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J Neurophysiol
87: 2664 –2675, 2002; 10.1152/jn.00453.2001.
Developmental Changes in the Density of Ionic Currents in
Antennal-Lobe Neurons of the Sphinx Moth, Manduca sexta
ALISON R. MERCER1,2 AND JOHN G. HILDEBRAND2
Department of Zoology, University of Otago, Dunedin, New Zealand; and 2Arizona Research Laboratories,
Division of Neurobiology, University of Arizona, Tucson, Arizona 85721
1
Received 1 June 2001; accepted in final form 25 January 2002
metamorphosis proceeds they become larger in amplitude and
shorter in duration, and cell-type–specific differences in the
response characteristics of the cells begin to emerge. Changes
in whole cell current profiles that coincide with developmental
changes in spike waveform have been described, but specific
currents contributing to these changes have yet to be identified.
This study describes voltage-gated and Ca2⫹-dependent currents in developing Manduca AL neurons and compares their
properties early (stage 5) and late (stage 14) in metamorphosis.
Two distinct cell types are examined in vitro: Rick Rack (RR)
neurons and Proximal Branching (PB) neurons, identified elsewhere as putative local AL interneurons and projection (output) neurons, respectively (Oland and Hayashi 1993; Oland et
al. 1992). Pharmacological manipulations, together with voltage inactivation and subtraction protocols, have enabled us to
identify six distinct current types in these cells. Changes in the
density of Na⫹, Ca2⫹, and K⫹ currents during adult metamorphosis are described, and currents that are likely to contribute
to developmental changes in action potential waveform in
Manduca AL neurons have been identified. The results provide
an important starting point for investigations into the development of electrical excitability in Manduca AL neurons, and its
role in the development of primary olfactory centers in the
brain of the moth.
INTRODUCTION
METHODS
Around stage 3 of the 18 stages of metamorphic adult
development of the sphinx moth, Manduca sexta, the arrival of
antennal sensory receptor axons in the antennal lobes (ALs) of
the brain triggers the formation of subunits of synaptic neuropil, called glomeruli (reviewed by Oland et al. 1998; Rössler et
al. 2000). Local AL interneurons, projection (output) neurons,
and centrifugal neurons that project to the ALs from elsewhere
in the brain undergo extensive growth and restructuring as
glomeruli develop in a lateral-to-medial wave across the AL
neuropil (reviewed by Hildebrand et al. 1997; Oland and
Tolbert 1996). Recordings from AL neurons in vitro and in situ
in semi-intact brain preparations reveal dramatic changes in the
electrical properties and response characteristics of the cells
during this period (see accompanying paper). Action potentials
elicited from Manduca AL neurons at the onset of metamorphosis are small in amplitude and long in duration, but as
Address for reprint requests: A. R. Mercer, Dept. of Zoology, University of
Otago, Dunedin, New Zealand (E-mail: [email protected]).
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Manduca sexta (Lepidoptera: Sphingidae) were reared on an artificial diet (modified from Bell and Joachim 1976) and maintained at
25°C under a long-day photoperiod regimen (17 h light/7 h dark).
Larvae hatch from eggs and pass through five larval instars before
undergoing metamorphosis from larva to pupa to adult. Metamorphic
adult development in the moth can be divided into 18 stages, each of
which lasts approximately 1 day and is accompanied by well-defined
changes in pupal morphology (Sanes and Hildebrand 1976a,b; Tolbert
et al. 1983). AL neurons from pupae at stages 2–16 were examined in
this study, but the study focuses on a comparison of currents recorded
in cells at pupal stages 5 and 14. In vitro analysis is used to examine
two morphologically distinct cell types: RR neurons, the out growing
neurites of which have a striking zig-zag appearance in vitro (Mercer
et al. 1996a; Oland and Hayashi 1993), and PB neurons, which
possess distinctive clusters of arbors that arise from the primary
neurite close to the cell body (Hayashi and Hildebrand 1990; Mercer
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0022-3077/02 $5.00 Copyright © 2002 The American Physiological Society
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Mercer, Alison R. and John G. Hildebrand. Developmental
changes in the density of ionic currents in antennal-lobe neurons of
the sphinx moth, Manduca sexta. J Neurophysiol 87: 2664 –2675,
2002; 10.1152/jn.00453.2001. Early in metamorphic adult development, action potentials elicited from Manduca sexta antennal lobe
neurons are small in amplitude, long in duration, and calcium dependent. As development proceeds, the action potential waveform becomes larger in amplitude, shorter in duration, and increasingly sodium dependent. Whole cell voltage-clamp analysis of Manduca
antennal-lobe neurons in vitro has been used to identify voltageactivated currents that contribute to developmental changes in the
electrical excitability of these cells. Proximal Branching neurons
[putative projection (output) neurons] and Rick Rack neurons (putative local antennal-lobe interneurons) are examined in detail early
(pupal stage 5) and late (pupal stage 14) in adult metamorphosis. In
both cell types, four voltage-gated and two calcium-dependent ionic
currents have been identified. Cell-type–specific changes in the density of sodium, calcium, and potassium currents correlate temporally
with changes in cell excitability and spike waveform. Developmental
changes in ionic current profiles are accompanied also by the emergence of cell-type–specific response characteristics in the cells. Together with the accompanying paper, this study provides an important
foundation for examining the impact of developmental changes in
electrical excitability on the growth, electrical properties and connectivity of neurons in central olfactory pathways of the moth.
IONIC CURRENTS IN MOTH CENTRAL OLFACTORY NEURONS
Cell culture
Cells were dispersed and cultured according to methods described
by Hayashi and Hildebrand (1990). Briefly, brains were removed from
cold-anesthetized pupae and placed into sterile culture saline. ALs
were transferred into Hanks’ Ca2⫹- and Mg2⫹-free buffered salt
solution containing 0.5 mg/ml collagenase and 2 mg/ml dispase for 2
min at 37°C to dissociate the tissue, which was then dispersed by
trituration with a fire-polished Pasteur pipette. Enzyme treatment was
terminated by centrifugation, first through culture saline and then
through culture medium (for recipes, see accompanying paper). Dissociated cells were allowed to settle and adhere to the surface of
culture dishes coated with Concanavalin A (200 ␮g/ml) and laminin
(2 ␮g/ml). Cultures were placed in a humidified incubator at 26°C and
maintained for 5– 6 days. Only cells without contacts to other neurons
were selected for recording.
Electrophysiological recording
Whole cell voltage-clamp recording techniques (Hamill et al. 1981)
were used to examine voltage-activated currents in neurons from
pupae at stages 2–16 of the 18 stages of metamorphic adult development. Electrodes with resistances of 1–2 M⍀ were made from borosilicate glass (VWR Scientific, West Chester, CA) and filled with a
solution containing (in mM) 150 K-aspartate, 5 NaCl, 2 MgCl2, 1
CaCl2, 11 EGTA, and 10 HEPES, adjusted to pH 7 and 330 mOsm.
