Institut für Neurobiologie, Freie Universität Berlin,
D-14195 Berlin, Germany
 |
INTRODUCTION |
Voltage-gated K+ currents of the A type are defined by
their relatively fast activation and inactivation and by their
sensitivity to 4-aminopyridine (4-AP). Since their first description in
the somata of Anisodoris neurons (Connor and Stevens
1971a
,b
) A currents were identified in a large variety of
systems (Adams and Galvan 1986
; Barry and
Nerbonne 1996
). They have been shown to influence major aspects
of electrical activity such as spike broadening during repetitive
firing (Ma and Koester 1995
, 1996
), firing frequency (Byrne 1980a
,b
; Tierney and Harris-Warrick
1992
), or synaptic transmission (Kaang et al.
1992
). Mutations affecting A currents lead to malfunctions of
the nervous system, as described for the shaker phenotype in
Drosophila (Salkoff and Wyman 1981
;
Tanouye et al. 1981
).
In arthropods, the
-subunits of channels underlying A currents are
encoded by the shaker or the shal gene. The
Drosophila shaker gene encodes a subfamily of K+
channel components that are produced by alternative splicing (Kamb et al. 1988
; Papazian et al. 1987
;
Pongs et al. 1988
; Schwarz et al. 1988
).
Most of these components give rise to fast-inactivating, voltage-gated
K+ currents (Iverson and Rudy 1990
;
Iverson et al. 1988
; Timpe et al. 1988
;
Wu et al. 1983
). Expression of the shal gene
may also yield a fast-inactivating, voltage-gated K+
current (Pak et al. 1991
; Tsunoda and Salkoff
1995
).
Immunocytochemical localization of the shaker gene products
in the brain of adult Drosophila revealed a nonuniform
distribution and indicated a high expression in the mushroom bodies
(MBs) (Rogero et al. 1997
; Schwarz et al.
1990
). The MBs are involved in higher functions of the insect
brain such as learning and memory, e.g., Drosophila
(Davis 1993
; de Belle and Heisenberg
1994
; Heisenberg et al. 1985
) and honeybee
(Erber et al. 1980
; Menzel et al.1974
). Each MB of the worker honeybee consists of ~170,000
(Witthöft 1967
) densely packed and parallel
arranged, intrinsic Kenyon cells. Kenyon cells are the third-order
interneurons of the olfactory pathway that converge on the MBs with
other sensory and modulatory pathways crucial for olfactory learning
(Hammer 1993
; Hammer and Menzel 1995
).
The MB Kenyon cells can be taken into primary cell culture
(Kreissl and Bicker 1992
). Thus native ionic currents can be studied in a well-defined type of neurons in the insect brain.
Descriptions of native neuronal shaker currents in insects are rare.
Neuronal shaker currents were first identified in a small subpopulation
of neurons that were dissociated from thoracic ganglia of pupal
Drosophila (Baker and Salkoff 1990
). A
detailed kinetic description of a native neuronal shaker current exists
for the photoreceptors of Drosophila (Hardie
1991
). A number of voltage-dependent ionic currents,
Na+, Ca2+, and K+ currents, was
identified in the Kenyon cells (Schäfer et al. 1994
). It was suggested that a prominent A-type K+
current might be a shaker-like current. Therefore we investigated the
kinetic properties of this A current in detail to enable a comprehensive comparison with shaker and shal currents described in
other systems. Our data on the kinetic properties of the A current of
honeybee Kenyon cells indicate that it is dominated by a shaker-like current.
Data on the A current was incorporated into a Hodgkin-and-Huxley-style
mathematical model that also contained the voltage-gated Na+ current and delayed rectifier K+ current
from the honeybee Kenyon cells. The aim of the model was to investigate
the role of the A current during action potential generation and its
interaction with other currents involved. A rapidly activating
K+ current may repolarize the action potential. However,
depending on its voltage-operating range and the resting potential of
the cell, an A current may also cause a delay in the initiation of an
action potential. The model presented predicts that the A current reduces the peak amplitude of the action potential and mediates the
repolarization phase.
