Chem. Senses 27: 599-610,
2002
© Oxford University Press 2002
Voltage- and Calcium-activated Currents in Cultured Olfactory Receptor Neurons of Male Mamestra brassicae (Lepidoptera)
INRA, Unité de Phytopharmacie et des Médiateurs Chimiques, Route de Saint Cyr, 78026 Versailles Cedex, France 1 CNRS, Laboratoire de Neurobiologie Cellulaire et Moléculaire, 91198 Gif-sur-Yvette, France
Correspondence to be sent to: Philippe Lucas, Unité de Phytopharmacie et des Médiateurs Chimiques, Route de Saint Cyr, 78026 Versailles Cedex, France. e-mail: plucas{at}versailles.inra.fr
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Abstract |
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Insect olfactory receptor neurons (ORNs) grown in primary cultures were studied using the patch-clamp technique in both conventional and amphotericin B perforated whole-cell configurations under voltage-clamp conditions. After 10-24 days in vitro, ORNs had a mean resting potential of -62 mV and an average input resistance of 3.2 G
. Five different voltage-dependent
ionic currents were isolated: one Na+, one Ca2+ and
three K+ currents. The Na+ current (35-300 pA) activated
between -50 and -30 mV and was sensitive to 1 µM tetrodotoxin (TTX). The
sustained Ca2+ current activated between -30 and -20 mV, reached a
maximum amplitude at 0 mV (-4.5 ± 6.0 pA) that increased when
Ba2+ was added to the bath and was blocked by 1 mM Co2+.
Total outward currents were composed of three K+ currents: a
Ca2+-activated K+ current activated between -40 and -30
mV and reached a maximum amplitude at +40 mV (605 ± 351 pA); a
delayed-rectifier K+ current activated between -30 and -10 mV, had
a mean amplitude of 111 ± 67 pA at +60 mV and was inhibited by 20 mM
tetraethylammonium (TEA); and, finally, more than half of ORNs exhibited an
A-like current strongly dependent on the holding potential and inhibited by 5
mM 4-aminopyridine (4-AP). Pheromone stimulation evoked inward current as
measured by single channel recordings. |
Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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For their survival, insects depend heavily on their capacity to monitor their external chemical environment. They have evolved very sensitive and specific olfactory receptor neurons (ORNs). This sensitivity and specificity of insect ORNs, in addition to the availability of active ligands, make insects useful models for the study of olfaction.
Insect ORNs are bipolar neurons located on the antennae. The dendrites
extend into cuticular structures—sensilla—and the axons project to
central nervous structures—the antennal lobes—where olfactory
information is processed. These sensillar structures are particularly suitable
for recording odorant responses extracellularly
(Kaissling, 1986
). An abundant
literature describes the high specialization of insect ORNs, the detection of
sex pheromone compounds by male moths being the prime example. However, in
contrast to vertebrate main olfactory epithelium or vomeronasal organ, where
sensory neurons can be either acutely isolated
(Maue and Dionne, 1987a;
Firestein and Werblin, 1989;
Liman and Corey, 1996) or
patch-clamp recorded from intact epithelial preparation
(Ma et al., 1999;
Leinders-Zufall et al.,
2000), the presence of the cuticle impedes direct access to the
ORNs in insects. There are thus very few descriptions of currents or ion
channels expressed in insect ORNs.
To elucidate the processes underlying olfactory transduction, it is
necessary to have an understanding of the electrophysiological properties of
ORNs. The activation of voltage-dependent ion channels, involved in the
generation of action potentials, constitutes an important step in ORN
response. They play important roles in shaping spike firing patterns and are
likely involved in the high sensitivity of ORNs. Adult insect ORNs could only
be isolated from Locusta migratoria
(Wegener et al.,
1992). High conductance Ca2+- and voltage-dependent
K+ channels, ATP blockable, present on the soma of these neurons,
were characterized in single channel recordings. In Lepidoptera, ORNs were
isolated from nymphs and before cuticle deposit
(Zufall et al.,
1991a) at an early stage of their development. These neurons
expressed only one voltage-gated current, a transient outward current with the
characteristics of an A-current. In situ patch-clamp recordings from
insect ORNs, either on extruded dendrite
(Zufall and Hatt, 1991) or
cell body (Dubin and Harris,
1997), are feasible but technically difficult due to the small
size of these sensory cells and the presence of closely enveloping accessory
cells and the cuticle. Moreover, in situ recordings limit the use of
pharmacological agents. The development of long-term ORN cultures
(Stengl and Hildebrand, 1990)
has helped to resolve many of these difficulties. Patch-clamp recordings
demonstrated the expression of four different types of voltage-gated channels
in cultured ORNs from Manduca sexta: one Na+ and three
K+ channels (Zufall et
al., 1991b). The sustained outward current had the
characteristics of the delayed rectifier. The transient outward current
presented the properties of the A-current. Single Ca2+-activated
K+ channels were described in inside-out recordings, but no
Ca2+-activated K+ current was characterized in
whole-cell recordings.