Cells were viewed under an Olympus IMT-2 inverted microscope
equipped with Hoffman modulation contrast optics. Prior to recording, culture medium was replaced with insect saline containing (in
mM) 100 NaCl, 4 KCl, 6 CaCl2, 5 D-glucose, and 10 HEPES (pH 7),
adjusted to 360 mOsm with mannitol to facilitate the formation of
high resistance (gigaohm) seals. Saline levels in the perfusion chamber were maintained as low as possible to reduce electrode capacitance transients and improve compensation. Junction potentials were
nullified prior to seal formation. To obtain whole cell recordings, light
suction and brief high-voltage pulses were used to rupture the cell
membrane beneath the recording electrode. Recordings were made
using an AxoPatch 1B amplifier (Axon Instruments), and data were
acquired and analyzed using pClamp6 software (Axon Instruments,
version 6.02) run on an i30486 microcomputer. Membrane currents
were sampled at intervals of 100 ␮s and were filtered at 2 kHz using
a low-pass 4-pole Bessel filter. Cell capacitance was determined from
the capacitative current elicited by a 10-mV depolarizing voltage step
following establishment of the whole cell clamp. The whole cell
J Neurophysiol • VOL
capacitative charge (calculated as the integral of the whole cell capacitative current transient) was divided by the applied voltage to
obtain cell capacitance. Membrane capacitance measurements of 86 ⫾
25 (SE) pF and 63 ⫾ 27 pF were recorded for RR neurons (n ⫽ 133)
and PB neurons (n ⫽ 89), respectively. No stage-related differences in
cell capacitance were observed. Linear leakage currents and residual
capacitance artifacts were subtracted using a P/4 protocol included in
the data acquisition software (Axon Instruments). All recordings were
performed at room temperature (20 –22°C).
Series resistance compensation was applied, where possible ⱕ80%.
However, large series resistance errors associated with the measurement of Na⫹ currents in mature neurons made it difficult to routinely
apply adequate compensation. The largest currents, which were recorded in stage 14 RR neurons, approached ⫺7.5 nA in some cells.
Electrode resistance was low (1–2 M⍀, see above), and series resistance was estimated to be ⬍4 M⍀, suggesting residual errors (with
70% compensation) of ⱕ9 mV in these cells. For this reason, Na⫹
currents were examined in detail only in immature neurons at pupal
stage 5, where residual errors were estimated to be 5 mV or less. For
all isolated currents other than INa, residual errors were estimated to be
⬍5 mV. Series resistance errors will result in underestimates of
developmental increases in current amplitudes and activation rates.
No correction has been made to plots showing current-voltage (I-V)
relations, or to estimates of time to peak current.
Current isolation
Components of the whole cell current profile were isolated using
routine pharmacological techniques and subtraction protocols described previously for Manduca neurons (Duch and Levine 2000;
Grünewald and Levine 1998; Hayashi and Hildebrand 1990; Hayashi
and Levine 1992; Kloppenburg et al. 1999a; Mercer et al. 1996b;
Zufall et al. 1991). Na⫹ currents were blocked with tetrodotoxin
(TTX, 10⫺8 M) and Ca2⫹ currents with 5 ⫻ 10⫺4 M CdCl2. K⫹
currents, collectively, were blocked by replacing KAsp in the electrode with CsCl. A-type K⫹ currents were blocked with 5 ⫻ 10⫺3 M
4-aminopyridine (4-AP), and 3 ⫻ 10⫺2 M tetraethylammonium chloride (TEA) was used to block non–A-type K⫹ currents. Ca2⫹-activated currents were blocked indirectly, by blocking Ca2⫹ currents
with Cd2⫹. In Na⫹-free solutions, NaCl was replaced with Tris-Cl. To
normalize for differences in cell size, peak current amplitudes (in pA)
were divided by cell capacitance (in pF). Measurements of activation
threshold, time-to-peak current, and maximum current density were
recorded for each cell. The significance of differences in the properties
of neurons at developmental stages 5 and 14 was tested by two-tailed
Student’s t-tests. For comparison of multiple group means, one-way
ANOVA was performed using Minitab 8.2 software (State College,
PA, 1991); post hoc comparisons were performed using Tukey tests.
A significance level of 0.05 was accepted for all tests. Data are
presented as means ⫾ SE.
RESULTS
Inward currents
To isolate Na⫹ currents in Manduca AL neurons, outward currents were blocked by substituting K⫹ in the
pipette solution with Cs⫹ and adding 3 ⫻ 10⫺2 M TEA and
5 ⫻ 10⫺3 M 4-AP to the extracellular solution. Ca2⫹ currents
were blocked with 5 ⫻ 10⫺4 M CdCl2. Large amplitude Na⫹
currents present in mature neurons made it difficult to obtain
adequate series resistance compensation. For this reason, Na⫹
currents were examined in detail only at pupal stage 5, which
falls within a narrow time window in which the percentage of
RR neurons exhibiting rapidly activating, transient Na⫹ current
(INa) increases significantly, but current amplitudes are still
NA⫹ CURRENTS.
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et al. 1996a; Oland and Hayashi 1993). PB neurons were derived from
the median group of AL neurons, a cluster of cells that contains
projection (output) neurons, but that lacks local neurons (Homberg et
al. 1989). RR neurons were derived from either, the lateral cell cluster,
or from dissociations of whole ALs. To avoid the possibility that small
differences in culture medium or developmental stage may affect the
results of comparisons between RR neurons and PB neurons, only
cells examined in parallel from the same sets of animals were used to
compare current densities in the two cell types.
Changes in the whole cell current profiles of these neurons during
adult metamorphosis have been described elsewhere (see accompanying paper). In RR neurons, these changes are represented by four
distinct types of whole cell current profile (types 1– 4), whereas for PB
neurons, three types of current profile have been described. Approximately 15% of the stage 5 RR neurons examined in this study
exhibited RR-type 1 whole cell current profiles, but the majority
exhibited profiles of types 2 and 3. All stage 14 RR neurons exhibited
RR-type 4 current profiles. In the case of PB neurons, stage 5 cells
displayed PB-type 1 or type 2 profiles, whereas only cells with
PB-type 3 profiles were included in the analysis of stage 14 PB
neurons. Descriptions of whole cell current profiles are provided in the
accompanying paper.
