saline containing TTX and Cd2⫹ to eliminate inward Na⫹ and Ca2⫹
currents as well as outward Ca2⫹-activated K⫹ currents (Saito
and Wu 1991). Two types of K⫹ currents with different inactivation kinetics can be seen in wild-type neurons (Fig. 6A) (cf.
Delgado et al. 1998). The decay of Type 1 currents could be
fitted by a two-exponential process with fast [␶1 ⫽ 68.3 ⫾ 4.1
(SE) ms] and slow (␶2 ⫽ 1,133 ⫾ 221 ms) components. Type
1 currents were encountered in the majority of giant neurons
(⬃60%, n ⫽ 59). Type 2 currents, found in the remaining cells
(⬃40%), inactivated with a single-exponential time course
(␶ ⫽ 1,066 ⫾ 485 ms). Both 4-aminopyridine (4-AP)- and
TEA-sensitive components were found in the two types of
currents. In general, Type 1 currents contained a greater 4-APsensitive, fast-inactivating component, and Type 2 currents
were more sensitive to TEA (data not shown).
We found similar fractions of neurons in wild-type and
mutant cultures expressing Type 1 versus Type 2 currents [wild
type (WT): 73 vs. 27%; ala1: 75 vs. 25%; ala2: 73 vs. 27%;
DC0X4: 62 vs. 38%, no significant differences under 1-sample
binomial tests]. In ala1 and ala2 but not in DC0X4 neurons, ␶1
⫹
FIG. 6. Altered decay kinetics of K
currents in ala, but not DC0X4,
neurons. A: 2 types of K⫹ currents can be identified based on their decay
kinetics. Type 1 can be fitted by 2 exponential processes, while Type 2 currents
can be fitted with a single exponential decay. K⫹ currents were elicited by
depolarization to Vm ⫽ ⫹40 mV from a holding potential of ⫺80 mV. B: ala
specifically accelerates the faster decay process (␶1) of Type 1 currents. DC0X4
mutants did not alter the kinetics of either type. The number of cells examined
is indicated in parentheses. In this and the following figures, TTX and Cd2⫹
were added to the saline to eliminate inward Na⫹ and Ca2⫹ currents and
outward Ca2⫹-activated K⫹ currents. Error bars indicate SE (not shown if
smaller than the symbol size). * P ⬍ 0.05; ** P ⬍ 0.01, Student’s t-test
(2-tailed) for the difference between WT and mutants.
Downloaded from http://jn.physiology.org/ by 10.220.33.3 on August 3, 2017
X4
FIG. 5. Instability of firing patterns in ala and DC0
neurons. A: 20
identical depolarizing current pulses (80 pA, 0.5 Hz) were delivered to WT,
ala1, ala2 (not shown), and DC0 neurons to test their reproductivity of firing
patterns. Four representative voltage responses from an example neuron of
each genotype are shown. Note the reproducible spike patterns in WT and the
varying patterns in mutants. B: coefficient of variance (CV) was determined for
each cell as SD/Mean, where Mean represents the average number of spikes
per spike train and SD the standard deviation of the 20 trials.
of the double-exponential approximation for Type 1 currents
was significantly reduced compared with WT neurons (Fig. 6).
The specific effect of ala mutations on the fast decay time
constant is consistent with previous pharmacological studies
that showed accelerated decay of K⫹ currents after CaMKII
inhibition (Peretz et al. 1999; Yao and Wu 1999b). However,
the slow inactivation time constant (␶2, not shown) in Type 1
currents and the decay rate (␶) of Type 2 currents did not
significantly differ among genotypes (Fig. 6). These results
suggest that CaMKII may differentially regulate certain channels with distinct kinetic properties in Drosophila neurons.
To examine how the amplitudes of IA and IK in the two
current types were affected in ala and DC0X4 neurons, we used
a prepulse paradigm to separate the early, inactivating currents
(IA) and the delayed, noninactivating currents (IK; Fig. 7A). We
found that both ala and DC0X4 mutations affected neurons
displaying Type 1 currents. In these cells, current density of IA
was reduced significantly in both ala and DC0X4 neurons (Fig.
7B). When the current-voltage relations (I-V curves) are normalized for the three genotypes (Fig. 7C), it is apparent that the
reductions in current amplitude were independent of membrane
potentials. The current density of IK was also reduced in ala
and DC0X4 neurons. However, there was an apparent voltage
dependence of this effect with more severe reduction at lower
DIFFERENTIAL REGULATION OF EXCITABILITY BY PKA AND CAMKII
1389
same cell may reveal how neurons of different firing patterns
express varying portions of CaMKII- and PKA-sensitive K⫹
current components.
membrane potentials (especially in ala neurons) (Fig. 7C),
which is also reflected in the significantly shifted half-activation voltage in ala and DC0X4 (Fig. 7D). The amplitudes of IA
and IK components of Type 2 currents were not significantly
different between WT and ala neurons [WT: IA ⫽ 13.17 ⫾
1.29, IK ⫽ 19.85 ⫾ 0.77 (n ⫽ 3); ala: IA ⫽ 13.59 ⫾ 4.32,
IK ⫽ 17.31 ⫾ 2.39 (n ⫽ 3); P ⬎ 0.05, t-tests; consistent results
were also obtained from nonseparated IA and IK (data not
shown). All currents were recorded at ⫹20 mV]. In contrast,
the DC0X4 mutation did not affect IA but decreased IK components of Type 2 current significantly [DC0X4: IA ⫽ 19.02 ⫾
2.78, IK ⫽ 9.01 ⫾ 2.09 (n ⫽ 5), P ⬍ 0.01]. Taken together, ala
affects both the kinetics and amplitude of Type 1 currents,
whereas DC0X4 selectively affects current amplitude but in
both Type 1 and Type 2 currents. Such distinct effects on
current density may contribute to the differences in the regulation of excitability and firing patterns by CaMKII and PKA.
