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The Journal of Neuroscience, April 1, 2003, 23(7):2932
Contribution of Excitatory Chloride Conductance in the
Determination of the Direction of Traveling Waves in an Olfactory
Center
Satoshi
Watanabe,
Tsuyoshi
Inoue, and
Yutaka
Kirino
Laboratory of Neurobiophysics, Graduate School of Pharmaceutical
Sciences, The University of Tokyo, Bunkyo-ku, Tokyo 113-0033, Japan
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ABSTRACT |
Traveling waves have been found in the CNS of vertebrates
and invertebrates. In the olfactory center [procerebrum (PC)] of the
terrestrial slug Limax, periodic waves travel from the
apex to the base with a frequency of ~0.7 Hz. The oscillation and
propagation of waves have been thought to be mediated by the mutual
connections of bursting neurons in the PC. The direction of the wave is
Cl dependent, because lowering the
Cl concentration in the medium reverses the
direction. The bursting neurons have a Cl
channel-coupled glutamate receptor (GluClR), and, using a calcium imaging technique, the receptor was found to be excitatory. Activation of the GluClR with its selective agonist ibotenate resulted in an
increased frequency of the oscillatory neural activity recorded as a
periodic local field potential. Depletion of cytoplasmic Cl with Cl-free saline
abolished all of the ibotenate-induced effects. Perforated-patch-clamp recording in single PC neurons revealed a spatial difference in the
Cl-dependent periodic depolarizations in the
bursting neurons, with a higher amplitude in the apical region. These
results suggest the involvement of excitatory GluClRs in the
unidirectional propagation of waves in the PC.
Key words:
neural oscillation; wave propagation; olfaction; mollusk; procerebrum; glutamate; ibotenate; perforated patch recording; fluorescent Ca2+ indicator
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Introduction |
In many CNSs, there are oscillatory
neural activities that are synchronized over a large area of the
network (for review, see Ritz and Sejnowski, 1997 ). Often these network
oscillations have phase lags within the network, resulting in traveling
waves. Traveling waves arise as a result of the mutual interactions of oscillatory local circuits (Ermentrout and Kleinfeld, 2001) and have
been found in olfactory centers (Kleinfeld et al., 1994; Lam et al.,
2000), in the visual cortex in vivo (Prechtl et al., 1997),
and in neocortical slices (Golomb and Amitai, 1997; Sanchez-Vives and
McCormick, 2000). The phase difference along the network changes as it
receives sensory inputs (Gervais et al., 1996). Such changes in the
spatiotemporal pattern could modify the relationship between individual
neurons constituting the network, making it possible to separate and
integrate the neural activities encoding different sensory signals
(Gelperin, 1999).
Analyses of mathematical network models with coupled oscillators have
clarified the basic properties of oscillatory networks. For example,
the local network with the highest intrinsic oscillatory frequency will
have the earliest phase (Ermentrout and Kleinfeld, 2001). However, to
fully understand the precise mechanisms underlying the traveling waves,
both the membrane properties of single neurons and synapses should be
characterized, but we have yet only partial knowledge about any of
these parameters. Therefore, we analyzed the ionic basis of traveling
waves in the olfactory center of the terrestrial mollusk
Limax.
The olfactory center of the slug Limax shows regular
oscillatory activity (Gelperin and Tank, 1990), and the oscillation has a phase lag along the procerebrum (PC), which is normally advanced in
the apical region (Kleinfeld et al., 1994; Kawahara et al., 1997; Inoue
et al., 1998). The direction of the wave propagation is therefore from
the apex to the base. However, lowering the Cl concentration in the medium reverses
the direction (Kleinfeld et al., 1994). Also, when the PC is locally
lesioned, the locus of the lesion becomes the origin of wave
propagation (Kleinfeld et al., 1994). These results suggest that the
network architecture itself allows bidirectional wave propagation, and
the direction depends on the difference in the excitability of the
local networks, which depends on Cl. The
oscillatory activity of the PC is produced by a synchronized oscillation in the bursting PC neurons, which constitute ~10% of the
total PC neurons and are presumed to be local inhibitory interneurons
(Kleinfeld et al., 1994; Watanabe et al., 1998). The activity level of
the bursting neurons determines the oscillation frequency (Watanabe et
al., 2001). The bursting neurons, but not the other type of PC neurons
(nonbursting neurons), have a type of glutamate receptor coupled to a
Cl channel (GluClR) that is activated by
ibotenate (Watanabe et al., 1999). Moreover, as has been found in some
mammalian neurons, Cl conductances could
have excitatory actions (Owens et al., 1996). Therefore, in the present
work, we examined how Cl-mediated
mechanisms are involved in the traveling waves.