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Prior to the appearance of fast, transient Na⫹
currents, small amplitude action potentials could be elicited
from Manduca AL neurons. Preliminary evidence suggests that
these are Ca2⫹ spikes (see accompanying paper). To examine
Ca2⫹ current densities in the cells, Na⫹ currents were abolished with TTX (10⫺8 M), and K⫹ currents were blocked by
replacing KAsp in the electrode with CsCl, and adding 4-AP
(5 ⫻ 10⫺3 M) and TEA (3 ⫻ 10⫺2 M) to the external medium.
ICa was identified at all stages of development examined in this
study and, as shown elsewhere (Hayashi and Levine 1992;
CA2⫹ CURRENTS.
J Neurophysiol • VOL
Mercer et al. 1995), ICa could be abolished by the addition of
5 ⫻ 10⫺4 M CdCl2 to the external medium. Recordings from
RR neurons revealed marked differences in the amplitude of
Ca2⫹ currents early (Fig. 2A1) and late (Fig. 2A2) in metamorphosis.
I-V curves were compiled to compare the voltage dependence of ICa at early (stage 5, n ⫽ 8) and late (stage 14, n ⫽
10) stages of metamorphosis. RR neurons were used for this
purpose. ICa density was higher in stage 14 RR neurons than in
stage 5 neurons (Fig. 2B1), but normalizing of I-V plots to
maximum current amplitudes in each cell (I/Imax; Fig. 2B2)
indicated that there was no difference in the voltage dependence of ICa early and late in adult metamorphosis. In RR
neurons, and in PB neurons, ICa activation occurred around
⫺45 mV (see also Mercer et al. 1995). In stage 5 RR neurons,
ICa peaked 12.1 ⫾ 8.5 ms after onset of a voltage step from
⫺70 to ⫺10 mV, compared with 10.6 ⫾ 2.4 ms in pupal stage
14 RR neurons. ICa density in PB neurons and RR neurons
(Fig. 2C) was similar at stage 5, but by stage 14, the density of
Ca2⫹ currents in RR neurons was significantly higher than in
PB neurons, and significantly higher also than in RR neurons at
stage 5 (df 3, F ⫽ 3.9, P ⫽ 0.02).
To examine ICa inactivation, cells were held at a resting
potential of ⫺70 mV and stepped to a test potential of ⫺10 mV
following a conditioning prepulse (Fig. 2D1). Ca2⫹ currents
elicited after each prepulse were normalized to current levels
recorded after a conditioning prepulse of ⫺100 mV. In Fig.
2D2 this value is expressed as a function of the prepulse
potential. The mean of five normalized inactivation curves was
fitted with a Boltzmann distribution to derive V1/2 (⫺42.55
mV). ICa inactivation was examined only in stage 5 RR neurons.
Contribution of Ca2⫹ currents to spike waveform
RR neurons were used to examine in detail the contribution
of Ca2⫹ currents to spike waveform. Brief (20-ms) injections
of depolarizing current (0.2–2 nA) were applied to elicit single
action potentials from cells early (stage 3, Fig. 3A1 and stage
5, Fig. 3A2) and late (stage 10, Fig. 3A3 and stage 14, Fig. 3A4)
in metamorphic adult development. Cells represented in Fig. 3,
A1—A4, exhibited whole cell current profiles of types 1– 4,
respectively (see accompanying paper). Figure 3, B1—B4,
shows the impact on spike waveform of blocking Ca2⫹ currents with Cd2⫹. Early in metamorphosis, depolarizing current
pulses failed to elicit action potentials in some RR neurons
(Fig. 3A1). The prominent sag to more hyperpolarized potentials that occurred in these cells during the depolarizing current
pulse (arrow, Fig. 3A1) was partially blocked by Cd2⫹ (Fig.
3B1). Small-amplitude spikes characteristic of RR neurons
early in adult metamorphosis (Fig. 3A2) were also blocked by
Cd2⫹ (Fig. 3B2). However, at later stages of development,
action potentials remained intact in Cd2⫹ but showed significant changes in spike waveform (Fig. 3, B3 and B4). Cd2⫹
reduced spike amplitude, increased spike duration, and reduced
spike afterhyperpolarization (Fig. 3B3). Cd2⫹-induced reduction of spike amplitude was significantly greater in RR neurons
with type 3 current profiles (31.7 ⫾ 4.2% reduction, e.g., Fig.
3, A3 and B3) than in cells exhibiting type 4 profiles (9.5 ⫾
0.7% reduction, e.g., Fig. 3, A4 and B4; T ⫽ 8.87, P ⫽ 0.012),
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relatively small (Fig. 1, A1—A4). Cells in which no sodium
current could be detected (n ⫽ 7) were discarded. In 2 of the
remaining 11 stage 5 RR neurons, no fast, transient Na⫹
currents could be detected, but a small amplitude sustained
inward current was observed (Fig. 1A1). The characteristics of
this current have yet to be examined in detail and are not
considered further in this report. In all remaining cells (including PB neurons), INa activated at voltages between ⫺50 and
⫺45 mV. Stage 5 RR neurons used to examine more closely
the activation kinetics of this current were grouped initially
into those showing high (INa max) and low (INa min) density
Na⫹ currents. From these groupings, it was apparent that
comparisons would be improved by introducing a third category, INa mid, in which INa densities were intermediate between the two extremes (Fig. 1, A2—A4). Normalizing for cell
size did not alter the distribution of recordings between these
three categories. The large range of Na⫹ current densities
found in stage 5 RR neurons is illustrated in Fig. 1B1. If peak
current amplitudes at each voltage step are expressed as fractions of the maximum INa amplitude observed in each cell
(I/Imax, Fig. 1B2), the normalized I-V plots indicate that despite
rapid changes in INa density around pupal stage 5, the voltage
dependence of INa remains unchanged. In the three groups of
stage 5 RR neurons shown in Fig. 1B, mean times to reach peak
current recorded during a depolarizing voltage step from ⫺70
to ⫺10 mV were 1.9 ⫾ 0.5 ms (INa min, n ⫽ 3), 1.2 ⫾ 0.2 ms
(INa mid, n ⫽ 4) and 1.5 ⫾ 0.2 ms (INa max, n ⫽ 2). Although INa
appeared to activate more slowly in cells exhibiting low density currents (Fig. 1A2), differences between the 3 groups in
mean time to peak current were not statistically significant (df
8, F ⫽ 1.71; P ⫽ 0.26).
To examine INa inactivation, cells were held at a resting
potential of ⫺70 mV and stepped to a test potential of ⫺15 mV
following a conditioning prepulse (Fig. 1C1). Na⫹ currents
elicited after each prepulse were normalized to the Na⫹ current
evoked when no conditioning prepulse was applied. In Fig.
1C2, this value is expressed as a function of the prepulse
potential. No significant differences in steady-state inactivation
were identified between the three groups of stage 5 RR neurons
examined. The data were pooled and fitted with a Boltzmann
distribution to determine V1/2 (⫺50.34 mV).