Biochemical (Boekhoff et al.,
1990a,
b;
Breer et al., 1990)
and electrophysiological data (Wegener
et al., 1993; Stengl,
1994) demonstrate the involvement of IP3 as the second
messenger of olfactory transduction in insects. An IP3-dependent
Ca2+ current, a Ca2+-dependent cation current and a
protein kinase C-dependent cation current are successively activated in
pheromone receptor neurons after pheromone stimulation
(Stengl, 1994). These three
inward currents may constitute the receptor potential that elicits a discharge
of action potentials encoding odor quantity and quality centrally. However, in
apparent contradiction with these observations, olfactory dendrites of
Antheraea polyphemus seem to be equipped with only one type of ion
channel, that is sex-pheromone-dependent
(Zufall and Hatt, 1991). These
ion channels were activated from the intracellular side by protein kinase C
activators such as diacylglycerol and phorbolester and by cGMP, but not by
Ca2+ or IP3. This discrepancy indicates that insect
olfactory transduction is still not fully elucidated.
We have developed a procedure to culture olfactory neurons
(Lucas and Nagnan-Le Meillour,
1997). These cell cultures provide a useful system for the study
of sensory signaling and its modulation in insects. As a first step, we
present in this paper new data concerning voltage-gated currents that are
expressed in insect ORNs. We found that cultured insect ORNs possess a variety
of voltage- and Ca2+-dependent currents, including an
Na+ current, a Ca2+ current, a Ca2+-dependent
K+ current, a delayed-rectifier K+ current and a rapidly
inactivating K+ current (A-current). Although they support previous
findings, several new results, including the characterization of a
voltage-gated Ca2+ current and a macroscopic
Ca2+-activated K+ current, are presented here for the
first time in insect ORNs. Moreover, we have begun to study the responsiveness
of insect ORNs in vitro. These results have already been presented in
abstract form (Lucas and Shimahara,
2001).
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Materials and methods |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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All culture media were purchased from Life Technologies In Vitro Gene and all chemicals from Sigma, except tetrodotoxin (TTX) which was purchased from Alomone Laboratories.
Insects
Mamestra brassicae (Lepidoptera, Noctuidae) were reared on a
semi-artificial medium (Poitout and Bues,
1974). Larvae were maintained at 20-24°C under a long
photo-period regimen (16 h light/8 h dark) and 40-50% humidity. Experimental
pupae were selected within 12 h after pupation and were kept at 23°C until
dissected to isolate their antennal flagella.
Primary cultures of antennal neurons
The cell culture protocol was slightly modified from the method previously
described (Lucas and Nagnan-Le Meillour,
1997) in order to improve the survival of antennal cells. All
steps of this protocol were carried out at room temperature. Briefly, antennal
flagella from 4-day-old male pupae were dissected in 3 + 2 medium (three parts
of Leibovitz's L15 medium and two parts of Grace's medium supplemented with
lactalbumin hydrolysate and yeastolate). The antennal tissue was placed in 1
mg/ml EGTA for 5 min and rinsed three times for 5-10 min in Hanks'
Ca2+- and Mg2+-free salt solution (HBSS). Then, flagella
were disrupted by 20 min of incubation in L-cystein-activated papain (1
mg/ml), rinsed three times in HBSS and carefully triturated with a
fire-polished Pasteur pipette. The resulting cell suspension was plated onto
uncoated 35 mm Falcon Petri dishes. Dissociated cells were allowed to settle
and to adhere to the surface of culture dishes for at least 30 min. The
culture medium was then replaced with 150 µl of a 2 + 1 mixture of L15
medium conditioned with 5% of fetal calf serum and Grace medium supplemented
with lactalbumin hydrolysate and yeastolate and conditioned on the embryonic
cell line MRRL-CH1 (Eide et al.,
1975; Stengl and Hildebrand,
1990). The cultures were then inverted to form a hanging column
and were maintained at 23°C in a humid atmosphere. Half of the culture
medium was changed every 4-7 days.
Typically, cells were found to survive in vitro for >6 weeks.
Coating of the dish surface was not necessary since non-neuronal cells,
probably glial cells (Lucas and Nagnan-Le
Meillour, 1997), multiplied and formed a continuous sheet to which
other cell types attached (Figure
1). ORN-like cells that were previously found to exhibit positive
anti-HRP staining (Lucas and Nagnan-Le
Meillour, 1997) were distinguished from other cell types on the
basis of their small cell bodies (5-8 µm) and their thin processes.