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Materials and Methods |
Slugs Limax valentianus were hatched and cultured at
our laboratory. For dissection, the slug (0.5-1.0 gm) was anesthetized by injection of Mg2+ buffer solution that
contained the following (in mM): 60 MgCl2, 5 glucose, and 5 HEPES, pH 7.6, into the
body cavity. The circumesophageal ganglia were isolated and further
dissected in a dish filled with Mg2+
buffer. The cerebral ganglion was isolated from the other ganglia, and
the sheath covering the PC was removed with fine forceps. All
recordings were made at room temperature (20-24°C).
The saline solution used for the recordings contained the following (in
mM): 70 NaCl, 2 KCl, 4.9 CaCl2, 4.7 MgCl2, 5 glucose, and 5 HEPES, pH 7.6. Drugs were
bath applied to the ganglion, which was placed in a chamber (~0.2 ml
in volume) that was continuously perfused. Because ibotenate is the
only agonist known to activate the GluClR in Limax PC
neurons (Watanabe et al., 1999), different batches of ibotenic acid
from three suppliers (Sigma, St. Louis, MO; Research
Biochemicals, Natick, MA; and Tocris Cookson, Bristol, UK) were used,
and we confirmed that all of these gave similar results. Quisqualic
acid was supplied by Sigma, and
L-glutamic acid sodium salt was supplied by
Nakalai Tesque (Kyoto, Japan). For the
Cl-free saline,
Cl was totally replaced by gluconate.
For the Na+-free saline,
Na+ was replaced by
tris[hydroxymethyl]aminomethane (Trizma base; Sigma).
The local field potential (LFP) of the PC was recorded from the
posterior surface of the PC using a glass electrode filled with saline
solution (Kawahara et al., 1997). The signal was differentially amplified and bandpass filtered at 0.5-30 Hz. The signals were recorded on a DAT recorder (PC204Ax; Sony, Tokyo,
Japan). The frequency of the LFP oscillation was evaluated using at
least five cycles of oscillations just before and 10 sec after the
onset of the perfusion with ibotenate. The frequency was calculated as
the inverse of the averaged cycle period.
Calcium imaging in single PC neurons was done in a dissociated culture
of PC neurons (Rhines et al., 1993). The PC was desheathed and isolated
in Mg2+ buffer, digested with 1% protease
(type IX; Sigma) at 34°C for 1 hr, and dissociated by
trituration. Fifty microliters of cell suspension was placed on a
poly-lysine-coated glass-bottomed dish (MatTek, Ashland,
MA) at the final density of two PCs per dish, and, 2 hr later, 2 ml of
saline solution was added to the dish, after which the cells were
further cultured for 1-2 d at 20°C. Cells were loaded for 12 min at
28°C with the AM of the Ca2+
indicator fura-2 (fura-2 AM; Dojindo, Kumamoto, Japan)
dissolved in saline solution at the final concentration of 5 µM. The cells were then postincubated in saline solution
for 20 min at 28°C. Image acquisition was performed with an imaging
system (MCID; Imaging Research, St. Catharines, Ontario,
Canada) equipped with a CCD camera (CCD-72; Dage-MTI,
Michigan City, IN) with an image intensifier head (C2400-68;
Hamamatsu Photonics, Hamamatsu, Japan) set on
an inverted microscope (IX 70; Olympus, Tokyo, Japan). A
filter set (XF04; Omega Optics, Brattleboro, VT) (excitation filters,
340 ± 7.5 and 380 ± 7.5 nm; dichroic mirror, 430 nm; and
emission filter, 510 ± 20 nm) was used for epifluorescent illumination, and the excitation filter was switched by a filter changer (Lambda-10; Sutter Instruments, Novato, CA). Image
sets were acquired every 1 or 2 sec during the session of 120 sec, for
the last 90 sec of which the drug solution was perfused. The typical
number of cells found in an image was between 100 and 1000. To detect
the cells showing a cytoplasmic Ca2+
concentration
([Ca2+]i) rise,
ratio
(F340/F380)
images at 0 and 80 sec were compared, and the cells showing an increase
in the ratio value by >0.2 were counted as responding cells.