Changes in Na⫹ current density between pupal stages 5 and
14 were more striking in RR neurons than in PB neurons.
Figure 1D compares peak current densities in these cells.
Significant differences were identified between the mean densities of INa in the four groups of cells examined (df 3, F ⫽ 4.1,
P ⫽ 0.03). While the mean density of INa in stage 14 RR
neurons was significantly higher than in RR neurons at pupal
stage 5, a similar trend in PB neurons was not statistically
significant.
IONIC CURRENTS IN MOTH CENTRAL OLFACTORY NEURONS
J Neurophysiol • VOL
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⫹
FIG. 1. A—C: Na currents in stage 5 Rick Rack (RR) neurons. Cells in which no inward current could be identified (n ⫽ 7)
were discarded. A: in 2 of 11 stage 5 RR neurons, no fast-activating, transient Na⫹ currents (INa) could be detected, but a small
amplitude, sustained inward current was apparent (A1). Those cells in which INa could be identified were divided into 3 groups (see
text); cells displaying low (INa min, A2), medium (INa mid, A3), and high (INa max, A4) density Na⫹ currents. B: properties of Na⫹
currents. B1: voltage dependence of INa. Peak current density plotted as a function of membrane potential in cells exhibiting low
(INa min, 䊐, n ⫽ 3), medium (INa mid, E, n ⫽ 4), and high (INa max, ‚, n ⫽ 2) density Na⫹ currents. Each point represents the mean
peak current density (⫾SE). B2: the same data as in B1 expressed as a percentage of the maximum peak current density in each
cell. Despite significant differences in current density, the voltage dependence of INa in these cells was similar. Series resistance
errors associated with measurements of the large amplitude Na⫹ currents in stage 14 RR neurons [and Proximal Branching (PB)
neurons] made it difficult to apply adequate compensation. For this reason, properties of this current were not examined in these
cells. C1: inactivation protocol. To examine INa inactivation, cells (n ⫽ 8) were held at a resting potential of ⫺70 mV and stepped
to a potential of ⫺15 mV following a conditioning prepulse, as shown. C2: Na⫹ currents elicited after each prepulse were
normalized to the Na⫹ current evoked when no conditioning prepulse was applied. This value is expressed here as a function of
the prepulse potential. As no significant differences in steady-state inactivation between cells exhibiting different INa densities were
identified, the data were combined and fitted with a Boltzmann distribution to determine V1/2 (⫺50.34 mV). D: comparisons of
mean peak current densities (⫾SE) in RR neurons and PB neurons early (stage 5, white bars) and late (stage 14, gray bars) in
metamorphosis. The significance of differences between groups is indicated by letters above the bars in each graph. Groups that
share a letter are not significantly different; groups that do not share a letter differ significantly (P ⬍ 0.05). The density of Na⫹
currents in stage 5 RR neurons was significantly lower than in RR neurons at pupal stage 14. A similar trend in PB neurons was
not statistically significant.
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suggesting that Ca2⫹ currents contribute progressively less to
spike amplitude as development proceeds. Ca2⫹ currents contribute to spike waveform in PB neurons also (Fig. 4A), but the
effects of Cd2⫹ in these cells were not examined in detail in
this study. That K⫹ currents in general contribute significantly
to repolarization of the action potential in PB neurons and RR
neurons is indicated by the effects of blocking outward currents
with 4-AP and TEA (e.g., Fig. 4B).
Outward currents
Previous studies have shown that Na⫹ currents, and Na⫹dependent action potentials, in Manduca AL neurons can be
blocked with TTX, or by perfusing cells in Na⫹-free medium
(Mercer et al. 1996b; see also accompanying paper). Interestingly, these treatments were found in the present study to have
a significant impact also on a rapidly activating, transient
outward current that was particularly prominent in RR neurons
(Fig. 5A1). As shown in Fig. 5, TTX not only abolished INa, but
also reduced the amplitude of this outward transient (Fig. 5A2).
The effects of TTX on INa and on the fast outward transient
were at least partially reversible (Fig. 5A3) and could be
mimicked by perfusing cells with Na⫹-free saline (Fig. 5B).
Neither of these treatments blocked Ca2⫹-independent K⫹
currents in Manduca AL neurons (IA and IKV) (Hayashi et al.
1992; Kloppenburg et al. 1999a; Mercer et al. 1996b; see
J Neurophysiol • VOL
following text). However, the complete abolition of total outward current by the specific K⫹ channel blockers, 4-AP and
TEA, together with internal CsCl (see METHODS), suggests that
the TTX-sensitive outward current identified in this study is
carried mostly by K⫹ ions. In RR neurons and PB neurons, the
current is also reduced significantly by the Ca2⫹ channel
blocker, Cd2⫹ (see following text).
CA2⫹-DEPENDENT K⫹ CURRENTS.
Changes in spike waveform
resulting from addition of Cd2⫹ to the external medium (Figs.
3 and 4) suggest that Ca2⫹-dependent outward currents contribute to membrane repolarization in Manduca AL neurons.
To examine Ca2⫹-dependent components of the outward current profile, whole cell current profiles recorded in the presence
of Cd2⫹ (Fig. 6, A2 and B2) were subtracted from those
recorded prior to Cd2⫹ treatment (Fig. 6, A1 and B1, respectively). Subtraction currents reveal the time course and amplitude of outward currents blocked by Cd2⫹ (Fig. 6, A3 and B3).
Cells examined in this way were analyzed under two different
sets of conditions: in the first group, Na⫹ currents were
blocked with TTX (10⫺8 M) prior to the application of Cd2⫹
(Fig. 6A), whereas in the second group, Na⫹ currents were left
intact (see Fig. 6B).
At the onset of metamorphosis (stages 2–3), the Cd2⫹sensitive component of the outward current profile was generally dominated by a small amplitude, sustained outward current
(IKCa sustained, Fig. 6D1), but in some cells a rapidly activating,
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2⫹
FIG. 2. Ca
currents recorded from RR
neurons. A: recordings from RR neurons early
(stage 5, A1) and late (stage 14, A2) in metamorphosis. B: properties of Ca2⫹ currents. B1:
voltage dependence of Ica in stage 5 (E, n ⫽ 8)
and stage 14 (●, n ⫽ 10) RR neurons. Peak
density is plotted as a function of membrane
potential. Each point represents the mean
(⫾SE) peak current density. B2: the same data
as in B1 expressed as a percentage of the maximum peak current amplitude in each cell suggests a similar voltage dependence. C: comparisons of peak current densities (⫾SE) in RR
neurons and PB neurons early (stage 5, white
bars) and late (stage 14, gray bars) in metamorphosis. The significance of differences between
groups is indicated by letters above the bars in
each graph. Groups that share a letter are not
significantly different; groups that do not share
a letter differ significantly (P ⬍ 0.05). Ca2⫹
current densities in stage 14 RR neurons were
significantly higher than those recorded at
stage 5. In contrast, there was no significant
difference in ICa densities of PB neurons at
stages 5 and 14. Ca2⫹ current densities in neurons examined at stage 14 were significantly
higher in RR neurons than in PB neurons. D1:
inactivation protocol. To examine ICa inactivation, cells were held at a resting potential of
⫺70 mV and stepped to a potential of ⫺10 mV
following a conditioning prepulse, as shown.