Further current- and voltage-clamp correlation analysis in the
⫹
FIG. 8. Modulation of K currents by CaMKII and PKA inhibitors in WT
neurons. CaMKII antagonists KN-93 and -62 suppressed both the transient and
sustained K⫹ currents and accelerated the decay process of the transient
component. The PKA inhibitor Rp-cAMPS reduced the transient and the
sustained K⫹ currents without affecting the current decay kinetics. K⫹ currents
were elicited by depolarization steps (1 s) from a holding potential of ⫺80 mV
to between ⫺60 and ⫹60 mV in 20-mV increments. The final drug concentrations in saline: KN-93, 1 ␮M; KN-62, 10 ␮M; Rp-cAMPS, 50 ␮M.
Downloaded from http://jn.physiology.org/ by 10.220.33.3 on August 3, 2017
⫹
FIG. 7. Reduced IA and IK of Type 1 K
currents in ala and DC0X4
neurons. A: the voltage protocol used to separate IA from IK. A 1-s preconditioning pulse to ⫺10 mV from a holding potential of ⫺80 mV was used to
inactivate IA while leaving IK intact (A2). Subtraction of the voltage-activated
current with a prepulse (A2) from the one without (A1) gives IA (A1 ⫺ A2). B:
I-V relation of IA and IK. C: relative reduction of IA and IK in ala2 and DC0X4
neurons. The IA and IK for mutants are normalized to the corresponding values
for WT, which are shown as a horizontal line at 1.0. D: activation Vm1/2 and
slopes for IA and IK. The membrane conductance (determined by the formula
G ⫽ I/(V ⫺ Vr), where I is the current density in pA/pF, and Vr the reversal
potential (⫺75 mV) was fit to the Boltzman relationship G ⫽ Go/{1 ⫹
exp[(Vm1/2 ⫺ V)/Vslope]}, where Go, Vm1/2, and Vslope are the maximum
conductance, half-activation voltage (mV), and slope (mV/e-fold), respectively.
Genetic dissection of CaMKII and PKA modulation
on K⫹ currents
Voltage-gated K⫹ channel ␣ subunits in Drosophila neurons, encoded by the identified genes Sh, Shal, Shab, and Shaw,
generate K⫹ currents with distinct kinetics and voltage dependence when expressed in heterologous systems (Butler et al.
1989; Iverson and Rudy 1990; Timpe et al. 1988). Viable point
mutations of Sh (Wu and Ganetzky 1992) and Shab (Singh and
Singh 1999) provide powerful tools to dissect the downstream
effectors of PKA and CaMKII for modulating K⫹ currents.
Highly selective pharmacological agents that inhibit CaMKII
and PKA are also available to enhance the genetic approach.
KN-62 and -93 are specific inhibitors of CaMKII (Sumi et
al. 1991; Tokumitsu et al. 1990) while Rp-cAMPS (adenosine
3⬘,5⬘-cyclic monophosphorothioate, Rp-Isomer; 50 ␮M) selectively inhibits PKA activity. When applied to the bath solution,
CaMKII inhibitors KN-62 (10 ␮M) and KN-93 (1 ␮M) and
PKA inhibitor Rp-cAMPS (50 ␮M) all reduced both the peak
and sustained K⫹ currents in WT neurons (Fig. 8).
Sh mutations alter A-type currents in muscle (Salkoff and
Wyman 1981; Wu and Haugland 1985) and in neurons (Baker
and Salkoff 1990). The null mutation ShM deletes all Sh subunits in neurons and could be used to test whether Sh subunits
are a target for CaMKII or PKA modulation. As shown in Figs.
9 and 10, the non-Sh currents that remain in ShM neurons,
including both the A-type and the sustained components, were
significantly affected by KN-93 in both amplitude and kinetics
to a degree similar to those of WT neurons. In contrast, the
non-Sh currents were apparently resistant to modulation by
Rp-cAMPS (Figs. 9B and 10B).
1390
W.-D. YAO AND C.-F. WU
⫹
FIG. 9. Modulation of K
currents by CaMKII and PKA inhibitors in
different K⫹ channel mutants and ShD transformants. A: modulatory effects of
KN-93 on K⫹ currents recorded from ShM and Shab1 mutants and on heatshock (HS)-induced ShD currents. The HS-induced slow-recovering ShD
currents were extracted by a protocol described previously (Renger et al. 1999;
Zhao et al. 1995). B: modulatory effects of Rp-cAMPS on K⫹ currents in ShM,
Shab, and ShD transformant cells. Voltage-activated K⫹ currents were elicited
by depolarizing the neurons to ⫹40 mV (1 s) from a holding potential of ⫺80
mV. The final concentrations of KN-93 and Rp-cAMPS in saline were 1 and
50 ␮M, respectively.
