Differential expression of voltage-sensitive K+ and Ca2+ currents in neurons of the honeybee olfactory pathway
Institut für Biologie, Neurobiologie, Freie Universität Berlin, Königin-Luise-Strasse 28/30, D-14195 Berlin, Germany
e-mail: gruenewa{at}neurobiologie.fu-berlin.de
Accepted 26 September 2002
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Summary |
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Key words: patch clamp, mushroom body, antennal lobe, insect, calcium-dependent K+ current, honeybee, Apis mellifera, neuron, olfactory
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Introduction |
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The ionic currents of postsynaptic Kenyon cells, by contrast, have been
analysed in vitro. They express functional nicotinic acetylcholine
receptors, which may mediate fast synaptic transmission between antennal lobe
projection neurons and mushroom body Kenyon cells
(Goldberg et al., 1999;
Deglise et al., 2002
). Several
voltage-sensitive ionic currents have been described
(Schäfer et al., 1994
;
Pelz et al., 1999
). Among the
three different voltage-sensitive K+ currents is a transient A-type
K+ current, which resembles the shaker-like current of other
systems and interacts with a fast voltage-sensitive Na+ current
during spike generation (Pelz et al.,
1999
). Similar voltage-sensitive currents have been described in
honeybee antennal motor neurons, both in vitro and in situ
(Kloppenburg et al.,
1999a
).
The antennal lobes and mushroom bodies are differentially involved in
memory formation in the honeybee (Erber et
al., 1980; Hammer and Menzel,
1998
; Menzel,
2001
) and our long-term goal is to analyse learning-related
changes of cell physiology in the different olfactory neurons, beginning with
an analysis of the ionic conductances of the different neurons types, antennal
lobe projection neurons and mushroom body Kenyon cells. This, however,
requires a staining technique that allows the identification of the neuron
type prior to recording. Whereas cultures of mushroom bodies comprise somata
of a homogeneous cell type (Kenyon cells), the dissociation of antennal lobes
yields a heterogeneous mixture of projection neurons and local interneurons
(Gascuel and Masson, 1991b
;
Devaud et al., 1994
;
Kirchhof and Mercer, 1997
). To
record from identified cell types, namely projection neurons and Kenyon cells,
we have developed a labelling technique that enabled us to identify neurons in
the culture dish prior to patch clamp recording. This study revealed
pronounced differences between projection neurons and Kenyon cells.
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Materials and methods |
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Projection neurons were identified by dye injection. For this,
dextran-coupled rhodamine (MW 3000, Molecular Probes Inc., Eugene, OR, USA)
was injected into the mushroom body calyces (see below), and the somata of
projection neurons from the antennal lobes were retrogradely labelled and
could easily be identified in vitro (see below). The procedure used
was a modification of a protocol developed for confocal microscopical analyses
of projection neuron morphology
(Schröter and Malun,
2000).
Pupal honeybees were decapitated and a small window was cut into the head cuticle frontally between the compound eyes, the bases of the antennae, and the ocelli. After removal of trachea and glands the brain, with the prominent mushroom body calyces, was clearly visible. The head was flooded with sterile standard external saline (see below) with gentamycin (150 µl/50 ml, Gibco Life Technologies, Karlsruhe, Germany) added. Using a sterile quartz glass capillary (1.0 mm o.d., 0.5 mm i.d.) pulled with a horizontal laser puller (P2000, Sutter Instruments, Novato, CA, USA), the lip region of the calyces was repeatedly punctured in order to damage the neurons and allow dye uptake. A saturated paste from the dextran-coupled rhodamine was prepared by adding one drop of sterile distilled water to the dry dextranrhodamine on a glass slide. Therefore, the exact dye concentration could not be determined. The lip regions of the calyces were briefly punctured with a sterile capillary, whose tip was coated with the paste, to insert a small amount of dye. After gently rinsing off excessive dye solution with standard external saline, the dye was allowed to diffuse to the somata for 3 h at room temperature.