D2: Ca2⫹ current inactivation in stage 5 RR
neurons. Data were fitted with a Boltzmann
distribution to determine V1/2 (⫺42.55 mV).
IONIC CURRENTS IN MOTH CENTRAL OLFACTORY NEURONS
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FIG. 3. Responses of RR neurons to depolarizing current pulses show
effects on spike waveform of blocking Ca2⫹ currents with Cd2⫹. A1—A4:
recordings in current-clamp mode from RR neurons that exhibited whole cell
current profiles of types 1– 4, respectively (see accompanying paper), illustrate
characteristic changes in spike waveform associated with metamorphic adult
development. B1—B4: recordings from the same cells as in A1—A4, respectively, 2–3 min after the addition of 5 ⫻ 10⫺4 M Cd2⫹ to the medium bathing
the cells. Cell membrane potential was held at the level recorded prior to Cd2⫹
treatment. A1 and B1: recordings from a stage 3 RR neuron. Prior to the
appearance of spike activity (A1), Cd2⫹ blocks a large component of the sag
(arrow) apparent in responses elicited by depolarizing current pulses (B1),
indicating that Ca2⫹-activated outward currents contribute to the sag. A2 and
B2: recordings from a stage 5 RR neuron. Ca2⫹ spikes apparent in RR neurons
prior to the appearance of Na⫹-dependent action potentials (arrow, A2) were
blocked by Cd2⫹ (B2), but not affected by TTX (see accompanying paper). A3
and B3: recordings from a stage 8 RR neuron. During development, the action
potential waveform became larger in amplitude, shorter in duration, and
increasingly Na⫹ dependent. Blocking Ca2⫹ currents with Cd2⫹ increased
spike duration and reduced the size of the afterhyperpolarization (B3). Cd2⫹
also reduced spike amplitude. A4 and B4: recordings from a stage 14 RR
neuron. The results suggest that Ca2⫹ currents contribute significantly to spike
waveform late in metamorphosis. However, the magnitude of the effects on
spike amplitude of blocking Ca2⫹ currents with Cd2⫹ was greater in cells
midway through metamorphosis (e.g., see B3) than in stage 14 RR neurons
(e.g., see B4, see text).
transient component (IKCa transient) was also apparent. IKCa
tranisent was prominent in the subtraction currents of cells in
which Na⫹ currents remained intact (Fig. 6, B3, D2, and D3).
Blocking Na⫹ currents with TTX prior to Cd2⫹ treatment
significantly reduced the amplitude of this current (Fig. 6A3),
suggesting that IKCa transient and the TTX-sensitive outward
component described above (Fig. 5) are one and the same
current. This is supported by time to peak current estimates
J Neurophysiol • VOL
recorded in stage 14 RR neurons during depolarizing voltage
steps from ⫺70 to ⫹20 mV. The mean time to reach the peak
of the IKCa transient (4.7 ⫾ 0.3 ms, n ⫽ 4), measured from
subtraction currents similar to those shown in Fig. 6B3, was
⫹
FIG. 5. A and B: Na -dependent outward current. A: a series of depolarizing voltage steps from ⫺70 to ⫹50 mV shows the whole cell current profile of
a stage 14 PB neuron exhibiting a PB-type 3 current profile (see accompanying
paper). No Cd2⫹ was present in the medium bathing these cells. A1: prior to
applying 10⫺8 M TTX to the cell (Control), a fast, transient outward component was apparent in the outward current profile (arrow). A2: in the presence
of 10⫺8 M TTX this outward component was blocked (TTX). A3: the effects
of TTX were partly reversed when cells were washed in TTX-free saline
(Wash). B1: single voltage steps from ⫺70 to ⫹40 mV are used to illustrate the
effects of perfusing cells in Na⫹-free saline. Recordings from a stage 14 RR
neuron in normal saline (gray arrow) and in Na⫹-free saline (arrow, Na⫹-free)
are superimposed to show that in Na⫹-free saline both, the fast transient inward
current, INa, and a fast, transient outward current disappear. B2: subtraction of
the current profile recorded in Na⫹-free saline from the current profile recorded
in normal saline reveals the 2 currents that are abolished in Na⫹-free saline.
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FIG. 4. Responses of PB neurons to brief (20-ms) depolarizing current
pulses (approximately 0.5 nA) show effects on spike waveform of (A) blocking
Ca2⫹ currents with Cd2⫹, and (B) blocking K⫹ currents with tetraethylammonium (TEA) and 4-aminopyridine (4-AP). A: recording in current-clamp mode
from a PB neuron that exhibited a PB-type 2 whole cell current profile (see
accompanying paper). Responses obtained before (Control) and after (⫹Cd2⫹)
the addition of 5 ⫻ 10⫺4 M Cd2⫹ are superimposed to show spike broadening
induced by Cd2⫹. B: effects on spike waveform of blocking outward currents
with 4-AP (5 ⫻ 10⫺3 M) and TEA (3 ⫻ 10⫺2 M). In the presence of these
specific K⫹ channel blockers, repolarization of the membrane (arrow) is
slowed dramatically.
2670
A. R. MERCER AND J. G. HILDEBRAND
similar to that of the Na⫹-dependent outward transient (4.8 ⫾
0.21 ms, n ⫽ 3), measured using whole cell current profiles
similar to those shown in Fig. 5B. The presence or absence of
this rapidly activating outward transient is strongly cell-type
specific.
Detailed analysis of the transient and sustained components
of IKCa was not possible, as neither current could be examined
in isolation. Plotting IKCa density against command voltage,
however, indicates that IKCa activation occurs at voltages
around ⫺10 mV (Fig. 6C). Significant differences in IKCa
density were apparent between the four groups of cells examined (IKCa transient, df 3, F ⫽ 4.5, P ⫽ 0.03; IKCa sustained, df 3,
F ⫽ 3.92, P ⫽ 0.04). In RR neurons, both the transient
component (Fig. 6E) and the sustained current (Fig. 6F) exhibited significantly higher densities at stage 14 than at pupal
stage 5, but this was not the case in PB neurons. Differences in
J Neurophysiol • VOL
the density of Ca2⫹-dependent K⫹ currents in PB neurons at
stages 5 and 14 were not statistically significant.