To directly assay the modulation of an identified Sh ␣
subunit by CaMKII kinase and PKA, we used ShD transgenic
strains in which a copy of ShD cDNA was inserted into the
first, second, or third chromosome in the ShM host background
to express ShD channels in neurons lacking endogenous Sh
channels. The expressed ShD current, induced by heat shock
(see METHODS), exhibited fast inactivation kinetics and extremely slow recovery from inactivation (Zhao et al. 1995).
These distinct kinetic properties allow the isolation of this
current from the native K⫹ currents by a twin pulse paradigm
(Renger et al. 1999; Zhao et al. 1995). As shown in Figs. 9 and
10, ShD current was inhibited by Rp-cAMPS but not by
KN-93. Taken together, the results support the idea that the Sh
product is a target for PKA, but not for CaMKII, modulation.
The Shab gene has been proposed to be responsible for the
major slowly inactivating delayed rectifier K⫹ currents in
Drosophila neurons (Tsunoda and Salkoff 1995b) and muscle
cells (Singh and Singh 1999). We found that a point mutation,
Shab1 (Singh and Singh 1999), eliminated most of the steadystate currents in giant neurons (Fig. 9). This mutation allowed
us to analyze further how CaMKII and PKA modulate the
different K⫹ currents in neurons. Figures 9 and 10 show that in
Shab-deficient neurons, the transient A current (more prominent due to the removal of the delayed rectifier) was still
⫹
FIG. 10. Summary of KN-93, KN-62, and Rp-cAMPS effects on K currents in WT and mutant neurons. A: KN-93 effects. The drug (1 ␮M) suppressed the peak currents in WT, ShM, and Shab1 neurons but did not affect
ShD current. The sustained components were also significantly inhibited in
WT and ShM and in Shab1 to a lesser extent. B: Rp-cAMPS effects. This drug
(50 ␮M) inhibited the WT A current but had no effects on currents in ShM
neurons. “Fraction remaining” is the proportion of current remaining after drug
treatments compared with that before treatments. * P ⬍ 0.05, ** P ⬍ 0.001,
1-sample 2-tailed t-tests.
Downloaded from http://jn.physiology.org/ by 10.220.33.3 on August 3, 2017
significantly modified by KN-93 and Rp-cAMPS. However,
these two drugs did not significantly affect the small sustained
non-Shab components remaining in Shab-deficient neurons.
The A current in Drosophila neurons is thought to be primarily composed of Shal currents (Tsunoda and Salkoff 1995a)
with a smaller contribution from Sh (Baker and Salkoff 1991).
The sustained current consists of a number of components
including the delayed rectifiers Shab (Tsunoda and Salkoff
1995a) and the steady-state components of A-type Sh and Shal
currents. Currents conferred by Shaw subunits have also been
implicated as part of the sustained current (Tsunoda and
Salkoff 1995a). The suppression of A-type currents in Shab1
neurons is consistent with the idea that CaMKII and PKA
modulate Shal and Sh channels, respectively (see preceding
text). In contrast, the lack of significant effects of CaMKII
inhibitors on the non-Shab sustained current in Shab1 neurons
suggests that modulation of the Shab current may be responsible for part of the reduction in sustained currents of WT
neurons by those drugs.
In summary, a working scheme about PKA and CaMKII
modulation of K⫹ channels can be proposed based on the
genetic and pharmacological data: modulation of A currents is
mediated by PKA on Sh channels and CaMKII on Shal channels (Fig. 11), whereas modulation of delayed rectifier K⫹
currents by CaMKII is mediated primarily by Shab. Future
work on Shaw mutants, when they become available, should
DIFFERENTIAL REGULATION OF EXCITABILITY BY PKA AND CAMKII
1391
X4
FIG. 11. Summary of DC0 and ala action potential phenotypes and a scheme incorporating the effects
on underlying K⫹ currents by PKA and CaMKII
modulation.
DISCUSSION
In this study, we report that CaMKII and PKA modulate
different aspects of neuronal firing properties, resulting at least
in part from differential targeting of voltage-gated K⫹ channels
by the two enzymes. Our results establish roles for the two
Ca2⫹/CaM-activated pathways in neuronal spike shaping and
firing patterning. These properties provide another regulatory
mechanism, in addition to modulation of synaptic efficacy,
which may contribute to behavioral modifications.
Differential regulation of neuronal function by PKA
and CaMKII
Neuronal firing patterns carry highly structured temporal
information. Firing rate and action potential shape are two
major parameters that dictate the timing and amount of neurotransmitter release. Neuronal electrical activity can be highly
plastic, being affected by both short- and long-term modulatory
mechanisms (Getting 1989; Marder et al. 1996; McCormick
1992; Turrigiano et al. 1994, 1995). Such functional plasticity
has important implications not only for behavioral modification
but also for activity-dependent refinement of proper neural
circuits during development (Budnick et al. 1990; Cline 1991;
Hubel and Wiesel 1970). Given the critical roles of PKA and
CaM kinase in behavioral plasticity, it is not surprising that at
the cellular level, both enzymes regulate neuronal firing properties. Comparisons of how the two signaling pathways modulate neuronal membrane properties can provide insights into
the cellular mechanisms underlying behavioral modifications
(Fig. 11).