Confocal microscopy
To confirm that rhodaminedextran labelled only projection neurons in
the antennal lobe, whole-mount preparations were analysed using confocal
microscopy. Brains were dissected out of the head capsule after labelling,
briefly rinsed with standard external saline, and fixed for 1 h in 4%
paraformaldehyde in 0.1 mol l-1 phosphate-buffered saline (PBS), pH
7.2 at room temperature. Subsequently, specimens were dehydrated in graded
ethanol and cleared in methyl salycilate. Whole-mounts were mounted in
Permount (Fisher Chemicals, Springfield, NJ, USA) on slides and viewed with a
confocal laser scanning microscope (Leica TCS-4D) equipped with a
krypton/argon laser light source. At primary magnifications between
10-40x, several optical sections were imaged at 1-5 µm intervals.
Series of images were stacked and two-dimensional projections of image stacks
generated using the extended focus function of Imaris software (version 2.7,
Bitplane).
Preparation of cell cultures and cell identification in vitro
Kenyon cells and projection neurons were dissected and cultured following a
modified protocol of Kreissl and Bicker
(1992). After the staining
procedure brains were removed from the head capsule and transferred into a
preparation medium (Leibovitz L15, Gibco BRL; see below). The glial sheath was
gently removed and the mushroom bodies or the antennal lobes dissected out of
the brains. After incubation (10 min) in calcium-free saline, mushroom bodies
were transferred back to the preparation medium (2 mushroom bodies or 10-15
antennal lobes per 100 µl medium) and dissociated by gentle trituration
with a 100 µl Eppendorf pipette. Cells were then plated in 10 µl samples
on Falcon plastic dishes coated with polylysine (polylysine-L-hydrobromide, MW
150-300 kDa, Sigma) and allowed to settle and adhere to the substrate for at
least 15 min. Thereafter, the dishes were filled with 2.5 ml of a supplemented
culture medium (see below) and were kept at 26°C in an incubator at high
humidity. Because the mushroom bodies can be mechanically dissected out of the
brain and contain somata of Kenyon cells exclusively, this procedure yielded a
culture of pure Kenyon cells. By contrast, antennal lobe neurons are a
heterogeneous population consisting of two major classes, projection neurons
and local interneurons. Thus, dissection of antennal lobes yielded cultures
containing neurons of different classes (cf.
Kirchhof and Mercer, 1997
) and
projection neurons needed to be identified in vitro. Since only
projection neurons were labelled by dextranrhodamine, their somata were
easily identified in the culture dish by their fluorescence. Labelled
projection neurons were located with epifluorescence illumination and
photographed using a Zeiss inverted microscope (Axiovert 10, Zeiss, Jena,
Germany), which was part of the patch clamp setup. Images were taken under
phase-contrast optics at 32x primary magnification with a PC-controled
digital camera (Olympus DP10; utility software C-2.1, Olympus). The digitized
images were analysed and processed with Adobe Photoshop (version 5.0, Adobe
Systems Inc.).
For electrophysiological measurements, cells were used between culture days 3 and 6. Processes of those cells chosen for recordings did not overlap with neighbouring neurites.
The contents of the cell culture solutions were similar to those used
during earlier physiological studies
(Goldberg et al., 1999;
Pelz et al., 1999
).
Preparation medium
To 1000ml of Leibovitz's L15 medium (Gibco) was added: sucrose, 30.0 g;
glucose, 4.0 g; fructose, 2.5 g; proline, 3.3 g. The medium was adjusted to pH
7.2 with NaOH and to 500 mosmol l-1; with sucrose.
Culture medium
Culture medium contained heat-inactivated fetal calf serum (Sigma, St
Louis, MO, USA), 13% (v/v); yeast hydrolysate (Sigma), 1.3% (v/v); L-15 powder
medium (Gibco BRL), 12.5% (w/v); glucose, 18.9 mmol l-1; fructose,
11.6 mmol l-1; proline, 3.3 mmol l-1; 93.5 mmol
l-1 sucrose; Pipes, 2.1 mmol l-1. The medium was
adjusted to pH 6.7 and 500 mosmol 1-1.