 |
METHODS |
Animals and cell preparation
Pupae of the honeybee, Apis mellifera, were collected
from the hive between days 4 and 6 of the pupal development, which
lasts 9 days under natural conditions. For dissection and culturing of
Kenyon cells the original protocol of Kreissl and Bicker (1992)
was
modified. Brains were removed from the head capsule in Leibowitz L15
medium (GIBCO-Bethesda Research Laboratory) supplemented with sucrose,
glucose, fructose, and proline (42.0, 4.0, 2.5, and 3.3 g/l; 500 mosm;
pH 7.2). The MBs were dissected out of the brains and incubated in
calcium-free saline containing (in mM) 130 NaCl, 5 KCl, 10 MgCl2, 25 glucose, 180 sucrose, and 10 HEPES, 500 mosm, pH
6.7 for 10 min. After transferring the MBs back to L15 medium (2 MBs/100 µl) the cells were dispersed by gentle trituration with a
100-µl siliconized Eppendorf pipette. Cells were then plated in 10 µl medium on poly-L-lysine-coated plastic dishes
(Falcon) and allowed to adhere to the substrate for 20 min. Thereafter the dish was filled with 2.5 ml of bee medium; 775 ml of this medium
consisted of 100 ml FCS (inactivated, Sigma), 10 ml yeast extract
(Sigma), 9.7 g L-15 (GIBCO), 2.66 g glucose, 1.67 g
fructose, 2.19 g proline, 25 g sucrose, and 0.5 g PIPES,
500 mosm, pH 6.7. The dishes were kept at 27°C in an incubator at
high humidity. Under these conditions the Kenyon cells started to grow
processes and survived for
2 wk. Cells were used for recordings
between 2 and 6 days in culture. Only large Kenyon cells with a soma
diameter of ~10 µm and with clearly visible processes were examined
but not small Kenyon cells with a diameter of ~6 µm or without
processes. The culture also contained a few glia cells, but they were
easily recognized because of their large size.
Electrophysiological techniques
Tight-seal whole cell recordings were performed after the
methods described by Hamill et al. (1981)
. All measurements were performed at room temperature. Recordings were made with an Axopatch 200 A amplifier (Axon Instruments). For pulse generation, data acquisition, and data analyses a TL-1 interface in conjunction with
pCLAMP software version 6.0 (Axon Instruments) was used. Pipette and
membrane capacitance were compensated, and series resistance
compensation (80%) was routinely employed. Signals were low-pass
filtered with a four-pole Bessel filter at 2 or 5 kHz and digitally
sampled at 5-50 kHz depending on the pulse protocol used. Liquid
junction potential was corrected, and on-line leakage currents were
compensated when necessary. Electrodes were pulled from borosilicate
glass capillaries (GC 150-15, Clark, Reading) and had resistances
between 3 and 5 M
in standard external saline. We used ORIGIN 4.1 (MicroCal) and IGOR pro 3.0 (WaveMetrics) to analyze the data. All data
are presented as means ± SE.
Solutions
The recording chamber was continuously perfused with saline. The
standard external saline consisted of (in mM) 130 NaCl, 6 KCl, 4 MgCl2, 5 CaCl2, 10 HEPES/NaOH, 25 glucose, and
160 sucrose, 500 mosm, pH 6.7. In addition during recording the
external saline contained 200 µM quinidine, 50 µM cadmium chloride,
and 100 nM TTX. In a few experiments the external solution contained
variable concentrations of K+ (2, 6, or 10 mM); in some
experiments 4-AP or agitoxin-2 (Alomone Labs) was added.
The pipettes were backfilled with a solution containing (in mM) 20 KCl,
115 K-gluconate, 40 KF, 3 Na2ATP, 3 MgCl2, 10 HEPES/Bis-Tris, 120 sucrose, 5 K-bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic
acid, 3 glutathione, and 0.1 GTP-Mg, 500 mosm, pH 6.7. All chemicals were purchased from Sigma unless otherwise stated.
Simulations
The equations originally developed by Hodgkin and Huxley (1952)
were modified to derive a set of exponential functions describing the
kinetics of the A current (see APPENDIX). These functions
were implemented with IGOR pro 3.0, with a least-squares fitting
algorithm. The degrees of freedom of the fits were reduced by using
fixed steady-state values, which were derived from the steady-state activation and steady-state inactivation. To determine the voltage dependency of the kinetic parameters, the various time constants were
fitted with a Boltzmann equation (see APPENDIX).
Simulations were run on a SunSPARC workstation with the simulation
software package SNNAP (Ziv et al. 1994
). The model that
was used for simulations under voltage-clamp conditions included only
the A current. For the simulation of action potentials the
voltage-gated Na+ current, the delayed rectifier
K+ current of the honeybee Kenyon cells, and a small
leakage current were added to the model. Data from Schäfer et al.