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Recording solutions
During recordings, cells were kept in 
1 ml of `extracellular' Ringer
solution containing (in mM): NaCl, 156; KCl, 4; CaCl2, 6; glucose,
5; and HEPES, 10, adjusted to pH = 7.4 with NaOH. In some recordings,
Ca2+ was replaced with Ba2+ (Ca2+-free
Ringer) or Na+ was replaced by choline (Na+-free
Ringer). To increase the Ca2+ current amplitude, in some recordings
Ba2+ (10, 20 or 40 mM) was added to Ca2+. NaCl
concentration was reduced according to the concentration of BaCl2
to maintain the osmotic pressure of the solution. Recordings were performed
for a maximum of 2 h after the replacement of the culture medium with the
extracellular recording solution. The `intracellular' pipette filling solution
used for whole-cell recordings contained (in mM); KCl, 150; NaCl, 5;
MgCl2, 2; CaCl2, 1; HEPES, 10; EGTA, 11; adjusted to pH
7.2 with KOH. The free Ca2+ concentration in the pipette solution
was calculated to be 18 nM. In some experiments, all K+ in the
pipette solution was replaced with Cs+ and 20 mM tetraethylammonium
(TEA) in order to block outward currents. Perforated patch-clamp recording
electrodes were filled with (in mM): Cs-MeSO4, 130; CsCl, 25;
CaCl2, 1; HEPES, 10; EGTA, 11; adjusted to pH 7.2 with CsOH. The
osmolarities of bath and intracellular solutions were adjusted when necessary
to 360 and 330 mosmol/l, respectively, by the addition of mannitol. The
compositions of the different solutions used are summarized in
Table 1.
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Drug and pheromone application
A gravity driven system was used to perfuse the cells at a flow rate of 1.5
ml/min. Different solutions could be perfused using a mechanical rotary valve.
For pheromone stimulation, a pipette was filled with 10 µM
(Z)-11-hexadecenyl acetate (Z11-16:Ac; synthesized in the
laboratory by M. Lettere). Z11-16:Ac is the main pheromone component
of M. brassicae (Bestmann et
al., 1978; Descoins
et al., 1978). It activates one of the two neurons housed
in each long sensillum trichodeum present on male antennae
(Den Otter and Van der Hagen,
1989; Renou and Lucas,
1994). Z11-16:Ac was dissolved in dimethyl sulfoxide
(DMSO), with the final concentration of DMSO in Ringer not exceeding 0.1%.
DMSO alone did not elicit any current on ORNs. Recombinant pheromone binding
protein Mbra-PBP1 (Campanacci et
al., 1999), which specifically binds in vitro
Z11-16:Ac (Maïbeche-Coisne
et al., 1997), was added at a concentration of 50 µM
to help solubilize hydrophobic pheromone and better activate pheromone
receptor proteins. Pheromone stimuli were pressure ejected towards the cell
body of the neuron being recorded using a picospritzer (General Valve company,
Fairfield, NJ). Fluorescein was used in a set of preliminary experiments to
establish the optimal placement of the stimulus pipette (50 µm from the
cell), its tip diameter (2 µm) and the ejection pressure (2 x
104 Pa). By measuring fluorescence dilution after injection of a
defined concentration of fluorescein in these conditions, the mean volume
ejected in 1 s was measured to be 10 nl. The deduced amount of pheromone
ejected in the direction of the recorded neuron was 10-14 mol.
During pheromone application, the membrane patch was kept at 0 mV in the
cell-attached configuration.
Patch-clamp technique and data analysis
All recordings were obtained at room temperature from ORNs that had been in
culture for 10-24 days. Neurons were identified on the basis of their
morphology at 600x magnification with an Olympus IX 70 inverted
microscope equipped with phase-contrast and Hoffmann modulation contrast
optics. Currents were recorded using conventional patch-clamp recording
methods (Hamill et al.,
1981), with an Axopatch 200 B amplifier (Axon Instruments, Union
City, CA). Electrodes were pulled from thick-wall borosilicate glass
capillaries (World Precision Instruments, GC 150-10) using a programmable
micropipette puller (model P97; Sutter Instruments, Novato, CA). The pipettes
were coated with Sylgard (GE Bayer Silicones, The Netherlands) and fire
polished. When filled with the physiological solutions, their tip resistance
was 4-6 M for whole-cell or perforated recordings and 7-10 M for
cell-attached recordings. The recording dish was grounded using an agar bridge
to an Ag/AgCl electrode. Pipette offset potential was compensated before
forming a seal. The cell was approached with positive pressure in the pipette
and the high resistance seal (>10 G and frequently >40 G)
was either formed spontaneously when removing the pressure or when gentle
suction was applied. Before breaking into the whole-cell configuration, the
pipette capacitance was compensated and a potential of -60 mV was applied in
the electrode, preventing the cell from experiencing a loss in its holding
potential. The whole-cell configuration was obtained by applying further
suction in the pipette.
For some recordings, the perforated patch-clamp technique with amphotericin
B was employed to prevent run-down of Ca2+ currents according to
the protocol described previously (Rae
et al., 1991). A stock solution of amphotericin B was
prepared daily by dissolving 3 mg of amphotericin B in 50 µl DMSO. The
recording pipette was filled by dipping its tip into the intracellular saline
solution for 10 s. The pipette was then back-filled with a solution
containing 4 µl of the stock solution of amphotericin B vigorously stirred
for 1 min with 1 ml of intracellular pipette solution. The amphotericin B
dilution was used for a maximum of 2 h. The access resistance to the cell
interior was monitored under pCLAMP 8 by applying 10 mV pulses. In most cases,
it reached a stable value between 15 and 40 M within 1-10 min.