Calcium-free saline
Calcium-free saline consisted of: NaCl, 147 mmol l-1; KCl, 5
mmol l-1; Hepes, 65 mmol l-1; pH 7.2, 392 mosmol
l-1.
Electrophysiology
Whole-cell seal recordings (G) were performed at room temperature
following the methods described by Hamill et al.
(1981
). Recordings were made
using a computer-controlled HEKA EPC9 patch-clamp amplifier (HEKA-Elektronik,
Lambrecht, Germany). Data acquisition and online analyses were performed with
PULSE software (version 8.50, Heka-Elektronik) running on a Pentium-based PC.
Data were sampled at 10-20 kHz and were low-pass filtered with a four-pole
Bessel filter. Voltages were corrected for liquid junction potential (4 mV);
leakage currents were not subtracted. Series resistances were 5-20 M
and were compensated at about 80%. The cell capacitance for each cell was
estimated from the capacitance compensation routine of the PULSE software. For
some cells, cell capacitances and time constants were calculated from
capacitive charging currents, which were measured during hyperpolarizing
voltage pulses to a nonactive region of the membrane potential (-80 to -100
mV) prior to capacitance compensation. Electrodes were pulled from
borosilicate glass capillaries (GB 150-8P, Science Products Germany) with a
horizontal puller (DMZ Zeitz Instruments, München, Germany), and had tip
resistances of 8-12 M
in standard external solution (see below). The
holding potential was -80 mV throughout.
Solutions
The bath was continuously perfused at about 2 ml min-1 with a
standard external solution that consisted of NaCl, 130 mmol l-1;
KCl, 6 mmol l-1; MgCl2, 4 mmol l-1;
CaCl2, 5 mmol l-1; sucrose, 160 mmol l-1;
glucose, 25 mmol l-1; Hepes, 10 mmol l-1. The external
saline was adjusted to pH 6.7 with NaOH and to 510 mosmol l-1. To
record currents through K+ channels, the standard saline was
replaced with one in which tetrodotoxin (TTX, 100 nmol l-1) was
added to block currents through the voltage-sensitive Na+ channels.
Some experiments were performed with additional CdCl2 (50 µmol
l-1) in the external solution to block voltage-sensitive
Ca2+ currents. The pipette solution contained: K-gluconate, 87 mmol
l-1; KF, 40 mmol l-1, KCl, 20 mmol l-1;
CaCl2, 0.2 mmol l-1; MgCl2, 3 mmol
l-1; K-EGTA, 10 mmol l-1; Na2ATP, 3 mmol
l-1; Mg-GTP, 0.1 mmol l-1; glutathione, 3 mmol
l-1; sucrose, 120 mmol l-1; Hepes/bis-Tris, 10 mmol
l-1; pH 6.7, 500 mosmol l-1. To record currents through
Ca2+ channels, tetraethylammonium chloride (TEA-Cl, 10 mmol
l-1) was added to the external standard saline. In the pipette
solution K+ ions were replaced by TEA or Cs2+;
Cs-gluconate, TEA-Cl, Cs-EGTA and CsF replaced the corresponding K+
salts. All chemicals were purchased from Sigma (St. Louis, MO, USA).
Data analyses
Patch clamp data were analysed with PulseFit software (version 8.5, Heka)
and Igor Pro (version 3.12; WaveMetrics Inc, OR, USA) on a Pentium-based PC.
Statistical analyses were performed using Statistica (version 5.1, StatSoft
Inc., Tulsa, OK, USA). Values are given as means ± standard errors of
means (S.E.M.). Statistical significances between means were tested using
Student's t-test; the level of significance was taken as
P=0.05.
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Results |
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Within the protocerebrum other mushroom body-extrinsic neurons, which
innervate the mushroom bodies are stained as well. Among these neurons,
feedback neurons from extensive branches within all calycal regions
(Grünewald, 1999a).