(1994)
were the basis for the data of the Na+ current and
the delayed rectifier current. The total number of recordings available
was increased by additional recordings. The parameters necessary for
the model were obtained from the original current traces by
reevaluation similar to the evaluation described for the A current. We
present these parameters in the APPENDIX because the
Na+ current and the delayed rectifier current were not in
the focus of this study, but they are necessary to document the model.
 |
RESULTS |
Isolation of the A current
Kenyon cells of the honeybee express a variety of voltage-gated
and calcium-activated ionic currents. To isolate the A current, the
Na+ current was blocked by 100 nm TTX, the Ca2+
current by 50 µM cadmium, and the delayed rectifier K+
current by adding 200 µM quinidine to the external solution. Under
these conditions a significant part of the delayed rectifier K+ current remains unblocked. Therefore a subtraction
procedure was used in addition to separate the delayed rectifier
K+ current from the A current. For this in a first pulse
protocol the command potential was preceded by a
125-mV prepulse of
3-s duration to completely remove inactivation of the A current (Fig. 1A). In a second pulse
protocol the prepulse contained a voltage step to
5 mV (120 ms) to
inactivate the A current and a after brief voltage step to
125 mV (26 ms) to deactivate the delayed rectifier K+ current (Fig.
1B). This last step is too short to allow for pronounced recovery from inactivation of the A current. Subtraction of the current
traces recorded with these two pulse protocols yielded the pure A
current (Fig. 1C). Under these conditions, depolarizing voltage commands of
35 mV and greater activated a transient outward current in all Kenyon cells recorded. It shows a fast voltage-dependent time course of activation and inactivation. A summary of the derived time constants and steady-state parameters of this A current is given
in Table 1.

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Fig. 1.
Isolation of the A current of the Kenyon cells was achieved by a
combination of pharmacological blockers and suitable pulse protocols.
By adding 100 nM TTX, 50 µM cadmium, and 200 µM quinidine to the
external solution the voltage-gated Na+ current, the
Ca2+ current and and the delayed rectifier K+
current were blocked. A: after a hyperpolarizing
prepulse to 125 mV for 3 s, the test potential is stepped to
voltages from 55 to +45 mV with increments of 20 mV. The resulting
outward current consists of the A-type current and the unblocked part
of the delayed rectifier K+ currents. B:
subsequently in a second pulse protocol the prepulse was interrupted by
a voltage step to 5 mV for 120 ms to inactivate the A current. The
resulting outward current consists of a relatively small part of the A
current that is not inactivated and the delayed rectifier
K+ current. C: subtracting the current
traces elicited by the second pulse protocol from the current traces
evoked by the first protocol (A and B)
yielded the pure A current.
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Approximately 50% of the A current was blocked by 5 mM 4-AP
(n = 14, Fig. 2).
Agitoxin-2 did not affect the native A current of the Kenyon cells at
concentrations of
100 nM (n = 6, data not shown).

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Fig. 2.
4-aminopyridine (5 mM) does not completely block the A current. The
current traces shown here were elicited by depolarizing voltage steps
to +45, +25, +5, and 15 mV with the pulse protocol described in the
text.
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Activation
To counter any effects the activation of the persistent delayed
rectifier K+ current might have on the traces of the A
current, we used the subtraction protocol described previously to
isolate the pure A current. An advantage of the subtraction procedure
is the elimination of capacitive transients at the beginning of the
test potential that might partially obscure the current activation
phase. The resulting data could be fitted best with an exponentially
rising function with the power of three. Functions with powers of four or powers of two did not yield an equally good fit of the experimental data. The time constant of activation (
m) is voltage
dependent (Fig. 3). A command potential
of +45 mV induced a current with an activation time constant of
0.4 ± 0.1 ms (n = 4) and a time-to-peak of
1.39 ± 0.6 ms (n = 8; data not shown).

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Fig. 3.
Time constants of activation and deactivation of the A current. At
voltages more positive than 35 mV the time constants are derived from
fitting the activation of the A current (see Fig. 1C)
with an exponential function of the form given in A4. The exponential
value n was 3. At more negative voltages deactivation is
the predominant process. Deactivation time constants were determined by
single exponential fits to tail currents (see Fig. 4). Small crosses
represent the determined time constants, large crosses represent the
mean values for a given potential, dotted lines connect these mean
values, and solid line depicts the Boltzmann fit to the mean values
used in the simulations.