Data were acquired and analyzed with the aid of pCLAMP 8 software (Axon
Instruments, Union City, CA). Cells were clamped at a holding potential of -60
or -80 mV and voltage steps were used to activate voltage-gated channels in
the neurons. For whole-cell recordings, a fractional (P/N) method, using four
fractionally scaled hyperpolarized sub-pulses, was used for online leak
compensation. Resting membrane potentials were measured as zero-current
potentials. Cell capacitance was calculated by membrane test algorithms of
pCLAMP 8 as the integration of the area under the capacitive current evoked by
10 mV voltage pulses from a holding potential of -60 mV. Membrane resistance
was either determined from the slope of the current—voltage
(I—V) relationship between -150 and -50 mV, where no
voltage-gated currents were elicited, or it was read from membrane test
protocols of pCLAMP 8 as solved iteratively using the Newton—Raphson
method. Currents were low-pass filtered at 2-5 kHz with a low-pass four-pole
Bessel filter and digitally sampled at 20 kHz. In whole-cell recordings,
series resistance (33.5 ± 13.0 M, n = 48) was
compensated (80%) when the whole-cell current was >300 pA. On average, the
remaining 20% of uncompensated series resistance will have caused an error
(underestimation) in command potential of 6.7 mV for each nanoampere of
whole-cell current. The amplitude of sustained currents was measured as the
mean amplitude of the current elicited during the second half of 100 or 200 ms
voltage steps. The amplitude of transient currents was measured as the maximum
of the current elicited within 5 ms (Na+ current) or 25 ms (A-like
current) after voltage steps.
Mean values ± standard deviations (SD) are given throughout and n indicates the number of neurons.
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Results |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Whole-cell passive properties
The duration of recordings was between 5 and 30 min. Whole-cell capacitance
ranged from 2.0 to 5.5 pF (3.6 ± 1.0 pF, n = 27). The resting
potential as measured immediately after breaking into the whole-cell mode
varied from -48 to -75 mV (-62 ± 7 mV, n = 42). The membrane
resistance of the cells ranged from 1.3 to 10.3 G (3.2 ± 2.2
G, n = 25). No correlation was found between age of neurons in
culture (10-24 days) and their membrane capacitance, resting potential or
input resistance.
Inward currents
Na+ current
A fast transient inward current activated between -50 and -30 mV and
reached a maximal amplitude that ranged from 35 to 300 pA at -10 or 0 mV
(Figure 2a). The amplitude of
the transient inward current was probably underestimated, since it declined
rapidly with time and because most cells were not well space clamped, as
indicated by the delay before recording inward current in response to steps to
-40 mV (Figure 2a). Despite
efforts to lower the access resistance to the cells, we were unable to improve
the space clamp. ORNs in culture were probably not electrically compact due to
their long and thin processes. The transient inward current was inhibited by
the addition of TTX (10-6 M). It was not observed when
Na+ was replaced with choline in the bath (n = 3). Thus,
this current presented the characteristics of an Na+ current. In
some recordings, trains of action potentials were observed
(Figure 2c). This incomplete
voltage clamp of the site of generation of action potentials indicates that
Na+ channels were probably not located on the soma, but rather on
neurites.
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Ca2+ current
In the search for voltage-dependent Ca2+ current, outward
currents were blocked by replacing K+ with Cs+ and by
adding 20 mM TEA into the patch pipette; Na+ current was inhibited
by the addition of 1 µM TTX into the bath. Due to its extremely small
amplitude and rapid run down, the sustained inward current was recorded using
the perforated patch-clamp technique with amphotericin B. Currents elicited by
steps were repeated three times for each potential and averaged to improve the
signal-to-noise ratio. Under these conditions, a sustained inward current was
elicited by depolarizing voltage steps
(Figure 3a). It activated
between -30 and -20 mV and reached a maximum amplitude (-4.5 ± 6.0 pA,
n = 18) at 0 mV. The amplitude of this inward current was correlated
to the concentration of Ba2+ present in the bath solution
(Figure 3b): -9.7 ± 8.4
pA (n = 7) at 0 mV in 10 mM Ba2+; -14.4 ± 10.1 pA
(n = 7) at 10 mV in 20 mM Ba2+; and -26.5 ± 12.2 pA
(n = 9) at 10 mV in 40 mM Ba2+. With Ba2+ in
the bath solution, this inward current was observed in all recorded ORNs
(n = 23) and it presented some inactivation during 100 ms voltage
steps (Figure 3c). This inward
current, recorded with 40 mM Ba2+, was blocked when 1 mM
Co2+ was added to the bath (n = 5;
Figure 3d). Taken together,
these results indicate that all ORNs expressed a sustained inward current
carried by Ca2+. Its little inactivation when recorded with
Ba2+ in the bath solution could indicate that more than one type of
Ca2+ channel underlies the Ca2+ current.
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In order to check for the presence of a low threshold T-type current, 100 ms depolarizing voltage steps were applied from a holding potential of -80 mV (n = 6). In no case was a rapidly decaying inward current evoked at potentials more negative than -40 mV, indicating that no T-type channel was expressed in cultured ORNs.
Outward currents
In standard Ringer, all ORNs from which recordings were made exhibited a
sustained voltage-activated outward current and 10% of ORNs (n =
12) exhibited a transient outward current after depolarizing voltage steps.