Accordingly, feedback neurons are well stained by rhodaminedextran; two
somata clusters were visible in most specimens. In addition, the dye labelled
numerous Kenyon cells in each mushroom body. Cultures prepared from mushroom
bodies contain only somata of Kenyon cells and some of them were labelled. In
cultures prepared from antennal lobes only projection neurons were labelled,
because only the antennal lobes were dissected out of the brain, and the
somata of other mushroom body-extrinsic neurons, such as feedback neurons,
were not cultured.
Outgrowth of olfactory projection neurons and Kenyon cells in
vitro
All cells were treated identically before, during and after the neurons
were cultured. For cultures of Kenyon cells the yield from one mushroom body
was plated onto 10 dishes, and the yield from 2-3 antennal lobes plated onto
one dish. Thus, the cell density of the Kenyon cell and the projection neuron
cultures were similar and adjusted so that the neurites of individual neurons
did not overlap. The labelling remained visible in cultured neurons, allowing
cell identification throughout the period of observation. Within each dish,
2-10 labelled projection neurons (or numerous Kenyon cells) were identified
after 2-6 days in vitro. Neurons started to sprout new and fine
processes after 1 day in vitro and continued to grow until day 6, the
last day of observation (Fig.
2). The somata diameters of cultured Kenyon cells measured
approximately 7-10 µm, as in the intact pupal mushroom body. The diameters
of somata from projection neurons were larger (10-25 µm), similar to their
size in the intact brain (Schröter
and Malun, 2000), and the area covered by their branches were
larger than those of Kenyon cells. Individual branches of cultured honeybee
neurons were very thin, especially those of Kenyon cells, with a diameter of
less than 1 µm. We did not quantify potential morphological differences
between the neuron types in vitro, because most neurons were used for
electrophysiological experiments before they grew elaborate neuritic
branches.
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Potassium currents
Whole-cell patch clamp recordings were performed from projection neurons,
identified from their fluorescence, and from Kenyon cells, which were derived
from pure mushroom body cultures. Cells were measured electrophysiologically
in vitro between days 3 and 5. Older neurons had rather elaborate
neuritic branches, which would compromise the control of the membrane
potential (space clamp). Thus, a total of 53 cells (22 Kenyon cells, 31
projection neurons) were measured. The capacitance of Kenyon cells was
4.1±0.27 pF (N=22) and 11.30±0.68 pF for projection
neurons (N=31). Kenyon cells have significantly smaller cell
capacitances because of their smaller somata diameters (P<0.001,
t=-8.56, d.f.=51, Student's t-test).
From the holding potential of -80 mV a hyperpolarizing prepulse was applied
to -120 mV to completely remove inactivation of the transient A type
K+ current (cf. Pelz et al.,
1999). Whole-cell membrane currents induced by depolarizing
voltage pulses of Kenyon cells and projection neurons were dominated by large
voltage-sensitive outward currents (Fig.
3). Depolarizing voltage pulses also induced a rapidly activating
transient inward current in most cells, which was blocked in all experiments
by switching to an external saline containing 100 nmol l-1 TTX.
Kenyon cells and projection neurons expressed different types of outward
K+ currents. Schäfer et al.
(1994
) identified that these
outward currents are carried by K+ ions, by altering the external
K+ concentration and determining the tail current reversal
potential. The outward K+ currents may comprise a rapidly
activating transient and a sustained component (delayed rectifier,
IK,V). The transient K+ current (A type current,
IK,A) of the Kenyon cells was more pronounced than the
IK,A of the projection neurons, as can be seen when comparing
traces of typical currents of two representative cells (Figs
3,
5). The transient current
component can be blocked in Kenyon cells by a depolarising prepulse to -20 mV
(Pelz et al., 1999
).