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Deactivation
To determine its deactivation, the A current was activated to
~75% by a brief voltage step to +45 mV (1.2 ms), after which the
cell was stepped to various deactivating test potentials between
105
mV and
45 mV (20 ms). The A current was isolated by a subtraction procedure that comprised the same prepulses as described previously. Inward tail currents were recorded in the range of
105 to
75 mV,
and outward tail currents were recorded in the range of
65 to
45 mV
(Fig. 4). The decay of the tail current
was fitted with a single exponential function. The time constant of
deactivation is voltage dependent (Fig. 3); at a test potential of
75
mV its value is 0.39 ± 0.1 ms (n = 5).

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Fig. 4.
To measure the time constant of deactivation we applied an activating
voltage pulse followed by a hyperpolarization. The A current was
activated by a brief voltage step to +45 mV (1.2 ms). The duration of
the depolarizing pulse was chosen to let the current activate to
~75%. Then the cell was stepped to various potentials ( 105 to 45
mV). Inward tail currents were recorded in the range of 105 to 75
mV; outward tail currents were recorded in the range of 65 to 45
mV. The component of the delayed rectifier K+ current was
eliminated by a subtraction procedure with suitable prepulses.
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Steady-state activation
Steady-state activation of the isolated A current (see Fig. 1) was
determined by measuring the peak current amplitude activated by
depolarizing voltage steps ranging from
55 to +45 mV. From these
values the relative conductance
(g/gmax) was calculated. For each
cell this normalized curve of the steady-state activation (Fig.
5A) was fitted separately with
a Boltzmann function, and the potential at which one-half of the
current is activated (V1/2) was determined. The
V1/2 values range from +11.9 to
12.1 mV; the
mean is
0.7 ± 2.9 mV (n = 8). The value for the
factor S, which determines the slope of the curve, is
16.1 ± 0.9. In addition, the curve of the mean
conductance-voltage relationship of all cells was fitted with the
Boltzmann function (Fig. 5B).

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Fig. 5.
A: steady-state activation and the respective
inactivation curves of 8 individual cells. The steady-state activation
curves represent the normalized peak conductances calculated from the
isolated A current as described in the text. For the steady-state
inactivation the normalized peak current was measured at a test pulse
potential of +45 mV after the different preconditioning voltage
commands indicated on the x-axis. For the elimination of
the delayed rectifier K+ current, the value of the
noninactivating component at the end of the test pulse was substracted
from every point of the current trace. B: mean values
and the SE of the steady-state activation and inactivation of the
different cells were fitted with a Boltzmann function.
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Inactivation
The inactivation time constant was determined by fitting a single
exponential function to the falling phase of the isolated A current
(see Fig. 1C). This yielded time constants of the
inactivation process that were strongly voltage dependent (Fig.
6). A command potential of +45 mV induced
a current with an inactivation time constant of 3.0 ± 1.6 ms
(n = 9). We also applied a series of double-exponential
fits. However, except for the current traces just above the activation
threshold where the signal-to-noise ratio is very small and possible
minor contaminations with other conductances would be relatively large,
we never observed significant improvements in the quality of the fits.

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Fig. 6.
Time constants of inactivation and recovery from inactivation. The
values in the voltage range between 125 and 60 mV represent time
constants of recovery from inactivation; values in the voltage range
between 35 and +45 mV represent those of inactivation. The time
constants of the inactivation are the same in A and
B. They were calculated from experimental data by
fitting a single exponential function to the falling phase of the
current in response to depolarizing pulses (see Fig. 1). The time
constants of recovery from inactivation were extracted from
double-pulse experiments (see Fig. 7). Small crosses represent the time
constants, large crosses represent their mean values for a given
potential, dotted line connects these mean values, and solid line
depicts the Boltzmann fit used in the simulations.
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Recovery from inactivation
To investigate the kinetics of the recovery from inactivation we
determined the time constants by application of double-pulse experiments (Fig. 7). The hyperpolarizing
(
125 mV) prepulse before the onset of the double-pulse protocol
lasted 5 s. In these experiments the unblocked part of the delayed
rectifier K+ current becomes apparent in the
noninactivating current at the end of the depolarizing pulses. This
current was measured at the end of the first depolarizing pulse and was
substracted from each point of the current trace. This method slightly
overestimates the delayed rectifier current. The ratio of the peak
current amplitudes elicited by the second and the first depolarizing
pulse indicates the extent of recovery at the given time interval. An
asymptotic level of 100% of recovery from inactivation is reached
within 3 s at an interpulse potential of
125 mV, whereas at
interpulse potentials between
110 and
60 mV asymptotic levels of
steady-state recovery of <100% were measured.

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Fig. 7.