The activity-dependent accumulation of Ca2⫹ could activate
both PKA and CaM kinase activity through a Ca2⫹/calmodulin-dependent mechanism (Fig. 11B). Direct binding of Ca2⫹/
CaM is required for CaMKII activation while Ca2⫹/CaMdependent activation of adenylyl cyclase stimulates cAMP
synthesis, which in turn activates PKA. DC0X4 neurons in
which the PKA catalytic subunit is defective showed aberrant
frequency coding, unstable firing patterns, enhanced firing
frequency, and decreased spike amplitude, but no changes in
action potential width (Figs. 1, 2, and 4, Table 1). These
phenotypes are in general consistent with the defects found in
dnc and rut neurons (Zhao and Wu 1997) which have a
defective cAMP phosphodiesterase and adenylyl cyclase, respectively. The exception is that dnc and rut neurons display
spontaneous activity not seen in DC0X4, which raises the
possibility of regulation mediated by direct cAMP binding to
ion channels (Delgado et al. 1991; Dhallan et al. 1990; Nakamura and Gold 1987) or via non-DC0-dependent PKA pathways. In comparison, neurons of ala1 and ala2 transformants,
which express a CaMKII inhibitory peptide (Griffith et al.
1993), exhibited unstable firing patterns in repeated stimulation
trials, but no fluctuations in spike frequency coding during
single current injections. Furthermore the action potential
waveform was prolonged in ala neurons. Because CaMKII is
highly abundant at the synapse (Kennedy 1997; Nairn et al.
1985), its regulation of action potential duration could influence the dynamics of Ca2⫹ influx and thus the amount of
transmitter release. Indeed, enhancement of synaptic currents
and irregularity of release patterns has been reported for the
larval neuromuscular junction of Drosophila ala transformants
(Wang et al. 1994).
The preceding observations suggest that different secondmessenger systems may regulate distinct aspects of neuronal
firing properties. This can enhance the capacity of the nervous
system to fulfill specific functional requirements for a variety
of behavioral tasks. Based on previous observations, cAMP
and CaM kinase pathways are known to exert different effects
on nerve terminal growth and synaptic transmission at the
larval neuromuscular junction (Wang et al. 1994; Zhong and
Wu 1991; Zhong et al. 1992), which demonstrates that differential regulation of different aspects of neuronal function by
the two signaling cascades can occur within the same cell. It
will be interesting to further investigate firing patterns between
these mutants in neurons associated with identified neural
circuits underlying distinct behaviors.
Correlation of K⫹ current modulation with firing patterns
The firing properties of Drosophila “giant” neurons are
shaped by a plethora of ionic currents, including voltageactivated outward K⫹ currents, inward Ca2⫹ and Na⫹ currents,
and Ca2⫹-activated outward K⫹ currents (Saito and Wu 1993).
IA has been shown in this preparation to modulate the onset,
the duration, and the frequency of action potentials whereas IK
primarily contributes to the repolarization of the action potential, determining its duration (Saito and Wu 1993; Yao and Wu
1999a; Zhao and Wu 1997). The distinct patterns of adaptive,
Downloaded from http://jn.physiology.org/ by 10.220.33.3 on August 3, 2017
provide further evidence concerning the genetic basis of
CaMKII and PKA modulation of K⫹ currents.
1392
W.-D. YAO AND C.-F. WU
Distinct regulation of K⫹ channel subunits by different
second-messenger systems
It has been established that a variety of voltage-gated K⫹
channels in Drosophila neurons are composed of a combination of pore-forming ␣ subunits, including Sh, Shal, Shab, and
Shaw, and auxiliary ␤ subunits such as hyperkinetic (Yao and
Wu 1999a). Based on the genetic and pharmacological analyses presented above, a scheme of K⫹ channel modulation by
PKA and CaMKII can be proposed (Fig. 11B).
Our data show that KN-93 affected IA in WT neurons and
non-Sh transient currents in ShM mutant neurons to a similar
degree, suggesting that Shal, but not Sh, is modulated by CaMKII.
This was supported by the fact that ShD current is resistant to
KN-93 treatment. Since Sh RNA undergoes extensive alternative
splicing (Kamb et al. 1988; Pongs et al. 1988; Schwarz et al.
1988), further tests of whether other Sh products are modulated by
CaMKII may be performed in future genetic and pharmacological
studies. On the other hand, our results show that Rp-cAMPS
modulated ShD currents in ShD transformant neurons but not the
non-Sh currents in ShM mutants, which is consistent with previous
findings: the Sh polypeptide contains PKA phosphorylation sites
(Schwarz et al. 1988) responsible for modulation of inactivation
of Sh channels expressed in Xenopus oocytes (Drain et al. 1994),
and in Drosophila muscle, the transient Sh current is modified by
dnc mutations and by acute application of cAMP analogs (Zhong
and Wu 1993). Taken together, these observations demonstrate
that Sh channels are a target of PKA but probably not of CaMKII.