Accordingly, inactivating prepulses reduced the amplitude of the transient
K+ currents in Kenyon cells, but did not affect whole cell
K+ currents of projection neurons
(Fig. 3). Subtraction of these
currents yielded the pure transient K+ current
(Fig. 3C). The IK,A
was a large portion of the total K+ current of Kenyon cells,
whereas such transient K+ currents were negligible in projection
neurons. The ratio of transient over sustained K+ current, as
revealed by calculating the quotient of the maximum current at the current
onset and at the end of the depolarizing pulse (command potential +50 mV), was
higher in Kenyon cells than in projection neurons (P<0.0001,
t=8.95, d.f.=30, Student's t-test, N=10 Kenyon
cells, 22 projection neurons; Fig.
4C).
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The K+ current amplitudes of the projection neurons were higher than those of Kenyon cells (Fig. 4A). Measured at a pulse potential of +50 mV, the mean peak outward current (measured a few ms after current onset) of projection neurons measured 7376.5±117.2 pA and was significantly higher than those of Kenyon cells (2907.2±67.1 pA; P<0.001, t=3.65, d.f.=30, Student's t-test). Similarly, when measured at the end of the depolarizing pulse, the amplitude of the sustained current component was higher in projection neurons (P<0.001). This was only partially due to the larger soma diameter of projection neurons, since the comparison of the current densities (pA/pF) revealed differences for the sustained K+ current (567.0±46.0 pA/pF for projection neurons versus 326.9±50.2 pA/pF for Kenyon cells, P<0.005, t=3.18, d.f.=29), but not for the transient K+ current (Fig. 4B, P=0.29). This indicates that the total K+ current consisted mainly of non-inactivating currents and only a small transient component, whereas that of Kenyon cells was a mixture of inactivating and non-inactivating currents with a pronounced transient current.
The I-V relationship of the outward currents showed current activation at approximately -45 mV (Figs 5C, 6). The I-V curves of projection neurons and Kenyon cells differed when whole-cell currents were measured without blockade of the calcium currents by external Cd2+. Kenyon cells expressed a linear voltage-dependency of K+ currents at positive membrane potentials. By contrast, projection neurons showed a pronounced N-shaped I-V curve with a local current peak at +60 mV and a local minimum at +80 mV (Figs 5C, 6). When voltage-sensitive Ca2+ currents were blocked with external 50 µmol l-1 Cd2+ the outward currents of projection neurons were significantly reduced. By contrast, Cd2+ did not affect or only slightly increased the outward K+ currents of Kenyon cells (Fig. 5B,C). In addition, external Cd2+ irreversibly transferred the N-shaped form of the I-V curve of projection neurons into a linear one (Fig. 5C), indicating that Cd2+ blocked a calcium-sensitive outward current in projection neurons but not in Kenyon cells. Typically, such a calcium-dependent N-shaped I-V curve is caused by the activation of calcium-dependent K+ currents.
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Calcium currents
The differences in Ca2+-dependent K+ currents may
reflect differences in the voltage-sensitive Ca2+ currents.
Therefore, we measured the voltage-sensitive Ca2+ currents of
Kenyon cells and projection neurons. For this, currents through calcium
channels were isolated by blocking outward K+ currents with
external TEA and Cs2+ in the patch pipette and voltage-sensitive
Na+ currents with external TTX. The remaining inward currents were
currents through Ca2+ channels
(Schäfer et al., 1994).
Under these conditions, depolarizing command potentials activated inward
currents at command potentials higher than -40 mV in both neuron types (Figs
7,
8). These currents through
calcium channels showed rapid activation and slow inactivation
(Fig. 7A). The peak current
amplitude ranged between -38.6 and -419.9 pA (mean -197.3±30.9 pA,
N=12) for Kenyon cells. Projection neurons expressed Ca2+
currents with peak amplitudes of -292.6 to -1166.2 pA (mean -683.0±30.9
pA, N=9, Fig. 8B).
When equimolar barium was used a charge carrier instead of calcium, the
whole-cell currents through calcium channels did not change substantially (not
shown). Instead, current amplitudes were only slightly increased and the
current decay during depolarizing was almost indistinguishable (not shown).