The resulting current traces elicited by the double-pulse protocol used
to measure recovery from inactivation. The first pulse leads to
complete activation and inactivation of the current. The percentage of
channels that recovers from inactivation before the second pulse
depends on the time elapsing (25-3,000 ms) as well as on the
interpulse potential ( 125 to 60 mV). The recordings shown were
conducted with an interpulse potential of 125 mV. For the 2 depolarizing pulses the potential was stepped to +45 mV. The unblocked
part of the delayed rectifier K+ currents (measured at the
end of the first depolarizing pulse) was eliminated by subtraction
before the ratio of recovery was calculated.
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Because the time course of recovery from inactivation cannot adequately
be fitted by a single exponential function, we used a
double-exponential function yielding two time constants indicative of
the contribution of a slow and a fast process. The ratio of the fast
and the slow recovery from inactivation varied only slightly over the
range of interpulse potentials, with ~30 and 70% respectively. Both
time constants were voltage dependent (Fig. 6). At an interpulse potential of
75 mV the fast time constant is 18 ± 3.1 ms, and the slow time constant is 745 ± 107 ms (n = 5).
Steady-state inactivation
Steady-state inactivation curves were obtained by measuring the
peak currents in response to a test pulse (+45 mV) that was preceded by
preconditioning voltage steps to various potentials between
125 and
5 mV (Fig. 8). These preconditioning
voltage steps were preceded by a hyperpolarizing prepulse of
125 mV
and 3-s duration. We cannot use the subtraction protocol to eliminate the unblocked part of the delayed rectifier K+ currents.
Therefore the amplitude of the noninactivating current at the end of
the test pulse was substracted from the current trace. This method
introduces a slight error because of an overestimation of the delayed
rectifier current. The peak currents were normalized (I/Imax), and the resulting
current-voltage relationship of the steady-state inactivation was
determined (Fig. 5). The curves of each cell were fitted separately
with the Boltzmann function. The measured V1/2
values range between
66.7 and
45.9 mV; the means are
54.7 ± 2.4 mV for V1/2 and 7.0 ± 0.2 for
S.

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Fig. 8.
Current traces elicited by a pulse protocol used to determine
steady-state inactivation. A hyperpolarizing prepulse ( 125 mV; 3 s) was used followed by a conditioning pulse to various command
potentials and by a test pulse to +45 mV (60 ms).
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Effects of external K+ ions
The K+ concentration of the external solution commonly
used in our experiments was 6 mM. To investigate the effects of
external K+ ions on the A current, external solutions with
K+ concentrations of 2, 6, and 10 mM were used. The
respective K+ equilibrium potentials were determined
according to the Nernst equation, assuming the K+
concentration within the cell to be identical to that of the pipette
filling solution. From current traces evoked with a simple activation
pulse protocol (a
125-mV prepulse preceding the test potential from
55 to +45 mV) the conductances under the various K+
concentrations were calculated (Fig.
9A). The conductance is increased by increased K+ concentrations. Scaling the
current traces obtained at the various K+ concentrations to
the same size reveals that the shape of the traces is not affected
(n = 9) (Fig. 9B). This shows that there are
no K+-dependent changes in the kinetics of A current
activation and inactivation. The voltage dependency of steady-state
activation and inactivation was also unaffected by the external
K+ ion concentration (n = 6, data not
shown).

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Fig. 9.
A: from current traces obtained under different
K+ concentrations of the external solution (2, 6, and 10 mM) with the pulse protocol described in Fig. 1A
conductance traces were calculated. The traces in response to test
pulses of +45, +25, +5, or 15 mV are shown. An increase in the
external K+ concentration leads to an increased
conductance. B: current traces in A were
scaled to the same size. The shape of the current traces is not
affected by the external K+ concentration, which indicates
that the time constants of activation and inactivation are not
affected.
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We further tested the influence of extracellular K+
concentration on the recovery from inactivation. A plot of the
normalized peak current (% recovery) versus the interpulse interval
(holding potential
125 mV) shows that a reduced extracellular
K+ concentration slows the recovery from inactivation (Fig.
10A). This is due to an
increment of the slow time constant (Fig. 10B). Its value
increases with a decreasing concentration of external K+
ions from 326 ± 38 ms (10 mM K+) to 539 ± 49 ms
(6 mM K+) to 660 ± 27 ms (2 mM K+)
(n = 6). The fast time constant is not affected.

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Fig. 10.
A: currents were measured by applying the double-pulse
protocol with an interpulse potential of 125 mV. The percentage of
recovery from inactivation at 2, 6, and 10 mM external K+
was determined as described in the text. Increasing external
K+ concentration accelerates the recovery from
inactivation. This can be attributed to an increase in the slow time
constant. B: effect of the external potassium
concentration on the slow time constant of recovery from inactivation
(n = 6).