Ca2⫹-independent K⫹ currents are likely to contribute also to membrane repolarization in
Manduca AL neurons (Kloppenburg et al. 1999a; Mercer et al.
1996b). In cells used to examine developmental changes in the
density of Ca2⫹-independent K⫹ currents, TTX (10⫺8 M) was
applied to block Na⫹ currents and Ca2⫹ currents were blocked
with 5 ⫻ 10⫺4 M CdCl2. Replacement of Ca2⫹ ions with Ba2⫹,
and removal of Ca2⫹ from the solution used to fill the recording electrodes have been used previously to confirm the Ca2⫹
independence of the K⫹ currents examined below (Mercer et
al. 1996b). Two distinct types of current contribute to the
Ca2⫹-independent K⫹ currents in Manduca AL neurons: rapidly activating, transient A-type current (IA) of which two
forms have been described, IA fast and IA slow, and a sustained
CA2⫹-INDEPENDENT K⫹ CURRENTS.
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2⫹
⫹
FIG. 6. Ca -dependent K currents (IKCa).
Whole cell current profiles of stage 14 RR neurons recorded in 10⫺8 M TTX (A) and in normal saline (B). A1 and B1: recordings made
prior to Cd2⫹ treatment. A2 and B2: effects of
applying 5 ⫻ 10⫺4 M Cd2⫹. A3 and B3: subtraction of current profiles recorded in the presence of Cd2⫹ (A2 and B2) from those recorded
prior to Cd2⫹ treatment (A1 and B1, respectively) reveal the amplitude and time course of
currents blocked by Cd2⫹. Cd2⫹-sensitive currents in cells not exposed to TTX (B3) displayed both, a fast transient outward component
(IKCa transient, arrow) and a sustained outward
component (IKCa sustained). In cells treated with
TTX (A3), the transient component was greatly
reduced in amplitude. C: peak current density
plotted as a function of membrane potential.
Na⫹ currents in these cells were blocked with
TTX. D: comparison of IKCa profiles recorded
(in the absence of TTX) from a stage 2 RR
neuron (D1), a stage 14 RR neuron (D2), and a
stage 5 PB neuron (D3). E and F: comparisons
of mean current densities (⫾SE) in RR neurons
and PB neurons early (stage 5, white bars) and
late (stage 14, gray bars) in metamorphosis.
Voltage steps from ⫺70 to ⫹40 mV were used
to examine the density of IKCa transient (E) and
IKCa sustained (F). The significance of differences
between groups is indicated by letters above the
bars in each graph. Groups that share a letter are
not significantly different; groups that do not
share a letter differ significantly (P ⬍ 0.05). E:
the density of IKCa transient was calculated from
Cd2⫹ subtraction currents recorded in TTX-free
saline (e.g., B3). A significant difference between the density of IKCa transient in RR neurons
at stages 5 and 14 was not apparent in PB
neurons. At stage 14 there was no significant
difference between RR neurons and PB neurons
in the density of IKCa transient. F: the density of
IKCa sustained was calculated from Cd2⫹ subtraction currents recorded in the presence of TTX
(e.g., A3). To minimize contamination of the
sustained current with IKCa transient, measurements of IKCa sustained were taken from the end
of the 100-ms depolarizing voltage step. IKCa
sustained density in RR neurons was significantly
higher at stage 5 than at 14. This was not the
case for PB neurons. Differences in IKCa sustained
density between RR neurons and PB neurons
were not significant.
IONIC CURRENTS IN MOTH CENTRAL OLFACTORY NEURONS
2671
component (IKV) that inactivates slowly, if at all, during a
200-ms depolarizing voltage step (Hayashi et al. 1992; Kloppenburg et al. 1999a; Mercer et al. 1996b).
For measurement of IA, 3 ⫻ 10⫺2 M
TEA was added to the saline bathing the cells to block the
sustained current, IKV. IA fast was identified in all (n ⫽ 5) of the
stage 5 RR neurons examined (Fig. 7A1), but in only three of
eight RR neurons at developmental stage 14. IA slow alone was
detected in the remaining stage 14 RR neurons (Fig. 7A2). IA
fast differed from IA slow in the following ways: first, peak
current density was consistently higher for IA fast than for IA
slow (Fig. 7B); second, I-V plots used to examine the voltage
dependence of IA (Fig. 7B) indicate that the threshold for
activation of IA fast occurs at a more hyperpolarized potential
(around ⫺45 mV) than for IA slow (around ⫺25 mV); and third,
between 0 and ⫹70 mV, time-to-peak was significantly faster
for IA fast than for IA slow. With a depolarizing voltage step from
⫺70 to ⫹20 mV, mean time to peak current was 8.0 ⫾ 1.3 ms
for IA fast and 15.9 ⫾ 1.7 ms for IA slow (T ⫽ 3.75, P ⫽ 0.005).
As the two A-type currents are difficult to separate, measurements comparing A-type current densities in RR neurons and
PB neurons (Fig. 7C) did not discriminate between these two
subtypes. ANOVA revealed significant differences between
the four groups of cells tested (df 3, F ⫽ 6.81, P ⫽ 0.005).
While the mean density of IA in stage 5 RR neurons appeared
lower than in stage 14 RR neurons, differences between these
two groups were not statistically significant. In contrast, the
mean density of IA in stage 14 PB neurons was significantly
higher than in stage 5 PB neurons (Fig. 7C). This is consistent
with the observation that, in marked contrast to RR neurons, IA
fast remained as prevalent in PB neurons at stage 14 as it was
at stage 5.
SUSTAINED K⫹ CURRENT. To measure IKV (Fig. 8A), A-type
K⫹ currents were blocked with 5 ⫻ 10⫺3 M 4-AP, or alternatively, measurements were taken from a holding potential of
⫺20 mV, where IA has been shown to be almost completely
inactivated (Kloppenburg et al. 1999a). I-V plots of data taken
from RR neurons indicate that there was no difference in the
voltage dependence of IKV between cells at stages 5 and 14 of
metamorphic adult development (Fig. 8B). IKV reached peak
current levels in 44.5 ⫾ 11 ms (stage 5 RR neurons) and
37.2 ⫾ 8.6 ms (stage 14 RR neurons) after the onset of
depolarizing voltage steps from ⫺70 to ⫹20 mV (T ⫽ 0.52;
P ⫽ 0.64). IKV densities in RR neurons and PB neurons were
similar (Fig. 8C), and neither cell type exhibited significant
differences in IKV density between pupal stages 5 and 14 (df 3,
F ⫽ 0.44, P ⫽ 0.73).