When K+ was replaced with Cs+ + TEA in the recording
pipette, the outward current was almost completely inhibited (11.9 ±
8.6 pA at + 60 mV, n = 18), indicating that the outward current was
carried by K+ ions. The analysis of outward current revealed three
different K+ currents: a Ca2+-activated K+
current, a delayed-rectifier K+ current and an A-like current.
Ca2+-activated K+ current
The sustained voltage-dependent outward current activated at -40 mV and
exhibited no or a weak decrement during the 100 or 200 ms voltage steps,
indicating a slow inactivation (Figure
4a). In every recorded ORN it showed an N-shaped
I—V curve (Figure
4c), as typically observed when a Ca2+-gated
K+ current is activated. The current peaked between 30 and 40 mV,
with an amplitude ranging from 202 to 2068 pA (734 ± 483 pA, n
= 30). The I—V curve became a simple rising function within a
minute after the addition to the bath of two Ca2+ channel
inhibitors: 1 mM Co2+ (n = 7;
Figure 4b, c) or 10 µM
Cd2+ (n = 2). Co2+ had no effect at 100 µM
(n = 2). In standard Ringer, the N shape of the I—V
curve faded spontaneously within a few minutes with a similar time course to
Ca2+ current rundown and progressively became a simple rising
function (Figure 4e). The
Ca2+-dependent component, calculated as the subtracted
Co2+-sensitive outward current, showed a bell-shaped
I—V curve (Figure
4d), whose maximal amplitude, at +40 mV, was 605 ± 351 pA
(n = 7). This sustained current was classified as a
Ca2+-dependent K+ current. The potential at which the
Ca2+-dependent K+ current was maximum was 40 mV higher
than the potential that elicited the maximum of Ca2+ current in
normal Ringer. Therefore, the Ca2+-dependent K+ current
was also dependent on the membrane potential.
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Two pharmacological agents were used to determine if intracellular
Ca2+ stores were involved in the generation of
Ca2+-dependent K+ current, as observed in vertebrate
ORNs (Zufall et al.,
2000). Thapsigargin, a potent inhibitor of Ca2+-ATPases
that mediate the uptake of cytosolic Ca2+ into endoplasmic
reticulum stores, and ryanodine, an inhibitor of Ca2+ release from
sarcoplasmic reticulum, were applied in the bath. Neither 1 µM thapsigargin
(n = 4) nor 20 µM ryanodine (n = 6) were found
significantly to modify the magnitude of the sustained outward current.
Therefore, the Ca2+-dependent K+ current did not depend
on the release of intracellular Ca2+.
Delayed-rectifier K+ current
In normal Ringer, depolarizations beyond potentials that activate
Ca2+-dependent K+ current (
80 mV) activated a
sustained current with a slower kinetics than weaker depolarizations
(Figure 5a). This slowly
activating Ca2+-independent K+ current was isolated
after the addition of 1 mM Co2+. It activated between -30 and -10
mV (Figure 5b) and had an
I—V relation that followed single exponential curves
(Figure 5c). The mean amplitude
of the current elicited by a step to +60 mV from a holding potential of -60 mV
at least 2 min after addition of 1 mM Co2+ in the bath was 111
± 67 pA (n = 7). In Ca2+-free Ringer, a similar
current had an amplitude of 124 ± 65 pA (n = 12). The
Ca2+-independent sustained outward current presented the
characteristics of the delayed-rectifier K+ current. This current
had no run down, was not inhibited by the addition of 5 mM 4-aminopyridine
(4-AP; n = 3) and was strongly reduced by the addition of 20 mM TEA
(n = 5).
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Transient K+ current (A-like current)
In standard Ringer, 10% of neurons (n = 12) exhibited a
transient outward current at the beginning of depolarizing voltage steps
(Figure 6a). This transient
outward current typically inactivated in <40 ms and was followed by a
sustained K+ current. The ratio between peak and sustained currents
varied considerably between individual ORNs. In more than half of the ORNs
where it appeared to be absent, a transient outward current was unmasked by
equimolar replacement of all external Ca2+ ions with
Ba2+ ions (9 out of 15 ORNs;
Figure 7b) or after the
addition of 10 µM Cd2+ in the bath (one out of two ORNs). The
transient outward current was completely inactivated when the holding
potential was set to values equal or more positive than -40 mV
(Figure 7d) and it was
inhibited by the addition of 5 mM 4-AP to the bath (n = 3;
Figure 7e). Thus, the transient
outward current presented similarities with the A-current first described in
gastropod neurons (Connor and Stevens,
1971). In contrast to Cd2+ treatment or replacement of
extracellular Ca2+ with Ba2+, the A-like current was
never unmasked by the addition of Co2+ to the bath. On the
contrary, when visible in standard extracellular solution, the A-like current
was inhibited by 1 mM Co2+ application (n = 2).
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Hyperpolarization-activated current
No inward current was activated by 200 ms hyperpolarization up to -150 mV.