The Ca2+ currents were sensitive to externally applied 50µmol
l-1 Cd2+, a concentration that was shown to be
sufficient to completely and irreversibly block Ca2+ currents in
Kenyon cells (Schäfer et al.,
1994
). However, in projection neurons, a small
Cd2+-resistant inward current remained unblocked at this
Cd2+ concentration (Fig.
7B).
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The voltage threshold for activation of inward Ca2+ currents was approximately -35 mV. The currentvoltage relationship of the currents through Ca2+ channels peaked at command potentials between 0 and 10 mV and had a reversal potential at approximately 35-45 mV (Figs 7C, 8A). The overall shapes of the voltage-sensitive Ca2+ currents and the I-V curves of this current in Kenyon cells and projection neurons were similar (Fig. 8A). The mean peak current amplitudes, however, were significantly higher in projection neurons than in Kenyon cells (P<0.0001, t=-5.57, d.f.=19, Student's t-test; Fig. 8B). This is probably due to their larger soma diameter, because the mean peak Ca2+ current densities (pA/pF) did not differ (P=0.29, t=-1.09, d.f.=19; Fig. 8C). Thus, Kenyon cells and projection neurons express similar voltage-sensitive Ca2+ currents with similar current densities.
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Discussion |
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Identification and culturing of projection neurons
In order to assign physiological properties to a certain neuron class it is
crucial to identify the neuron type. This is particularly true for antennal
lobe cultures, which contain different types of neuron. Cultured projection
neurons or Kenyon cells do not retain their in vivo morphology. This
is probably not a consequence of the labelling procedure, because it has been
described previously for cultures of unlabelled Kenyon cells
(Kreissl and Bicker, 1992) and
unidentified antennal lobe neurons (Devaud
et al., 1994
; Kirchhof and
Mercer, 1997
). Also, in the present study branching patterns of
labelled and unlabelled neurons within a given culture dish were
indistinguishable. Thus, in contrast to cultures of, for example, antennal
lobe neurons of the moth Manduca sexta
(Hayashi and Hildebrand, 1990
;
Oland et al., 1996
; Mercer and
Hildebrand,
2002a
,b
),
labelling is required to differentiate between cultured local interneuron and
projection neurons in the honeybee. The identification of antennal lobe
projection neurons was achieved by rhodamine dye injections into the mushroom
body calyces. The staining within the antennal lobes presented here is very
similar to previous stainings observed with rhodaminedextran into the
mushroom body (Schröter and Malun,
2000
). The morphology of the projection neurons within the
honeybee brain has been described in detail elsewhere
(Homberg, 1984
;
Gascuel and Masson, 1991a
;
Fonta et al., 1993
;
Schröter and Malun, 2000
;
Abel et al., 2001
). The
staining procedure that we used labelled other neurons as well as antennal
lobe projection neurons and Kenyon cells, including several known mushroom
body extrinsic neurons (Mobbs,
1982
,
1985
;
Rybak and Menzel, 1993
),
mushroom body feedback neurons (Bicker et
al., 1985
; Grünewald,
1999a
) and, occasionally, a few somata within the suboesophageal
ganglia. However, staining of these neurons did not interfere with the
identification of projection neurons in vitro, because in the
antennal lobes only projection neurons could take up the tracer and were,
therefore, the only labelled cells in these cultures.
Cultured dextran-labelled neurons have been used successfully for patch
clamp recordings; their viability has repeatedly been noted (e.g.
Kloppenburg and Hörner,
1998; Kloppenburg et al.,
1999a
; Dugladze et al.,
2001
) and was confirmed here. Projection neurons grew extensive
neurites in culture throughout the period of observation (3-6 days), as has
been described for unidentified antennal lobe neuron cultures
(Gascuel and Masson, 1991a
;
Devaud et al., 1994
;
Kirchhof and Mercer, 1997
),
which suggests that they were healthy and that the dye did not impair the
viability of the cells. The applied mass staining technique is therefore a
very reliable method for the identification of honeybee antennal lobe
projection neurons in vitro and may be used in the future for
analyses of other identified neurons of the bee brain, such as mushroom body
feedback neurons.