Calcium imaging in the intact PC was performed using the AM of the
calcium indicator dye rhod-2 (rhod-2 AM; Dojindo) by the method described previously (Inoue et al., 1998). The cerebral ganglion
was incubated with 50 µM rhod-2 AM and 0.025% cremophor EL (a dispersing reagent; Sigma) for 40 min at 28°C.
Optical recording was performed using a MOS-based camera system
(HR-Deltaron 1700, Fuji Photo Film, Tokyo, Japan) and an
inverted microscope (IX-70; Olympus) equipped with a 10×
(numerical aperture, 0.40) or 20× (numerical aperture, 0.75) objective
lens and a 100 W halogen lamp with a stabilized DC power supply. A
filer set (U-MNG; Olympus) with an excitation filter
(530-550 nm), a dichroic mirror (570 nm), and an emission filter
(>590 nm) was used. The images were acquired at the rate of 76.8 msec/frame. The original images (128 × 128 pixels) were converted
off-line into ratio ( F/F) images by
dividing each image by the time-averaged image, using a custom-made program for Matlab (MathWorks, Natick, MA). The peak
response normalized by the prestimulus amplitude of
Ca2+ oscillation was calculated from the
peak (Rpeak) and bottom
(Rbottom) ratio values before the
onset of perfusion with ibotenate and the peak ratio value during the
perfusion (Rpeak,IA) as
(Rpeak,IA  Rbottom)/(Rpeak Rbottom). The frequency of the
calcium oscillation was calculated in the same way as the LFP oscillation.
Nystatin perforated-patch recording of the bursting PC neuron was made
essentially by the method described previously (Watanabe et al., 1999).
We used nystatin as the ionophore because nystatin produces a fairly
low and stable access resistance. Whole-cell recording was not used in
the present study because of the low success rate. The channel formed
by nystatin permeates Cl (Horn and
Marty, 1988), and this allowed us to manipulate the cytoplasmic
Cl concentration
([Cl]i). With
the Cl-impermeable ionophore gramicidin
(Ebihara et al., 1995), we could not obtain an access resistance low
enough for analysis. First, the cerebral ganglion was isolated and the
sheath covering the PC was removed mechanically. The preparation was
placed on a microscope (BX50WI; Olympus) equipped with a
40× water immersion objective, and the electrode was visually guided
onto a bursting neuron. Bursting neurons were identified on the basis
of their soma size, which is larger than that of nonbursting neurons
(Watanabe et al., 1998). To characterize the contribution of
Cl conductance in the spontaneous
activity of bursting neurons, electrode solutions with two different
compositions were used. The high Cl
solution (80 mM Cl)
contained the following: 70 mM KCl, 5 mM
MgCl2, 5 mM HEPES, pH 7.6, and 250 µg/ml nystatin. The low Cl solution
(10 mM Cl) contained the
following: 70 mM K-gluconate, 5 mM
MgCl2, 5 mM HEPES, pH 7.6, and 250 µg/ml nystatin. The electrode resistance was 10-12 M , and
the access resistance was <200 M. As long as these recording
conditions are followed, the cytoplasmic
Cl concentration has been shown to
become quickly equilibrated with the electrode solution (Watanabe et
al., 1999). The recording was made in the current-clamp mode using a
patch-clamp amplifier (EPC-7; List Electronic, Darmstadt-Eberstadt,
Germany), and a negative DC current (<40 pA in amplitude) was injected
to the electrode to keep the membrane hyperpolarized to a fixed value (90 mV). The amplitudes of five cycles of depolarizations were measured in each recording, and their average was plotted against the
position of the cell body along the base-apex axis. The liquid junction potential (6 mV for the high Cl
solution and 19 mV for the low Cl
solution) was subtracted.