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When repetitive pulses (1 Hz, 20 ms, +45 mV, interpulse potential
85
mV) were applied, the A current cumulatively inactivated because of its
slow recovery from inactivation. Because the slow time constant of
recovery from inactivation is affected by the concentration of external
K+ ions, repetitive pulses result in different degrees of
cumulative inactivation depending on the concentration; the lower the
K+ concentration the stronger is the reduction of the peak
current (n = 7) (Fig.
11). The current traces recorded at a
given K+ concentration were normalized to the peak current
of the first pulse, so the measurements are independent of the shift of
the electromotive force because of the varying external K+
concentrations.

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Fig. 11.
Repetitive depolarizing pulses (1 Hz, test potential +45 mV, 20-ms
duration, interpulse potential 85 mV) led to cumulative inactivation.
The peak current was determined for each pulse normalized to the peak
current of the first pulse. After 20 pulses the cell was held at a
potential of 85 mV for ~30 s to allow recovery from inactivation,
and then another set of 20 pulses was applied. The peak currents evoked
by the 2 sets of pulses are similar. Therefore reduction of the peak
current in the course of repetitive depolarizing pulses is not due to
so-called run down but to cumulative inactivation. The degree of
cumulative inactivation depends on the external K+
concentration.
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Simulations
For the simulation of the A current in a voltage-clamp situation
the same voltage steps as in the physiological experiments (a
125-mV
prepulse preceding the test potential from
55 to +45 mV) were
applied. Comparison of a recorded whole cell A current with simulated
traces of the A current shows reasonable matching (Fig.
12). This confirms the validity of the
parameters determined by Hodgkin-Huxley-derived equations.

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Fig. 12.
Whole cell A current (  ) and mathematical simulation of the
experimental data (- - - ) under voltage-clamp conditions. The
kinetic parameters used for the simulation were extracted separately
from individual cells. The simulation is based on the mean values of
these parameters and was compared with the averaged current traces from
the patch-clamp experiments. The matching reconfirms the validity of
the kinetic parameters determined.
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For the simulation of action potentials we used a model that contained
the A current, a voltage-gated Na+ current, a delayed
rectifier K+ current, and a leakage current. Before each
simulated experiment the cell model was clamped to a holding potential
of
70 mV to let it reach a steady state. After termination of the
voltage clamp the cell model reached a stable resting potential of
63.2 mV within 21 s in the free running mode (data not shown).
On current injection (60 pA) a single action potential was generated
(Fig. 13, top). Current injections of <60 pA lead to
subthreshold activation of the
Na+ current. Injecting currents of >60 pA did not trigger
additional action potentials.

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Fig. 13.
Adding a voltage-gated Na+ current and a delayed rectifier
K+ current of the Kenyon cells (Schäfer et al.
1994 ) and a leakage current to the simulation enables us to
test how these currents interact in the generation of the action
potential. Without current injection the model produced a stable
resting potential. Top: current injection triggered a
single action potential. Middle: in the course of the
action potential the A current reaches its peak 200 µs later than the
Na+ current. K+ flux carried by the A current
overlaps the diminishing Na+ current.
Bottom: Na+ conductance and the conductance
of the A current reach their peaks at the same time as the respective
currents. Peak conductance of the Na+ current is twice as
large as the conductance of the A current.
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The peak of the action potential reached +9.5 mV, which is clearly
below the Na+ equilibrium potential. During the action
potential the A current reaches its maximum value just 200 µs later
than the Na+ current; the time frames of these two currents
show marked overlap (Fig. 13, middle). The same holds true
for the conductivity of these currents (Fig. 13, bottom).
The afterhyperpolarization of the action potential reached
74.2 mV at
its maximum and lasted for ~150 ms.
 |
DISCUSSION |
In honeybees it is possible to prepare a pure culture of the
intrinsic MB neurons, the Kenyon cells. The A current of these Kenyon
cells shows a pronounced voltage dependence of the inactivation time
course, which is a distinct property of shaker currents studied in
heterologous expression systems (Timpe et al. 1988
;
Wei et al. 1990
; Wittka et al. 1991
),
transgene giant neurons (Zhao et al. 1995
), myotubes
(Solc et al. 1987
), and photoreceptor cells (Hardie 1991
; Hevers and Hardie 1995
). By
contrast the inactivation kinetics of native shal currents
(Tsunoda and Salkoff 1995
) as well as shal currents
studied in heterologous expression systems (Pak et al.