The sustained K⫹ current consists of multiple components
as discussed in RESULTS, but its major component, the delayed
rectifier current, is thought to be mediated by the Shab subunit
(Fig. 9) (cf. Singh and Singh 1999; Tsunoda and Salkoff
1995b). The fact that the sustained currents in both WT and
ShM neurons were substantially suppressed, following KN-93
treatments, to a level comparable to the steady-state currents in
Shab1 neurons (see preceding text) (cf. Peretz et al. 1998;
Singh and Singh 1999; Yao and Wu 1999b) strongly suggests
that the delayed rectifier Shab channel mediates the majority of
CaMKII effects on sustained K⫹ currents.
The differential effects of CaMKII and PKA on distinct K⫹
channel subunits discussed in the preceding text may contribute to the action potential phenotypes observed in ala and
DC0X4 neurons. While the modulation of Sh currents by PKA
appears to be important for generating regular firing patterns
for reliable frequency coding, modulation of non-Sh currents
by CaMKII may contribute to the control of action potential
repolarization and duration. Both PKA and CaMKII, however,
may regulate the stability of firing patterns through possible
effects on multiple channel types (see preceding text), which
may be critical for reliability and precision in signal processing
in the nervous system.
In conclusion, PKA and CaMKII target different K⫹ channel
subunits to confer differential regulation of neuronal excitability and spike patterning. The two second-messenger systems
may modulate separate parameters of neuronal function, including nerve terminal outgrowth (Wang et al. 1994; Zhong et
al. 1992), synaptic function and plasticity (Wang et al. 1994;
Zhong and Wu 1991), and neuronal firing patterns (Zhao and
Wu 1997; this study). These parameters might be conditioned
differentially during nervous system activity and thus may
underlie specific behavioral modifications by different stimulus
paradigms and in different model systems.
We thank Dr. Jeff Engel for comments on the manuscript.
This work was supported by National Institutes of Health grants to
C.-F. Wu.
Present address of W.-D. Yao: HHMI/Dept. of Cell Biology, Duke University Medical Center, Durham, NC 27710.
REFERENCES
ACEVES-PIÑA EO, BOOKER R, DUERR JS, LIVINGSTONE MS, QUINN WG, SMITH
RF, SZIBER PP, TEMPEL BL, AND TULLY T. Learning and memory in
Drosophila, studied with mutants. Cold Spring Harb Symp Quant Biol 48:
831– 839, 1983.
ACEVES-PIÑA EO AND QUINN WG. Learning in normal and mutant Drosophila
larvae. Science 206: 93–96, 1979.
BAILEY CH AND KANDEL ER. Structural changes accompanying memory
storage. Annu Rev Physiol 55: 397– 426, 1993.
BAKER K AND SALKOFF L. The Drosophila Shaker gene codes for a distinctive
K⫹ current in a subset of neurons. Neuron 4: 129 –140, 1990.
BLISS TV AND COLLINGRIDGE GL. A synaptic model of memory: long-term
potentiation in the hippocampus. Nature 361: 31–39, 1993.
BUDNICK V, ZHONG Y, AND WU CF. Morphological plasticity of motor axons
in Drosophila mutants with altered excitability. J Neurosci 10: 3754 –3768,
1990.
BUTLER A, WEI A, BAKER K, AND SALKOFF L. A family of putative potassium
channel genes in Drosophila. Science 243: 943–947, 1989.
Downloaded from http://jn.physiology.org/ by 10.220.33.3 on August 3, 2017
tonic, and delayed firing reflect differences in expression of
different K⫹ currents (Zhao and Wu 1997). Consistent with
this notion, our preliminary results suggest that Type 1 currents
can support all three types of firing patterns while Type 2
neurons only fire adaptive spikes (unpublished observations).
Although a complete explanation of mutant firing patterns
will require knowledge of both inward and outward currents,
some insights can be obtained by an initial analysis of K⫹
currents. For example, kinase inhibitors and K⫹ channel blockers can be used to explore the contributions of pharmacologically and kinetically distinct K⫹ current components. Specifically, 4-AP has been shown to increase the firing frequency
and shorten the latency to the onset of spikes, which is particularly striking for delayed type neurons, whereas TEA broadens the duration of action potentials without changing the firing
rate, which is most obvious for adaptive type neurons (Zhao
and Wu 1997). Current- and voltage-clamp correlation studies
from the same cells demonstrated that K⫹ currents in adaptive
cells have a large TEA-sensitive component and delayed cells
a large 4-AP-sensitive component (Zhao and Wu 1997). In our
preliminary experiments we found that KN-93 prolonged action potential duration especially in adaptive cells and RpcAMPS greatly reduced the latency and turned adaptive cells
into fast-spiking cells (Yao and Wu, Peng and Wu, unpublished observations). These observations are consistent with
the hypothesis that PKA has a major effect on Sh IA currents
and CaM kinase affects non-Sh currents including Shab IK.
However, the kinase inhibitors did not completely mimic the
modulation of either IA or IK channel blockers (data not
shown), suggesting that kinase inhibitors may affect firing
patterns in a more complicated manner. For example, INa and
ICa are known to affect the initiation and duration of action
potentials and may be modulated by protein kinases. A more
complete understanding of the ionic basis of firing patterns
would require voltage-and current-clamp correlation analysis
in the same cells of each firing pattern and should include not
only K⫹ currents but also other ionic currents.
DIFFERENTIAL REGULATION OF EXCITABILITY BY PKA AND CAMKII
IVERSON LE AND RUDY B. The role of the divergent amino and carboxyl
domains on the inactivation properties of potassium channels derived from
the Shaker gene of Drosophila. J Neurosci 10: 2903–2916, 1990.