Isolation of ionic currents in identified neurons
The major finding of this study is that antennal lobe projection neurons
and mushroom body Kenyon cells express different sets of ionic currents.
Kenyon cells express at least three different voltage-gated outward
K+ currents. The transient component comprises a rapidly
inactivating (A-type) current and a slowly inactivating current, whose kinetic
parameters have been described in great detail
(Pelz et al., 1999). The
amplitude of the sustained (delayed rectifier) current is relatively small
compared to the transient K+ currents, as described in various
insect or crustacean neurons (e.g. Byerly
and Leung, 1988
; Saito and Wu,
1991
; Hayashi and Levine,
1992
; Delgado et al.,
1998
; Kloppenburg and
Hörner, 1998
; Benkenstein
et al., 1999
; Kloppenburg et
al., 1999b
; Schmidt et al.,
2000
(for a review, see Wicher
et al., 2001
) and in antennal motoneurons within the honeybee
deutocerebrum (Kloppenburg et al.,
1999a
). By contrast, projection neurons do not express such
prominent transient K+ currents. This is interesting, because
computer simulations using HodgkinHuxley-derived equations indicated
that A-type K+ currents are mainly responsible for the membrane
repolarisation during an action potential in honeybee Kenyon cells
(Pelz et al., 1999
;
Ikeno and Usui, 1999
).
Similarly, shaker mutations in Drosophila impair spike
repolarisation (Tanouye and Ferrus,
1985
). Therefore, other outward currents (calcium-dependent or
delayed rectifier currents) must be responsible for spike repolarisation in
honeybee projection neurons. Supporting evidence for different expressions of
K+ channels comes from immunohistochemical studies of the
Drosophila brain showing that shaker channel proteins are highly
expressed in the mushroom body neuropil, but not in the antennal lobes
(Rogero et al., 1997
).
Furthermore, axons of motoneurons and mechanosensory neurons within the
antennal nerve of the fly are shaker-immunoreactive, consistent with the
presence of transient K+ currents in antennal motoneurons in
honeybees (Kloppenburg et al.,
1999a
).
Ca2+-dependent K+ currents have been described in a
variety of insect neurons (e.g. Thomas,
1984; Nightingale and Pitman,
1989
; David and Pitman,
1995
; Grolleau and Lapied,
1995
; Mills and Pitman,
1999
; Hewes, 1999
)
(for reviews, see Saito and Wu,
1991
; Wei et al.,
1994
; Grolleau and Lapied,
2000
; Wicher et al.,
2001
). They may provide many functional roles within the nervous
system, including spike repolarisation (e.g.
Lapied et al., 1989
) and
afterhyper-polarisation (Saito and Wu,
1993
; Hu et al.,
2001
). Ca2+-dependent K+ currents can be
blocked by Ca2+ channel blockers (typically Cd2+ in
insect neurons). This treatment reduced the amplitude of outward currents in
projection neurons, but not in Kenyon cells. In addition projection neurons,
which express voltage and Ca2+-dependent K+ currents,
show a nonlinear I-V relationship at positive command potentials, because the
K+ current amplitude is influenced by the activation of
voltage-sensitive Ca2+ channels. Assigning the honeybee
Ca2+-dependent K+ current to any of the identified
insect or vertebrate channels is currently not possible. It remains to be
analysed whether the currents are both calcium- and voltage-dependent and
whether honeybees express two separate Ca2+-dependent K+
channels as in DUM neurons of Periplaneta
(Grolleau and Lapied, 1995
) or
Drosophila `giant' neurons (Saito
and Wu, 1991
).