However, since inward rectifying K+ current (Ih) activates
slowly in frog ORNs (Trotier and
Døving, 1996), we also tested the effects of longer
hyperpolarization pulses. Still, no current was activated during 5 s
hyperpolarizing pulses from -70 to -110 mV in 10 mV increments (n =
5). The mean current measured during the second half of the 5 s pulses to -110
mV was -2.3 ± 2.7 pA.
Responses to olfactory stimulation
Cultured neurons were tested for responses to pheromone stimulation. A mixture of the major component of the female sex pheromone, Z11-16:Ac, and the PBP that specifically binds this compound in vitro, MbraPBP1, was used as pheromone stimulus. In the cell-attached configuration at 0 mV holding potential, no ion channel openings were recorded from the soma before stimulation (Figure 8a). After pheromone stimulation, inwardly directed ion channel openings with rapid flickering activity were recorded in 3 out of 14 cells (Figure 8b). In the absence of bath exchange, this ion channel activity continued after the end of pheromone application. The amplitude histogram of channel openings indicated that only one channel was activated by pheromone stimulation (Figure 8c). In the absence of pheromone application, such channel openings had never been observed in the cell attached configuration (n > 100).
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Our observations indicate that ORNs isolated from 4-day-old pupae and grown in culture for at least 10 days expressed at least five different types of voltage-gated currents. Furthermore, these cultured neurons could respond to the main component of the pheromone by opening pheromone-gated ion channels. These are indications of preserved physiological functions in vitro.
The measured resting potential of M. brassicae ORNs, -62 ±
7 mV, was remarkably similar to the values measured from other insect ORNs:
-62 mV from M. sexta cultured ORNs
(Zufall et al.,
1991b) and -70 mV in situ from Drosophila ORNs
(Dubin and Harris, 1997).
Current clamped ORNs usually displayed spontaneous fluctuations in membrane
potentials that probably resulted from spontaneous channel activity. As
already observed in ORNs, a large measured input resistance (3.2 ± 2.2
G) implies that very few ion channels were open at rest. This confers a
very high sensitivity to these neurons, as in vertebrate ORNs where action
potential firing could be elicited by injections of only 3 pA
(Firestein and Werblin,
1987).
A transient inward current was blocked by TTX and was not observed in
Na+-free Ringer. It was classified as an Na+ current
(INa). The morphology of M. brassicae ORNs in
vitro, with long (several hundreds of micrometers) and thin processes
prevented good space clamping of these neurons, similarly to in situ
whole-cell recordings of Drosophila ORNs
(Dubin and Harris, 1997).
Therefore, INa amplitude (between 35 and 300 pA) was
probably underestimated. Action potentials recorded from some neurons under
voltage-clamp whole-cell conditions indicate that the site of generation of
action potentials is not located on the cell body. This is consistent with the
findings that Na+ channels were never observed on the soma of
M. sexta cultured ORNs (Zufall
et al., 1991b) or isolated mouse ORNs
(Maue and Dionne, 1987b).
Furthermore, in isolated lobster ORNs, the amplitude of Na+ current
depended on the presence of a remnant axon and not on the size of the soma
(McClintock and Ache,
1989).
A sustained TTX-resistant inward current was blocked by Co2+.
This current could also be carried by Ba2+ and exhibited a rapid
run-down. It was classified as Ca2+ current
(ICa). This current activated between -40 and -30 mV and
had a maximum amplitude at 0 mV that was shifted to +10 mV when 20 or 40 mM
Ba2+ was added to the bath. The activation threshold of
ICa was closer to that of high-voltage-activated (HVA)
Ca2+ currents (-30 mV) than to that of low-voltage-activated
(LVA) Ca2+ currents (-60 mV)
(Wicher et al.,
2001). Moreover, it presented a slow inactivation during 100 ms
steps. Thus, ICa presented characteristics close to HVA
Ca2+ currents. No LVA (T-type) Ca2+ current was
discovered. A similar small voltage-gated Ca2+ current, with peak
Ca2+ current <10 pA, has been described in fish
(Corotto et al.,
1996). This is much smaller than in toad ORNs, where
Ca2+ currents were reported to be >200 pA
(Delgado and Labarca, 1993).
Despite its small amplitude under physiological conditions (-4.5 ± 6 pA
at 0 mV), ICa plays an important role in M.
brassicae ORNs by activating a large Ca2+-dependent
K+ current. Voltage-gated Ca2+ currents have been
characterized in a number of vertebrate ORNs, as well as in squid and lobster
ORNs (Schild and Restrepo,
1998), but had never been described in insects. The
Ca2+ current may have been overlooked due to its small amplitude in
insects. In recordings from M. sexta ORNs
(Zufall et al.,
1991b), voltage-gated Ca2+ channels may have been
inhibited by concanavalin A, used to coat the substrate on which ORNs were
cultured (Stengl and Hildebrand,
1990). Such an inhibition has been demonstrated in a ciliate
(Ivens and Deitmer, 1986).