JAN LY AND JAN YN. Properties of the larval neuromuscular junction in
Drosophila melanogaster. J Physiol (Lond) 262: 215–236, 1976.
KAMB A, TSENG-CRANK J, AND TANOUYE MA. Multiple products of Drosophila
Shaker gene may contribute to potassium channel diversity. Neuron 1:
421– 430, 1988.
KENNEDY MB. The postsynaptic density at glutamatergic synapses. Trends
Neurosci 20: 264 –268, 1997.
LEVIN LR, HAN PL, HWANG PM, FEINSTEIN PG, DAVIS RL, AND REED RR. The
Drosophila learning and memory gene rutabaga encodes a Ca2⫹/calmodulin-responsive adenylyl cyclase. Cell 68: 479 – 489, 1992.
LI W, TULLY T, AND KALDERON D. Effects of a conditional Drosophila PKA
mutant on olfactory learning and memory. Learn Mem 2: 320 –333, 1995.
LIVINGSTON MS, SZIBER PP, AND QUINN WG. Loss of Ca2⫹/Calmodulin
responsiveness in adenylate cyclase of rutabaga, a Drosophila learning
mutant. Cell 37: 205–215, 1984.
MALENKA RC, KAUER JA, PERKEL DJ, MAUK MD, KELLEY PT, NICOLL RA,
AND WAXHAM MN. An essential role for post synaptic calmodulin and
protein kinase activity in long-term potentiation. Nature 340: 554 –557,
1989.
MALINOW R, SCHULMAN H, AND TSIEN RW. Inhibition of postsynaptic PKC or
CaMKII blocks induction but not expression of LTP. Science 245: 862– 866,
1989.
MARDER E, ABBOTT LF, TURRIGIANO GG, LIU Z, AND GOLOWASCH J. Memory
from the dynamics of intrinsic membrane currents. Proc Natl Acad Sci USA
93: 13481–13486, 1996.
MAYFORD M, BACH ME, HUANG Y-Y, WANG L, HAWKINS RD, AND KANDEL
ER. Control of memory formation through regulated expression of a
CaMKII transgene. Science 274: 1678 –1682, 1996.
MCCORMICK DA. Cellular mechanisms underlying cholinergic and noradrenergic modulation of neuronal firing mode in the cat and gunia pig dorsal
lateral geniculate nucleus. J Neurosci 12: 278 –289, 1992.
NAIRN AC, HEMMINGS HC, AND GREENGARD P. Protein kinases in the brain.
Annu Rev Biochem 54: 931–976, 1985.
PERETZ A, ABITBOL I, SOBKO A, WU CF, AND ATTALI B. A Ca2⫹/calmodulindependent protein kinase modulates Drosophila photoreceptor K⫹ currents:
a role in shaping the photoreceptor potential. J Neurosci 18: 9153–9162,
1998.
PONGS O, KECSKEMETHY N, MULLER R, KRAH-JENTGENS I, BAUMANN A, KILTZ
HH, CANAL I, LLAMAZARES S, AND FERRUS A. Shaker encodes a family of
putative potassium channel proteins in the nerve system of Drosophila.
EMBO J 7: 1087–1096, 1988.
QUINN WG, HARRIS WA, AND BENZER S. Conditioned behavior in Drosophila
melanogaster. Proc Natl Acad Sci USA 71: 707–712, 1974.
RENGER JJ, YAO WD, SOKOLOWSKI MB, AND WU CF. Neuronal polymorphism
among natural alleles of a cGMP-dependent kinase gene, foraging, in
Drosophila. J Neurosci 19: RC28 (1– 8), 1999.
RUDY B. Diversity and ubiquity of K⫹ channels. Neuroscience 25: 729 –749,
1988.
SAITO M AND WU C-F. Expression of ion channels and mutational effects in
giant Drosophila neurons differentiated from cell division arrested embryonic neuroblasts. J Neurosci 11: 2135–2150, 1991.
SAITO M AND WU C-F. Ionic channels in cultured Drosophila neurons. In:
Comparative Molecular Neurobiology, edited by Pichon Y. Basel:
Birkhauer, 1993, p. 366 –389.
SALKOFF L AND WYMAN R. Genetic modification of potassium channels in
Drosophila Shaker mutants. Nature 293: 228 –230, 1981.
SCHWARZ TL, TEMPEL BL, PAPAZIAN DM, JAN Y-N, AND JAN LY. Multiple
potassium-channel components are produced by alternative splicing at the
Shaker locus in Drosophila. Nature 331: 137–142, 1988.
SIEGEL RW AND HALL J. Conditioned responses in courtship behavior of
normal and mutant Drosophila. Proc Natl Acad Sci USA 76: 3430 –3434,
1979.
SILVA AJ, PAYLOR R, WEHNER JM, AND TONEGAWA S. Deficient hippocampal
long-term potentiation in a calcium-calmodulin kinase II mutant mice.
Science 257: 201–206, 1992a.
SILVA AJ, STEVENS CF, TONEGAWA S, AND WANG Y. Impaired spatial learning in
␣-calcium-calmodulin kinase II mutant mice. Science 257: 206 –211, 1992b.