Probably all insect neurons express voltage-sensitive Ca2+
channels. These currents may contribute to action potential generation,
synaptic transmission or neuromodulation (for reviews, see
Jeziorski et al., 2000;
Wicher et al., 2001
). The
voltage-sensitive Ca2+ currents of the honeybee projection neurons
and Kenyon cells are similar (with respect to steady-state activation,
Cd2+-sensitivity and inactivation) to those described in other
insect preparations, e.g. Drosophila
(Byerly and Leung, 1988
;
Saito and Wu, 1993
;
Schmidt et al., 2000
),
Periplaneta (Grolleau and Lapied,
1996
; Wicher and Penzlin,
1997
; Mills and Pitman,
1997
), Manduca
(Hayashi and Levine, 1992
),
Gryllus (Kloppenburg and
Hörner, 1998
) and locusts
(Laurent et al., 1993
;
Pearson et al., 1993
). They
activate rapidly and show a slow inactivation. The functional properties of
the Ca2+ currents of Kenyon cells and projection neurons presented
here are thus consistent with earlier descriptions of Kenyon cells
(Schäfer et al., 1994
).
The Ca2+ current densities of Kenyon cells and projection neurons
are similar, which implies that the observed differences in
Ca2+-dependent K+ currents are not due to differing
densities of Ca2+ channels, but may indeed represent differential
expression of Ca2+-dependent K+ channel proteins in
Kenyon cells and projection neurons.
Although the present results are largely consistent with earlier
investigations (Schäfer et al.,
1994), there are differences as well. The finding that Kenyon
cells did not show pronounced calcium-dependent K+ currents,
contradicts the report of these currents by Schäfer et al.
(1994
). What could be the
reasons for this discrepancy? We exclude differences in Ca2+
channel expression between both studies, because the Ca2+ currents
are very similar with respect to activation threshold (approximately -40 mV)
and peak current amplitudes (means -160 pA and -197 pA). The culture
conditions have been changed since the study by Schäfer et al.
(1994
), e.g. the components
and pH of the medium. However, it is implausible that these changes abolish
Ca2+-dependent K+ currents only in Kenyon cells and not
in projection neurons, because both neuron types were cultured and measured in
parallel under the same conditions. The recordings by Schäfer et al.
(1994
) were performed solely
from the somata, because only cells without any processes were used, and the
neurons were cultured for only 12-36 h. In the present study neurons were
allowed to grow for 3-6 days and formed small neurites. Therefore, the whole
cell currents may comprise somatic and neuritic currents. Accordingly, Kenyon
cells may express somatic, but not neuritic Ca2+-dependent
K+ currents. It is interesting to note here that Pelz et al.
(1999
) also observed
differences in the pharmacology and kinetics of the A type K+
current as compared to Schäfer et al.
(1994
), but the ultimate
reasons for these differences remain unclear.
Functional roles of differential current expression
Differences in the ionic currents between Kenyon cells and projection
neurons indicate that a specific expression pattern is maintained throughout
the culturing procedure and for several days in vitro. Therefore, the
observed differences may in fact represent physiological differences of these
neuron types in vivo. In the living honeybee brain neither the
function nor the dendritic or axonal localization of the various
voltage-sensitive ionic currents has yet been satisfyingly unraveled. Both
Kenyon cells and projection neurons generate action potentials
(Homberg, 1984;
Hammer and Menzel, 1995
;
Abel et al., 2001
;
Müller et al., 2002
), but
differ with respect to the outward currents, which probably mediate spike
repolarisation. Thus, different ionic currents may interact during spike
generation in the two neuron classes. Both the antennal lobe and the mushroom
body are involved in olfactory learning and memory formation
(Masuhr and Menzel, 1972
;
Hammer and Menzel, 1998
;
Menzel, 1999
) and both are
innervated by modulatory octopaminergic neurons (Hammer,
1993
,
1997
;
Kreissl et al., 1994
).
Additional work is required to reveal the functional significance of the
differential expression of K+ currents of the different neuron
types within the central olfactory pathway of the honeybee and whether these
currents are differentially modulated during olfactory learning.
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Acknowledgments |
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References |
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