Outward currents were much larger than inward currents and were entirely
blocked by loading the cell with Cs+, indicating that they were
carried by K+. We have identified three types of K+
current on the basis of their kinetics and pharmacology: (i) a
Ca2+-dependent K+ current (IK(Ca));
(ii) a delayed-rectifier K+ current (IK,DR);
and (iii) a rapidly inactivating K+ current
(IA). The relative amplitudes of these three K+
currents varied considerably from cell to cell, but the main component of the
sustained outward current was always IK,Ca. The
association of these three different K+ currents or ion channels in
ORNs appears to be conserved from vertebrates [rodents
(Maue and Dionne, 1987b; Lynch
and Barry, 1991a,
b), fish
(Miyamoto et al.,
1992; Corotto et al.,
1996) and amphibians
(Firestein and Werblin, 1987;
Schild, 1989)] to
invertebrates [squid (Lucero and Chen,
1997) and insects (Zufall
et al., 1991b)].
Our observation of IK(Ca) in M. brassicae is
the only characterization of the macroscopic Ca2+-dependent
K+ current in insect ORNs. This current was absent in
Ca2+-free Ringer and was completely inhibited by 1 mM
Co2+. By contrast, in M. Sexta cultured ORNs,
IK(Ca) is not blocked by Cs+ or any other
conventional blockers (Stengl,
1993), suggesting differences between moth species. In M.
brassicae, IK(Ca) activated very fast, suggesting that
Ca2+-dependent K+ channels are colocalized with
Ca2+ channels. The absence of effect of thapsigargin and ryanodine
on outward current amplitude indicates that IK(Ca) did not
depend on intracellular Ca2+ stores. IK(Ca) was
dependent both on potential and Ca2+, as indicated by the shift to
the right by +40 mV of I-V curves of IK(Ca)
compared to those of ICa. Potassium channels that may
account for IK(Ca) have been described in invertebrate
ORNs. In Locusta migratoria ORNs, a K+ channel located on
soma and dependent both on membrane potential and intracellular
Ca2+ concentration, was classified as a maxi-K+ channel
due to its high conductance (180 pS) and strong K+ selectivity
(Wegener et al.,
1992). This channel was half-activated for a free Ca2+
concentration of 10-7 M. A similar Ca2+ and
voltage-dependent K+ channel, with a mean conductance of 215 pS,
was identified in lobster ORNs (McClintock
and Ache, 1989). By contrast, in M. sexta a
Ca2+-activated K+ channel with a smaller conductance, 66
pS, showed little voltage dependence
(Zufall et al.,
1991b). In mouse ORNs, two different Ca2+-activated
K+ channels have been characterized, one voltage-dependent and the
other voltage-insensitive (Maue and
Dionne, 1987b). While the macroscopic current activated in M.
brassicae ORNs demonstrates the presence of voltage-dependent
Ca2+-gated K+ channels, it does not rule out the
expression of their voltage-insensitive counter-parts. The expression of a
large and fast activating IK(Ca) in M. brassicae
ORNs suggests that this current contributes to the fast repolarization that
allows insect ORNs to respond to rapid stimulations
(Rumbo and Klaissling, 1989;
Marion-Poll and Tobin, 1992).
The sensillum lymph that bath outer dendritic segments in situ has an
unusually high (200 mM) K+ concentration
(Kaissling and Thorson, 1980).
Thus, to be involved in repolarization, K+ channels must be located
in membranes exposed to low external K+ levels, such as the inner
dendritic segment, the soma or the axon. In bursting neurons,
Ca2+-activated K+ channels have also been implicated in
the control of interspike interval and firing frequency
(Hille, 1992).
A slowly activating K+ current was isolated in all neurons by
blocking IK(Ca) either with 1 mM Co2+ or the
replacement of external Ca2+ with Ba2+. Both procedures
selectively blocked IK(Ca) without much effect on the
remaining sustained outward current that had the characteristics of a
delayed-rectifier K+ current, IK,DR. It
activated between -30 and -10 mV. At +60 mV, the amplitude of
IK,DR averaged 111 ± 67 pA after addition of 1 mM
Co2+ and 124 ± 65 pA in Ca2+-free Ringer.
Delayed-rectifier K+ channels were characterized from invertebrate
ORNs. These 30 pS channels were the most common in cell-attached recordings
from the soma of cultured M. sexta ORNs
(Zufall et al.,
1991b). In lobster ORNs, delayed-rectifier K+ channels
had a lower conductance of 9.7 pS
(McClintock and Ache,
1989).
A transient outward current was clearly visible in 10% of ORNs when
recorded in standard external Ringer. The same transient current, with
inactivation kinetics, voltage dependence and 4-AP sensitivity similar to an
A-current (Hille, 1992;
Wicher et al., 2001)
was unmasked in more than half of ORNs after inhibition of
IK(Ca) in Ca2+-free Ringer. The relative
amplitude of IA to the complete outward current was highly
variable. This current was blocked by 1 mM Co2+. However,
IA was not dependent on Ca2+ concentration,
since it could be observed in Ca2+-free Ringer. Therefore, the
effect of Co2+ was most likely direct on transient K+
channels. IA is the first voltage-dependent current
expressed in immature insect ORNs (Zufall
et al., 1991a). However, it remains unclear if all
immature ORNs express this current. It was isolated from 50% of ORNs in larval
salamanders (Firestein and Werblin,
1987) and was not found in toad
(Delgado and Labarca, 1993) or
lobster ORNs (McClintock and Ache,
1989). Our sampling was too small to study a correlation between
IA amplitude and the age of ORNs. Developmental studies
are needed to determine if two different types of neurons exist or if
IA amplitude decreases to zero during neuron lifetime.