SINGH A AND SINGH S. Unmasking of a novel potassium current in Drosophila
by a mutation and drugs. J Neurosci 19: 6838 – 6843, 1999.
SPATZ H-C, EMANNS A, AND REICHERT H. Associative learning of Drosophila
melanogaster. Nature 248: 359 –361, 1974.
Downloaded from http://jn.physiology.org/ by 10.220.33.3 on August 3, 2017
BUTLER LS, SILVA AJ, ABELIOVICH A, WATANABE Y, TONEGAWA S, AND
MCNAMARA JO. Limbic epilepsy in transgenic mice carrying a Ca2⫹/
calmodulin-dependent kinase II alpha-subunit mutation. Proc Natl Acad Sci
USA 92: 6852– 6855, 1995.
BYERS D, DAVIS RL, AND KIGER JA JR. Defect in cyclic AMP phosphodiesterase due to the dunce mutation of learning in Drosophila melanogaster.
Nature 289: 79 – 81, 1981.
BYRNE JH AND KANDEL ER. Presynaptic facilitation revisited: state and time
dependence. J Neurosci 16: 425– 435, 1996.
CHEN C AND TONEGAWA S. Molecular genetic analysis of synaptic plasticity,
activity-dependent neural development, learning and memory in the mammalian brain. Annu Rev Neurosci 20: 157–184, 1997.
CLINE HT. Activity-dependent plasticity in the visual systems of frogs and fish.
Trends Neurosci 14: 104 –111, 1991.
DAVIS RL. Physiology and biochemistry of Drosophila learning mutants.
Physiol Rev 76: 299 –317, 1996.
DELGADO R, DAVIS R, BONO MR, LATORRE R, AND LABARCA P. Outward
currents in Drosophila larval neurons: dunce lacks a maintained outward
current component downregulated by cAMP. J Neurosci 18: 1399 –1407,
1998.
DELGADO R, HIDALGO P, DIAZ F, LATORRE R, AND LABARCA P. A cyclic
AMP-activated K⫹ channel in Drosophila larval muscle is persistently
activated in dunce. Proc Natl Acad Sci USA 88: 557–560, 1991.
DHALLAN RS, YAU KW, SCHRADER KA, AND REED RR. Primary structure and
functional expression of a cyclic nucleotide-activated channel from olfactory neurons. Nature 347: 184 –187, 1990.
DRAIN P, DUBIN AE, AND ALDRICH RW. Regulation of Shaker K⫹ channel
inactivation gating by the cAMP-dependent protein kinase. Neuron 12:
1097–1109, 1994.
DRAIN P, FOLKERS E, AND QUINN WG. cAMP-dependent protein kinase and the
disruption of learning in transgenic flies. Neuron 6: 71– 82, 1991.
DUDAI Y, JAN Y-N, BYERS D, QUINN WG, AND BENZER S. dunce, a mutant of
Drosophila deficient in learning. Proc Natl Acad Sci USA 73: 1686 –1688,
1976.
DUERR JS AND QUINN WG. Three Drosophila mutations that block associative
learning also affect habituation and sensitization. Proc Natl Acad Sci USA
79: 3646 –3650, 1982.
ENGEL JE AND WU C-F. Alteration of non-associative conditioning of an
identified escape circuit in Drosophila memory mutants. J Neurosci 16:
3486 –3499, 1996.
ENGEL JE AND WU C-F. Genetic dissection of functional contributions of
specific potassium channel subunits in habituation of an escape circuit in
Drosophila. J Neurosci 18: 2254 –2267, 1998.
GETTING PA. Emerging principles governing the operation of neural networks.
Annu Rev Neurosci 12: 185–204, 1989.
GOODWIN SF, DEL VECCHIO M, VELINZON K, HOGEL C, RUSSELL SRH, TULLY
T, AND KAISER K. Defective learning in mutants of the Drosophila gene for
a regulatory subunit of cAMP-dependent protein kinase. J Neurosci 17:
8817– 8827, 1997.
GRIFFITH LC, VERSELIS LM, AITKEN KM, KYRIACOU CP, DANHO W, AND
GREENSPAN RJ. Inhibition of calcium/calmodulin-dependent protein kinase
in Drosophila disrupts behavioral plasticity. Neuron 10: 501–509, 1993.
GRIFFITH LC, WANG J, ZHONG Y, WU C-F, AND GREENSPAN RJ. Calcium/
Calmodulin-dependent protein kinase II and K⫹ channel subunit Eag similarly affect plasticity in Drosophila. Proc Natl Acad Sci USA 91: 10044 –
10048, 1994.
HARRIS-WARRICK RM AND MARDER E. Modulation of neural networks for
behavior. Annu Rev Neurosci 14: 39 –57, 1991.
HEISENBERG M, BORST A, WAGNER S, AND BYERS D. Drosophila mushroom
body mutants are deficient in olfactory learning. J Neurogenet 2: 1–30,
1985.
HEISENBERG M AND WOLF R. Vision in Drosophila: Genetics of Microbehavior. Berlin: Springer-Verlag, 1984.
HILLE B. Ionic Channels of Excitable Membranes. Sunderland, MA: Sinauer,
1992.
HUANG YY, KANDEL ER, VARSHAVSKY L, BRANDON EP, QI M, IDZERDA RL,
MCKNIGHT GS, AND BOURTCHOULADZE R. A genetic test of the effects of
mutations in PKA on mossy fiber LTP and its relation to spatial and
contextual learning. Cell 83: 1211–1222, 1995.