1991
) are relatively voltage independent.
The expression of the shaker gene in the CNS of Drosophila
was indicated by means of in situ hybridization (Pongs et al.
1988
) and of immunocytochemistry (Rogero et al.
1997
; Schwarz et al. 1990
). Rogero et al. (1997)
detected shaker channels in the neuropil but not in the somata of MBs,
and it appears that in most systems shal channels are underlying the
somatic current (Baro et al. 1997
;
Maletic-Savatic et al. 1995
; Seridio et al.
1994
; Sheng et al. 1992
; Song et al.
1998
; Tsunoda and Salkoff 1995
). Therefore we
must take into account that we are recording from a mixture of somatic
shal channels and of shaker channels that were incorporated into the
space-clamped compartments under cell culture conditions. Nevertheless,
the A current of the Kenyon cells appears to be dominated by a single
type of channel. Evidences for this derive from the following
observations. 1) Fitting the inactivation kinetics with a
double-exponential function instead of a single exponential function
did not improve the quality of the fit. 2) Elevating extracellular K+ concentrations increased the conductance
of the A current. If the A current comprised two components, increasing
the conductance of only one current component would result in an
altered shape of the normalized trace. However, this was not observed.
The A current of Kenyon cells is modulated by extracellular
K+ ions. The whole cell conductance is increased,
cumulative inactivation is decreased, and recovery from inactivation is
accelerated at higher external K+ concentration. Fitting
the time course of recovery from inactivation requires a function with
two exponentials, which indicates two processes that may represent the
recovery from N- and C-type inactivation as described for shaker
currents (Iverson and Rudy 1990
; Iverson et al.
1988
). Only the time constant of the slow process is affected by increasing the extracellular K+ concentration.
This kind of modulatory action of K+ is described for
several genetically identified shaker and shaker-like currents for
different concentration ranges: 2-500 mM (Demo and Yellen
1991
), 1 µM to 20 mM (Pardo et al. 1992
),
2-40 mM (Tseng and Tseng-Crank 1992
), 2-10 mM
(Baukrowitz and Yellen 1995
), and 5-150 mM (Levy
and Deutsch 1996
). In all these systems increasing
K+ concentrations accelerates recovery from inactivation.
By contrast Jerng and Covarrubias (1997)
described a retardation of
recovery from inactivation in shal-like mKv4.1 K+ channels
in mice in the concentration range of 5-98 mM. The effect of elevating
extracellular K+ on invertebrate shal channels was not yet examined.
Agitoxin-2 is a selective and highly potent blocker of shaker and
shaker-like currents in heterologous expression systems (e.g.,
Ki = 0.64 nM for shaker B) (Garcia et al.
1994
) but did not affect the A current of honeybee Kenyon
cells. This may be due to different pharmacological properties of
shaker currents in homologous and heterologous expression systems.
Zagotta et al. (1989)
reported that the expression of shaker B cDNA in
Xenopus oocytes gave rise to a current that was sensitive to
50 µM charybdotoxin, whereas its expression in myotubes resulted in
an charybdotoxin-insensitive current. Agitoxin-2 as well as
charybdotoxin binds to the outer vestibule of the channel
(Durell and Guy 1996
).
Schäfer et al. (1994)
described that the A current was almost
completely blocked by 5 mM 4-AP and showed half-maximal steady-state activation at 10.7 mV and half-maximal steady-state inactivation at
42.33 mV. By contrast we find a 50% block of the A current by 5 mM
4-AP, half-maximal steady-state activation at
0.7 mV, and
half-maximal steady-state inactivation at
54.7 mV. This contradiction is probably due to the different methods used. 1) We
improved the composition of the culture media and used the more
physiological pH of 6.7 instead of 7.2 in the culture media and during
recording. 2) We allowed the neurons more time to develop in
the culture dish. 3) We selected for cells that had grown
clearly visible processes. Therefore we assume that we are recording an
A current that differs in its underlying channels from the A current
described by Schäfer et al. (1994)
. The A current described in
our study is more likely to reflect the axonal or neuritic A current of the Kenyon cells, whereas in the previous study the A current more
likely reflects a somatic current.