TRANSIENT K⫹ CURRENT.
The principal aim of this study was to identify ionic currents
that contribute to changes in the electrical excitability of
Manduca AL neurons during metamorphic adult development
of the moth (see accompanying paper). The results show significant changes in the density of inward and outward ionic
currents in Manduca AL neurons that correlate temporally with
developmental changes in spike waveform and cell excitability. It is unlikely that we have isolated all voltage-gated currents present in these neurons, but the study provides important
first steps toward understanding the functional development
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DISCUSSION
2⫹
⫹
FIG. 7. Ca -independent K current, IA. Previous analyses have revealed
2 distinct forms of A-type current in Manduca AL neurons, IA fast and IA slow
(Mercer et al. 1996b). In the present study, IA fast (A1) was prominent in all
stage 5 RR neurons (n ⫽ 5), whereas IA slow only (A2) was observed in 6 of 8
stage 14 RR neurons. B1: voltage dependence of IA fast (Œ) and IA slow (‚). Peak
current density is plotted as a function of membrane potential. Activation of IA
fast occurs around ⫺45 mV. In contrast, IA slow activation occurs at a voltage
closer to ⫺25 mV. C: comparisons of mean current densities (⫾SE) in RR
neurons and PB neurons early (stage 5, white bars) and late (stage 14, gray
bars) in metamorphosis. Voltage steps from ⫺70 to ⫹40 mV were used. The
significance of differences between groups is indicated by letters above the
bars in each graph. Groups that share a letter are not significantly different;
groups that do not share a letter differ significantly (P ⬍ 0.05). In PB neurons,
A-type current densities at stage 14 were significantly higher than at stage 5,
but this was not the case for RR neurons. Interestingly, at stage 14, IA densities
were significantly higher in PB neurons than in RR neurons.
87 • JUNE 2002 •
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2672
A. R. MERCER AND J. G. HILDEBRAND
Cell-type specific differences in the appearance of INa
2⫹
⫹
FIG. 8. A Ca -independent K current, IKV. Recording from a stage 5 RR
neuron. B: voltage dependence of IKV in RR neurons at stage 5 (E) and stage
14 (●). Peak current density is plotted as a function of membrane potential.
Activation of IKV occurs around ⫺15 mV. C: comparisons of mean current
densities (⫾SE) in RR neurons and PB neurons early (stage 5, white bars) and
late (stage 14, gray bars) in metamorphosis using voltage steps from ⫺70 to
⫹40 mV. The significance of differences between groups is indicated by letters
above the bars in each graph. Groups that share a letter are not significantly
different; groups that do not share a letter differ significantly (P ⬍ 0.05). There
was no significant difference between the density of IKV in cells from stage 14
pupae and stage 5 pupae, either in PB neurons or in RR neurons.
and membrane biophysics of cells in central olfactory pathways of the moth.
Action potentials in Manduca AL neurons are initially
Ca2⫹ dependent
Early in metamorphosis, action potentials in Manduca AL
neurons are Ca2⫹ dependent, but they become increasingly
Na⫹ dependent during the course of adult metamorphosis.
Developmental changes in the ionic dependence of action
J Neurophysiol • VOL
The inward and outward currents identified in Manduca AL
neurons (Hayashi and Hildebrand 1990; Kloppenburg et al.
1999a; Mercer et al. 1995, 1996b; present investigation) are
similar to those described in other insect neurons (e.g., Baines
and Bate 1998; Baker and Salkoff 1990; Byerly and Leung
1988; Grünewald and Levine 1998; Hayashi and Levine 1992;
Kloppenburg et al. 1999b; Laurent 1991; O’Dowd and Aldrich
1988; Saito and Wu 1991; Schäfer et al. 1994; Solc and
Aldrich 1988). Voltage-gated currents appear in developing
central neurons in a predictable sequence, the order of which
varies between different types of neurons (Baines and Bate
1998; O’Dowd et al. 1988; Ribera and Spitzer 1990; Spitzer
1994). In Manduca AL neurons, A-type currents can be detected prior to the appearance of fast transient Na⫹ currents in
the cells, whereas in central neurons of the Drosophila embryo
the opposite is reported (Baines and Bate 1998). Interestingly,
blockade of synaptic transmission in Drosophila embryonic
motor neurons results in significant increases in voltage-gated
Na⫹ (and K⫹) currents in the cells (Baines et al. 2001). That
the expression of voltage-gated currents can be influenced by
local cues is suggested also by results showing the effects of
the steroid hormone 20-hydroxyecdysone (20-HE) on Ca2⫹
currents in Manduca motor neurons. Grünewald and Levine
(1998) have shown that pupal leg motor neurons exposed in
vitro to 20-HE exhibit significantly larger Ca2⫹ current levels
than untreated cells. Effects of 20-HE on the density of cur-
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potentials have been described in many neuronal systems (e.g.,
Baccaglini and Spitzer 1977; Blair 1983; Mori-Okamoto et al.
1983; Willard 1980). While events that trigger such changes
have yet to be fully elucidated, there is increasing evidence that
early forms of electrical excitability have a developmental
function (reviewed by Spitzer 1991, 1994). Manduca AL neurons exhibit spontaneous activity in vitro. This activity is
particularly prevalent prior to and during the transition from
Ca2⫹- to Na⫹-dependent spikes (see accompanying paper), and
recent studies show that the cells exhibit distinctive plateau
properties during this period (Mercer et al. 2000). The shift to
Na⫹-dependent action potentials is not accompanied by reductions in Ca2⫹ current density. On the contrary, in RR neurons
the density of Ca2⫹ currents was significantly higher at stage
14 than at pupal stage 5. In mature neurons it is likely that
Ca2⫹ spikes are masked by the large K⫹ currents present in
these cells.
Ca2⫹ spikes have been identified also in the Manduca motor
neuron, MN5, and may play a role in the postembryonic
dendritic remodelling of this cell (Duch and Levine 2000).
Duch and Levine (2000) show that the transient occurrence of
Ca2⫹ spikes correlates temporally with cessation of the formation of higher-order branches in MN5. In embryonic spinal
neurons it has been shown also that the frequency of growth
cone Ca2⫹ transients is inversely proportional to the rate of
axon outgrowth (Gomez and Spitzer 1999; Lautermilch and
Spitzer 2000). The role of Ca2⫹ spikes in Manduca AL neurons has yet to be determined, but it is interesting to note in this
regard that the neuromodulator 5-HT not only promotes neurite
outgrowth of immature RR neurons in vitro (Mercer et al.
1996a), but also significantly reduces Ca2⫹ currents in these
cells (Mercer et al. 1995).