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Results |
Effect of GluClR activation on the local field potential of
the PC
In normal saline, a regular LFP oscillation of ~0.7 Hz was
recorded from the surface of the PC. The oscillation in the LFP is
thought to arise mainly from IPSPs in nonbursting neurons, which
are presumably caused by periodic bursting in bursting neurons. Perfusion with glutamate (50 µM to 1 mM)
suppressed the LFP oscillation. This is consistent with a previous
report (Gelperin et al., 1993) and has been explained as a tonic
hyperpolarization of the PC neurons attributable to the effect
of glutamate on quisqualate-sensitive receptors (Watanabe et
al., 1999). In two of nine preparations, however, glutamate slightly
enhanced the oscillatory frequency before the oscillation was
suppressed (Fig. 1A).
This could be attributable to activation of a different subtype of
glutamate receptor, presumably the GluClR. In fact, perfusion with
50-250 µM ibotenate augmented the frequency of
the LFP oscillation in all of the six preparations tested (Figs.
1B,
2B), possibly because of its selective effect on excitatory receptors. The LFP oscillation recovered to its initial level after the washout of ibotenate. These
results suggest that the effect of glutamate on LFP oscillation is both
excitatory and inhibitory, and the excitatory effect is mediated by the
GluClR.
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Figure 1.
Effect of glutamate receptor agonists on the LFP
oscillation of the PC. A, Perfusion with 1 mM glutamate (duration, 3 sec; indicated by the
horizontal bar) slightly enhanced the LFP frequency
(arrow), followed by a complete suppression. After the
washout for 30 sec, the oscillation recovered. B,
Perfusion with 200 µM ibotenate (duration, 3 sec;
indicated by the horizontal bar) strongly enhanced the
frequency of the LFP oscillation (arrow). After the
washout for 30 sec, the oscillation recovered to the initial
level.
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Figure 2.
Effect of ibotenate on the LFP oscillation is
Cl dependent. A, Perfusion with
ibotenate (indicated by the horizontal bar) in
Cl-free saline did not change the frequency of the
LFP oscillation. B, Frequency changes caused by
ibotenate in normal saline and in Cl-free saline.
Control and Ibotenate indicate the
frequencies before and during the perfusion with ibotenate. For the
Cl-free condition, only those preparations that
showed regular oscillations were included. C, Summary of
the frequency changes with ibotenate in normal saline and in
Cl-free saline. The frequency changes under these
two conditions were significantly different (t test;
*p < 0.05).
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Because the ibotenate-induced current in the bursting neurons is
carried by Cl ions, the effects of
ibotenate on the LFP oscillation were confirmed in a preparation
incubated with Cl-free saline. Because
[Cl]i in neurons
easily becomes equilibrated with the external solution (Alvarez-Leefmans et al., 1988), the
[Cl]i of the
neurons under this condition is supposed to be virtually zero and the
Cl conductance to be no longer
functional. In control preparations in normal saline, ibotenate caused
a 20-580% increase in the oscillation frequency compared with the
resting frequency (n = 6). In the preparation that had
been incubated with Cl-free saline, the
resting LFP oscillation tended to be slower and often irregular, and
only those preparations showing regular oscillations were used for
analysis. In these preparations, ibotenate did not cause >15% changes
in the oscillatory frequency (n = 5) (Fig. 2). In three
other preparations that showed irregular oscillations, no changes were
observed in their excitability. Therefore, the effects of ibotenate are
mediated by Cl and unlikely to be
mediated by unidentified receptors permeable to other ions.
Effect of GluClR activation in isolated PC neurons
Because the network activity is a product of various types of
neural interaction, we next asked whether individual PC neurons show
excitation to ibotenate. We used a dissociated culture of PC neurons to
observe the direct effect of ibotenate on isolated PC neurons.
Perfusion with ibotenate evoked an increase in
[Ca2+]i in
4.0 ± 1.4% of the cells (mean ± SEM; summary of seven
populations of cells; a total of 2968 cells were measured) (Fig.
3). Although a small fraction of the
cells also showed a rise in
[Ca2+]i in control
experiments (1.0 ± 0.5%; n = 4) attributable to spontaneous activity, the fraction of cells showing a rise in [Ca2+]i was
significantly higher than in the control (t test;
p < 0.05). Perfusion with quisqualate or glutamate
evoked a rise in
[Ca2+]i in a much
smaller fraction of the cells (0.6 ± 0.3%, n = 3; or 0.2 ± 0.1%, n = 3, respectively).