IA currents are classically described as being involved in
shaping action potentials and in modulating spike frequencies
(Connor and Stevens, 1971;
Hille, 1992). It may also
contribute, in cooperation with IK(Ca), to the
repolarization of action potentials, provided that the IA
channels are located in situ in the inner dendritic segment, the soma
or the axon.
A Cl- channel, independent from the membrane potential, was
characterized in lobster ORNs (McClintock
and Ache, 1989). In vertebrate ORNs, the cAMP-mediated olfactory
transduction cascade involves entry of Ca2+ through
cyclic-nucleotide-gated channels and leads to the opening of
Ca2+-gated chloride channels
(Kleene and Gesteland, 1991;
Kleene, 1993;
Lowe and Gold, 1993). Opening
of these channels depolarizes the cell, since the reversal potential for
Cl- is more positive than the resting potential. In our recording
conditions, when internal K+ was replaced by Cs+ and
TEA, a small sustained outward current remained. This outward current could be
carried either by K+, due to incomplete dialysis of the
Cs+ pipette solution with the cytosol, or by Cs+ if its
permeability through K+ channels is different from zero, as has
been already demonstrated in squid ORNs
(Lucero and Chen, 1997). A
third possibility would be that this outward current was carried by
Cl-. However, because of its small amplitude (11.9 ± 8.6 pA
at +60 mV, n = 18), we did not attempt to block this current with
Cl- channel blockers. Regardless, under the conditions tested,
M. brassicae ORNs did not display a significant Cl-
conductance.
In contrast to vertebrate (Lynch and
Barry, 1991c; Miyamoto et
al., 1992; Trotier et
al., 1993; Trotier and
Døving, 1996) and lobster ORNs
(Corotto and Michel, 1994),
neither an inwardly rectifying K+ current, nor a
hyperpolarization-activated cation conductance were observed in M.
brassicae ORNs. A cAMP- and hyperpolarization-activated ion channel, with
a weak selectivity for K+ over Na+, has been cloned from
a cDNA library of Heliothis virescens
(Krieger et al.,
1999). In situ hybridization revealed a localization of
this channel in cell bodies beneath sensilla, possibly ORNs. Our experiments
showed no indication of such a channel in M. brassicae ORNs. If it is
present, this channel must be expressed at too low a density to elicit any
macroscopic current. By contrast, it is tempting to speculate that this ion
channel is, rather, expressed in accessory cells. This would be consistent
with our findings of hyperpolarization-activated conductances in accessory
cells (Lucas, 1999).
We have begun to study the responsiveness of cultured ORNs to pheromones.
The major component of the pheromone of M. brassicae, Z11-16:Ac,
elicited a response in 3 out of 14 ORNs. Non-responding neurons were perhaps
sensitive to minor pheromone components or to heterospecific compounds that
inhibit male attraction to pheromone sources. In situ, these
compounds activate ORNs insensitive to Z11-16:Ac
(Renou and Lucas, 1994).
Responses consisted of the opening of inwardly directed ion channel openings
after pheromone application. This is in good agreement with previous
recordings of olfactory responses from M. Sexta cultured ORNs
(Stengl et al.,
1992). In the absence of bath exchange, channel activation by
pheromone application lasted for several minutes and did not show any sign of
adaptation. Channels opened in long bursts and presented a flickering activity
similar to pheromone-dependent cationic channels recorded from the dendritic
membranes of ORNs (Zufall and Hatt,
1991) or from the soma of cultured ORNs
(Stengl, 1993). It is
conceivable that ion channels located in dendritic membranes of ORNs in
situ might be expressed on the soma of cultured neurons. This has been
demonstrated in cultured lobster ORNs, where odors evoked currents in ORNs
without processes, suggesting that these neurons could in vitro
insert all the elements of the transduction cascade into their soma, including
those normally confined to processes (Fadool et al., 1993). Our
in vitro preparation will allow the study of the role and specificity
of PBPs in pheromone activation.
This work extends the number of invertebrate species, that included one single moth species, in which voltage-gated currents were characterized in ORNs. This in vitro preparation and these results will give the basis for future studies on olfactory transduction, as well as for dissecting the mechanisms of action of olfactory modulators or blockers of pheromone responses in insects.
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Acknowledgments |
|---|
The authors would like to thank Monika Stengl for her generous gift of the cell line MRRL-CH1 used to condition culture medium, Patricia Nagnan-Le Meillour for her gift of recombinant Mbra-PBP1, Jan Dolzer, Frédéric Marion-Poll, Michel Renou, Didier Trotier, Kyrill Ukhanov, Jürgen Ziesmann and Frank Zufall for help with technical problems and for valuable discussions or comment on a previous version of the manuscript, Kevin Kelliher for English correction and Taylor Quadjovie for insect rearing.
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References |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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