We observed some variations among different cells in the kinetic
parameters of the Kenyon cells A current. This may be due to
differences of the time spent in culture or different Kenyon cell
types. Although Kenyon cells share a common gross morphology, they may
differ with respect to dendritic and axonal morphology, sensory input,
and the distribution of transmitters within the MBs (Menzel et
al. 1974
; Mobbs 1982
). Yang et al. (1995)
used the enhancer trap technique to distinguish subpopulations of Kenyon cells. However, it is not known whether different subpopulations of
Kenyon cells with different voltage-gated currents exist. Preliminary data do not allow to group Kenyon cells according to single biophysical properties, e.g., half-maximal inactivation of the A current. Rather,
there seems to be a continuum of biophysical properties of this
current. Moreover, these properties are likely to undergo modulation in
a cell type that is involved in learning and memory. Nevertheless, the
A current of the Kenyon cells is relatively homogenous with respect to
its fast activation and inactivation (in each recording adequately
fitted by a single exponential function), its recovery from
inactivation (in each recording adequately fitted by a
double-exponential function), and its modulation by extracellular K+.
As described previously, Kenyon cells do not fall into distinct groups
with respect to the properties of the A current. Therefore multiple
simulations based on single experiments would simply reproduce the
bandwith of the variation among cells. Instead, we present a reduced
model of the average Kenyon cell, which has the advantage to reduce
errors caused by noise. The reduced model of Kenyon cells is capable of
producing single action potentials. The peak of the action potential
does not reach the Na+ equilibrium potential in the
simulation as well as in soma recordings from cultured Kenyon cells
(Kreissl 1992
). The simulation shows that the
fast-activating A current counteracts the depolarization caused by the
Na+ current during the rising phase of the action
potential. The A current is mainly responsible for the repolarization
because the delayed rectifier current does not contribute markedly to this process because of its slow activation.
The model does not produce a train of action potentials, most likely
because the afterhyperpolarization does not reach sufficiently negative
values to allow for fast deinactivation of the Na+ current.
This is due to the fast inactivation of the A current. In current-clamp
recordings from cultured Kenyon cells the afterhyperpolarization is
more pronounced, and some of the cells generate trains of action potentials in vitro (Kreissl 1992
) and in vivo
(Hammer and Menzel 1995
). This difference is probably
due to the presence of voltage-sensitive Ca2+ currents and
Ca2+-activated K+ currents
(Schäfer et al. 1994
) in these cells. These
currents are usually very slow with respect to the Na+
current (Hille 1992
). By contrast the A current under
investigation is just slightly slower than the Na+ current.
Therefore voltage-sensitive Ca2+ currents and
Ca2+-activated K+ currents should play only a
minor role during a single action potential but become more important
in a train of action potentials. There are not enough data available
from Schäfer et al. (1994)
to incorporate the Ca2+
current and the Ca2+-dependent K+ current into
the model. The experiments necessary to model these currents in their
full complexity would be beyond the scope of this study.
There is some evidence that shaker currents are involved in the process
of olfactory learning in Drosophila (Davis
1996
). Cowan and Siegel (1986)
reported a shaker mutant line
that showed deficiencies in an olfactory learning paradigm. Shaker
K+ channels are found at high levels in the MBs of
Drosophila (Rogero et al. 1997
;
Schwarz et al. 1990
), a neuropile that is supposed to
play an essential role in olfactory learning in insects (de Belle and Heisenberg 1994
; Heisenberg et
al.1985
; Menzel et al. 1974
). The molecular
basis of native shaker currents differs from that derived from
heterologous expression systems with respect to the heteromultimeric
composition, which may include multiple splice variants of the shaker
gene (Isacoff et al. 1990
; McCormack et al.
1990
), products from the eag gene (Zhong and Wu
1991
), and auxiliary cytosolic
-subunits (Chouinard
et al. 1995
; Wang and Wu 1996
). Because all
these various subunits could be subject to modulation, e.g.,
phosphorylation, the A current of the honeybee Kenyon cells is a highly
interesting preparation to investigate modulatory processes potentially
underlying olfactory learning.
The model of the Kenyon cell's currents was constructed with
equations that follow the Hodgkin-Huxley model of voltage dependence of
activation and inactivation and their kinetics. We did not use
equations for the rate of forward or backward reactions. The A current,
the voltage-gated Na+ current, and the delayed rectifier
K+ current were modeled with the following equation
A similar function was chosen to describe the time constant-voltage
relationship
The exponential relaxation of the current caused by the inactivation
process was described by a single exponential function
We thank Dr. Bernd Grünewald for many fruitful discussions
and comments on the manuscript and M. Ganz for expert cell culturing support.
Address for reprint requests: C. Pelz, Institut für
Neurobiologie, Freie Universität Berlin, Königin-Luise-Str.
28-30, D-14195 Berlin, Germany.
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.