Perfusion with high K+ saline solution
(containing 10 mM
K+) evoked a rise in
[Ca2+]i in
80.7 ± 5.4% of the cells (n = 6). These results
indicate that ibotenate causes a rise in
[Ca2+]i in a small
fraction of PC cells, whereas glutamate and quisqualate have no
excitatory effects.
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Figure 3.
Changes in
[Ca2+]i with ibotenate in cultured PC
neurons revealed by calcium imaging. A-C, Example of
four neurons that showed [Ca2+]i rises
in response to ibotenate (A). Traces of the ratio
(F340/F380)
values are shown. Subsequently, the same neurons were stimulated with
quisqualate (B) and high K+
saline (C). D, The percentage of
the cells that showed an increase in the ratio values by >0.2 in
response to various stimulus conditions. Note the small fraction of
cells in the control group, which are spontaneously active cells often
showing slow oscillations. Ibotenate caused
[Ca2+]i elevations in a significantly
higher fraction of the neurons than the control in normal saline
(t test; *p < 0.05), whereas
ibotenate had no significant (N.S.) effect on the cells
under the Cl-free condition.
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To confirm that the
[Ca2+]i elevation
induced by ibotenate is mediated by Cl,
intracellular Cl was depleted by
incubating the cells in Cl-free saline.
In the Cl-free control, 1.9 ± 0.9% of the cells (n = 6) showed spontaneous rises in
[Ca2+]i, whereas
1.2 ± 0.5% of the cells (n = 6) showed a rise in
[Ca2+]i with
ibotenate, which was not significantly different from the control.
Perfusion with high K+ saline still evoked
a [Ca2+]i
elevation in 68.9 ± 5.5% of the cells (n = 4) in
Cl-free saline. These results indicate
that the [Ca2+]i
elevation with ibotenate requires functioning
Cl conductance.
Effect of GluClR activation in the whole PC
Calcium imaging in the intact PC revealed periodic
[Ca2+]i waves
propagating in the apex-to-base direction (Fig.
4). These
[Ca2+]i waves have
been shown to be synchronous with the membrane potential oscillation
(Inoue et al., 1998), and the
[Ca2+]i signal is
thought to arise mainly from bursting neurons because they always burst
periodically. Calcium imaging with a single neuron resolution has
revealed periodic
[Ca2+]i
oscillations in bursting neurons (Wang et al., 2001). Perfusion with
ibotenate elevated the
[Ca2+]i level as
well as the frequency of
[Ca2+]i
oscillation (Fig. 5). The
ibotenate-induced
[Ca2+]i elevation
was higher in the apical region of the PC than in the basal region when
normalized by the amplitudes of the periodic events before stimulation
(29.6 ± 8.5% for the regions in the apical half and 11.6 ± 6.1% for the regions in the basal half; paired t test;
p < 0.05; n = 10, only preparations
with stable baseline fluorescence were included; total number of
experiments, 13). The frequency measured at the apical region rose by
135 ± 55% (n = 13). At the peak of the
ibotenate-induced
[Ca2+]i elevation,
the oscillation frequency was often greater in the apical region than
in the basal region, indicating that some of the
[Ca2+]i waves
initiated at the apical site failed to propagate to the basal region.
Such locally restricted events were observed in 7 of 13 preparations.
Local events were also observed in one of four other preparations in
which recording was made only for part of the period of ibotenate
perfusion. This could be attributable to a limited capability of the
neural connections to transmit excitation. The events that failed to
propagate to the basal site did not seem like the double events
described previously (Kleinfeld et al., 1994), because the intervals
between the events were still fairly uniform rather than being an
alternation of long and short intervals.
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Figure 4.
Calcium oscillation in the PC. A,
Fluorescence image of the PC stained with rhod-2 AM, observed from the
dorsal side. CM, Cell mass; TM, terminal
mass; IM, internal mass. Scale bar, 400 µm. The
schemes on the right indicate the location of the PC in
the cerebral ganglion (top) and the direction of the
observation of the PC (arrow in the
bottom), which visualizes the three layers of the PC.