IONIC CURRENTS IN MOTH CENTRAL OLFACTORY NEURONS
Development of outward currents
The dramatic effects on action potential duration of reducing
outward currents with K⫹ channel blockers indicate that K⫹
currents contribute significantly to action potential repolarization in Manduca AL neurons. In addition to shaping action
potential waveforms, K⫹ currents play an important role in
setting resting membrane potentials, and in modulating the
frequency of neuronal firing (reviewed by Salkoff et al. 1992).
Increases in the density of K⫹ currents in AL neurons during
adult metamorphosis are consistent with K⫹ currents contributing not only to decreases in action potential duration, but also
to changes in cell excitability during this period. There was no
evidence in these neurons of developmental shifts in the I-V
curves of outward currents, suggesting that action potential
waveforms between pupal stages 5 and 14 are not affected by
this parameter. Moreover, no significant changes in times to
peak current were identified.
Complex changes in K⫹ current profiles
Changes in K⫹ current densities that coincide with adult
metamorphosis are complex and cell-type specific. In RR neurons, the percentage of cells exhibiting rapidly activating,
transient A-type current (IA fast) fell as development progressed, but the density of Ca2⫹-dependent K⫹ currents in
these cells increased. In contrast, analysis of K⫹ current densities in PB neurons at pupal stages 5 and 14 revealed significant increases in the density of IA. The persistence of IA fast in
PB neurons correlates well with the identification of large
amplitude currents of this type in projection neurons of the
adult moth (Kloppenburg et al. 1999a). K⫹ currents in stage 14
PB neurons were similar in amplitude to those seen in adult
projection neurons, suggesting that by pupal stage 14 the K⫹
current profile of projection neurons may be close to being
J Neurophysiol • VOL
fully mature. The contribution that each of the K⫹ currents
identified in this study makes to developmental changes in cell
excitability has yet to be determined. However, a lack of
change in peak IKV current densities between pupal stages 5
and 14, suggests that in RR neurons and PB neurons, IKV does
not contribute significantly to changes in spike waveform during this period. This is in marked contrast to Xenopus spinal
neurons, where delayed rectifier K⫹ current plays a major role
in the developmental shortening of the action potential (reviewed by Spitzer 1994; Spitzer and Ribera 1998). However,
effects on spike waveform of blocking Ca2⫹ currents in AL
neurons with Cd2⫹, and large increases in the density of
Ca2⫹-dependent currents apparent in RR neurons, suggest that
Ca2⫹-dependent K⫹ currents contribute significantly to repolarization of the action potential in mature AL neurons of the
moth. Both sustained and transient Ca2⫹-dependent K⫹ currents have been described previously in insect neurons (Schäfer
et al. 1994; Thomas 1984; Torkkeli and French 1995; Wegener
et al. 1992; Zufall et al. 1991), and evidence suggests that
Ca2⫹-dependent K⫹ currents contribute to repolarization of the
action potential both in vertebrate and invertebrate neurons
(Adams et al. 1982; Gao and Ziskind-Conhaim 1998; MacDermott and Weight 1982; Storm 1987; Walsh and Byrne 1989).
In Manduca AL neurons, removal of Na⫹ ions from the external medium, or blocking Na⫹ influx with TTX, reduces the
transient component, while leaving IKCa sustained intact.
Na⫹-dependent outward current in Manduca AL neurons
K⫹ channels activated by Na⫹ have been reported in many
types of neuronal membranes (Bischoff et al. 1998; Dryer
1991, 1994; Egan et al. 1992a,b). Transient, Na⫹-activated K⫹
current has been identified also in dorsal unpaired medium
(DUM) neurons of the cockroach, Periplaneta americana
(Grolleau and Lapied 1994), and a large sustained Na⫹- and
voltage-dependent K⫹ current has been reported in spinal
neurons of the frog embryo (Dale 1993). The functions of
Na⫹-activated K⫹ channels are not well understood. KNa channels in vertebrate neurons appear not to contribute to action
potential waveform (Safronov and Vogel 1996), but their activation in rat motoneurons during repetitive firing of action
potentials leads to pronounced slow hyperpolarization of the
cells. Transient Na⫹-activated K⫹ current identified in insect
DUM neurons can be elicited in Ca2⫹-free medium or in
solutions containing CoCl2 (Grolleau and Lapied 1994), but in
Manduca AL neurons the Na⫹-dependent outward current is
blocked by Cd2⫹ (see earlier), suggesting that IKNa and the
transient component of IKCa in these cells may be one and the
same current. A better understanding of the macroscopic K⫹
currents identified in Manduca AL neurons awaits analysis at
a single-channel level.
The diversity of ionic currents in Manduca AL neurons, and
the possibility of cell-type–specific differences in their developmental regulation, is revealed for the first time in this study.
The functional significance of developmental changes in electrical excitability is the subject of intense interest and investigation. Manduca AL neurons provide an experimentally tractable system to explore the impact of such changes on the
growth, electrical properties, and connectivity of developing
central neurons.
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rents in developing Manduca antennal-lobe neurons have yet to
be determined.
INa could not be detected in all stage 5 RR neurons. This is
consistent with the finding (see accompanying paper) that a
lower percentage of RR neurons exhibit Na⫹-dependent action
potentials at early stages of metamorphosis than PB neurons.
The earlier development of voltage-dependent ionic currents in
PB neurons may explain why differences in the density of ionic
currents (present investigation), and changes in spike waveform and cell excitability (accompanying paper), between cells
at pupal stages 5 and 14 were less pronounced in PB neurons
than in RR neurons. Developmental changes in the morphology of projection (output) neurons, such as PB neurons, also
occur slightly ahead of changes in local AL interneurons.
Dendritic processes of projection neurons invade developing
glomeruli before the processes of multiglomerular local AL
interneurons (Malun et al. 1994; Oland et al. 1990). Increases
in Na⫹ current density during adult metamorphosis are consistent with observed increases in spike amplitude during this
period (see accompanying paper). However, the absence of
differences in the voltage dependence or activation kinetics of
Na⫹ currents in RR neurons exhibiting different Na⫹ current
densities, suggests that these parameters do not contribute
significantly to changes in spike waveform in these cells at the
stages of metamorphosis examined in this study.
2673
2674
A. R. MERCER AND J. G. HILDEBRAND
We thank C. Turner and K. Miller for generous technical support, Drs. Peter
Kloppenburg, Richard Levine, and Lynne Oland for many helpful discussions
throughout the course of this work, and A. A. Osman for rearing of Manduca.
This work was supported by National Institutes of Health Grants NS-28485
and AI-23253 to J. G. Hildebrand and by University of Otago Grants MFZ B22
and B12 to A. R. Mercer. The contents of this paper are solely the responsibility of the authors and do not necessarily represent the official views of the
National Institutes of Health.
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