HUBEL DH AND WIESEL TN. The period of susceptibility to the physiological
effects of unilateral eye closure in kittens. J Physiol (Lond) 206: 419 – 436,
1970.
1393
1394
W.-D. YAO AND C.-F. WU
WU C-F AND HAUGLAND FN. Voltage clamp analysis of membrane currents in
larval muscle fibers of Drosophila: alteration of potassium currents in
Shaker mutants. J Neurosci 5: 2626 –2640, 1985.
WU C-F, SAKAI K, SAITO M, AND HOTTA Y. Giant Drosophila neurons
differentiated from cytokinesis-arrested embryonic neuroblasts. J Neurobiol
21: 499 –507, 1990.
WUSTMANN G, REIN K, WOLF R, AND HEISENBERG M. A new paradigm for
operant conditioning of Drosophila melanogaster. J Comp Physiol [A] 179:
429 – 436, 1996.
YAO W-D AND WU C-F. Auxiliary Hyperkinetic beta subunit of K⫹ channels:
regulation of firing properties and K⫹ currents in Drosophila neurons.
J Neurophysiol 81: 2472–2484, 1999a.
YAO W-D AND WU C-F. Regulation of firing pattern through modulation
of non-Sh K⫹ currents by calcium/calmodulin-dependent protein kinase II
in Drosophila embryonic neurons. Ann NY Acad Sci 868: 450 – 453,
1999b.
ZHAO M-L, SABLE E, IVERSON L, AND WU C-F. Functional expression of
Shaker K⫹ channels in cultured Drosophila neurons derived from Sh cDNA
transformants: distinct properties, distribution, and turn over. J Neurosci 15:
1406 –1418, 1995.
ZHAO M-L AND WU C-F. Alterations in frequency coding and activity dependence of excitability in cultured neurons of Drosophila memory mutants.
J Neurosci 17: 2187–2199, 1997.
ZHONG Y, BUDNIK V, AND WU C-F. Synaptic plasticity in Drosophila memory
and hyperexcitable mutants: role of cAMP cascade. J Neurosci 12: 644 –
651, 1992.
ZHONG Y AND WU C-F. Altered synaptic plasticity in Drosophila memory mutants with a defective cyclic AMP cascade. Science 251: 198 –201,
1991.
ZHONG Y AND WU C-F. Differential modulation of potassium currents by
cAMP and its long-term and short-term effects: dunce and rutabaga mutants
of Drosophila. J Neurogenet 9: 15–27, 1993.
Downloaded from http://jn.physiology.org/ by 10.220.33.3 on August 3, 2017
SUMI M, KIUCHI K, ISHIKAWA T, ISHII A, HAGIWARA M, NAGATSU T, AND
HIDAKA H. The newly synthesized selective Ca2⫹/Calmodulin-dependent
protein kinase II inhibitor Kn-93 reduces dopamine contents in PC12h cells.
Biochem Biophys Res Commun 181: 968 –975, 1991.
TIMPE IC, JAN YN, AND JAN L. Four cDNA clones from the Sh locus of
Drosophila induce kinetically distinct A-type potassium currents in Xenopus
oocytes. Neuron 1: 659 – 667, 1988.
TOKUMITSU H, CHIJIWA T, HAGIWARA M, MIZUTANI A, TERASAWA M, AND
HIDAKA H. Kn-62, 1-[N, O-bis(5-isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4-phenylpiperazine, a specific inhibitor of Ca2⫹/calmodulin-dependent protein kinase II. J Biol Chem 265: 4315– 4320, 1990.
TSUNODA S AND SALKOFF L. Genetic analysis of Drosophila neurons: Shal,
Shaw and Shab encode most embryonic potassium currents. J Neurosci 15:
1741–1754, 1995a.
TSUNODA S AND SALKOFF L. The major delayed rectifier in both Drosophila
neurons and muscle is encoded by Shab. J Neurosci 15: 5209 –5221, 1995b.
TULLY T, BOLWIG G, CHRISTENSEN J, CONNOLLY J, DELVECCHIO M, DEZAZZO
J, DUBNAU J, JONES C, PINTO S, REGULSKI M, SVEDBERG B, AND VELINZON
K. A return to genetic dissection of memory in Drosophila. Cold Spring
Harb Symp Quant Biol 61: 207–218, 1996.
TURRIGIANO GG, ABBOTT LF, AND MARDER E. Activity changes the intrinsic
properties of cultured neurons. Science 264: 974 –976, 1994.
TURRIGIANO G, LEMASSON G, AND MARDER E. Selective regulation of current
densities underlies spontaneous changes in the activity of cultured neurons.
J Neurosci 15: 3640 –3652, 1995.
WANG J, RENGER JJ, GRIFFITH LC, GREENSPAN RJ, AND WU C-F. Concomitant
alterations of physiological and developmental plasticity in Drosophila CaM
kinase II-inhibited synapses. Neuron 13: 1373–1784, 1994.
WU C-F AND GANETZKY B. Neurogenetic studies of ion channels in Drosophila. In: Ion Channels, edited by Narahashi T. New York: Plenum, 1992, vol.
3, p. 261–314.