TN, Tentacle nerve. B, Time courses of
the relative fluorescence (F/F)
at the three outlined regions shown in the image in
A, indicating the time lag in the
[Ca2+]i signals between the regions.
C, A series of images taken every 76.8 msec. The
pseudocolor indicates the relative fluorescence. Note that the strong
[Ca2+]i rise is restricted to the cell
mass, the layer that contains the cell bodies of the PC neurons, and
the neurites of the bursting neurons project therein.
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Figure 5.
Calcium elevation with ibotenate in the intact PC.
A, Fluorescence image of the PC used for the recording.
Scale bar, 400 µm. B, Time courses of normalized
fluorescence for the three outlined regions shown in
A. The traces have been normalized to give the same
resting amplitudes of the [Ca2+]i
events. The apical region (region 1) shows the highest
[Ca2+]i rise, and one event in
region 1, indicated by the asterisk,
failed to propagate to the basal regions (regions 2 and
3).
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In Cl-free saline, spontaneous
[Ca2+]i
oscillation still occurred in the PC. However, the direction of wave
propagation was reversed and became base-to-apex (n = 3), and the frequency tended to be lower than in normal saline (Fig.
6B). Perfusion with
ibotenate (200 µM) in
Cl-free saline had little or no effect
on the [Ca2+]i
oscillation in any of the three preparations (Fig. 6C). The direction of wave propagation was still in the base-to-apex direction. These results also indicate that Cl is
required for the
[Ca2+]i elevation
caused by ibotenate in the intact PC. In
Na+-free saline, no spontaneous activity
was observed, indicating that spontaneous oscillatory activity requires
extracellular Na+ (data not shown).
Perfusion with ibotenate caused no
[Ca2+] increase in the
Na+-free condition.
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Figure 6.
Effect of ibotenate on
[Ca2+]i in
Cl-free saline. A, Fluorescence
image of the PC, showing the regions selected for the traces in
B and C. Scale bar, 200 µm.
B, Time course of a
[Ca2+]i event (relative fluorescence)
in Cl-free saline. These traces are from the
three outlined regions shown in A and
also an expansion of the event with the asterisk in
C. The [Ca2+]i rise
occurs earlier in the basal region than in the apical region.
C, Time course of the relative fluorescence in the
selected regions in a slower scan, indicating that ibotenate has no
effect in the absence of Cl.
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Cl-dependent potentials in the
bursting neuron
To clarify the contribution of Cl
conductance to the propagation of waves, the activities of single
bursting neurons in the PC were recorded by patch-clamp recording using
electrodes containing a high (80 mM) or low (10 mM) concentration of Cl.
When recorded with the high Cl electrode
and a hyperpolarizing DC current to keep the membrane potential
hyperpolarized to 90 mV, subthreshold depolarizing potentials were
recorded. The amplitudes of these potentials were highly variable
between different neurons, and some bursting neurons showed very large
amplitudes (21.8 ± 2.7 mV; n = 16) (Fig.
7A). In these neurons, the
potentials tended to become larger at a more hyperpolarized potential
with a larger current injection, suggesting that these potentials are
mainly synaptic potentials. In the bursting neurons showing relatively
small potentials, on the other hand, the amplitudes did not depend much
on the membrane potential. With the low
Cl electrode (Fig. 7B), the
amplitudes of the spontaneous potentials were generally small
(12.3 ± 1.3 mV; n = 16), and the amplitudes showed little voltage dependence. These results suggest that, in
bursting neurons, Cl channels are
activated periodically to cause periodic depolarizations, although Cl-independent components also
exist.
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Figure 7.
Spontaneous activities of bursting neurons
recorded with the current-clamp mode of perforated patch recording. A
negative DC current was injected to adjust the bottom level of the
potential to 90 mV. A, An example of a bursting neuron
recorded with a high Cl electrode. This neuron was
positioned at 30% from the apex of the PC. B, An
example of a bursting neuron recorded with a low
Cl electrode. This neuron was located at 42% from
the apex of the PC. C, A plot of the amplitude of the
periodic depolarizations against the location in the PC recorded with a
high Cl electrode. Neurons in the apical area
showed larger amplitudes, and the correlation was significant
(p < 0.05). Each point is
the average of the amplitudes of five events in a neuron. The waveforms
of the depolarizations were highly regular, and the SEMs for each data
point never exceeded 1 mV (data not indicated). D, A
plot of the amplitude of the periodic depolarizations against the
location in the PC recorded with a low Cl
electrode. Neurons in the apical area showed smaller amplitudes, and
the correlation was significant (p < 0.05).
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Interestingly, bursting neurons in the apical area showed larger
periodic depolarizations than did those in the basal region when
recorded with the high Cl electrode
(Fig. 7C). The correlation between the amplitude and the
location of the cell was statistically significant (Pearson's correlation coefficient; r = 0.531; p < 0.05). In contrast, the apical bursting neurons showed slightly
smaller depolarizations than the basal bursting neurons when the
neurons were recorded with the low Cl
electrode (r = 0.612; p < 0.05) (Fig.
7D). In the neurons located in the apical half of the PC,
the amplitudes of the depolarizations recorded with the high
Cl and low
Cl electrodes were significantly
different (26.2 ± 3.6 mV for high Cl and 9.9 ± 0.9 mV for low
Cl; t test; p < 0.01). In contrast, there was no significant difference in the
amplitudes in the bursting neurons in the basal half of the PC between
the high Cl and low
Cl conditions (17.3 ± 2.8 mV for
high Cl and 15.8 ± 2.2 mV for low
Cl; p > 0.1). These are
consistent with the results above, indicating that the direction of
wave propagation depends on Cl-dependent
excitability and is possibly related to the spatial difference in
excitability or the intrinsic oscillation frequency of the local circuits.
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Discussion |
In the present work, we presented evidence that GluClR acts as an
excitatory channel in the Limax PC neurons and that it is involved in the periodic wave propagation in the apex-to-base direction. To summarize the results, (1) ibotenate evoked a
[Ca2+]i elevation
in both isolated PC neurons (Fig. 3) and in the intact PC (Fig. 5), and
this elevation was dependent on extracellular Cl; (2) ibotenate evoked a stronger
excitation in the apical region of the PC (Fig. 5); (3) normally, the
wave propagates from the apex to the base, whereas in the absence of
functional Cl conductances, the wave
propagates from the base to the apex (Fig. 6); and (4) apical bursting
neurons showed larger EPSP-like potentials whose amplitude depended on
the Cl concentration of the electrode,
whereas no Cl-dependent potentials were
observed in the basal bursting neurons (Fig. 7). All of these results
suggest that the excitatory Cl
conductance localized in the apical region is responsible for the
higher excitability (and hence the higher intrinsic oscillation frequency) of the apical network.
The difference in the intrinsic local frequency explains the direction
of wave propagation in a well documented manner (Ermentrout et al.,
1998). A detailed oscillator model of the PC is also consistent with
the propagation of waves in experiments under various conditions, including the low Cl condition, which
evokes a reversal in the direction of wave propagation (Ermentrout et
al., 1998). Consistent with the theory, the small pieces of local
circuits cut out from the PC have different intrinsic oscillatory
frequencies, with the highest frequency at the apical site and the
lowest frequency at the base (Ermentrout et al., 1998). Stronger
excitatory connections within the local circuit will result in a higher
frequency of the network.
The reversed wave propagation under the
Cl-free condition may be attributable to
a spatial gradient in the density of synaptic interactions mediated by
mechanisms other than GluClRs, more of which might be functioning in
the basal network. The Cl-independent
component of the periodic depolarizations could be mediated by
electrical synapses (Wang et al., 2000), as well as other chemical
synaptic mechanisms, including cholinergic transmissions (Watanabe et
al., 2001). The scheme explained above is summarized in Figure
8.
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Figure 8.
Scheme of the suggested mechanism for the
propagation of waves in the PC. Left, Under normal
conditions, the apical region has a higher intrinsic oscillatory
frequency attributable to a higher excitability caused by
Cl-mediated synaptic transmission. This results in
waves propagating in the apex-to-base direction. Right,
Under the Cl-free condition, in which
Cl-dependent synaptic transmission is blocked, the
basal region has a higher excitability, and this results in waves
propagating in the base-to-apex direction.
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In the mammalian neocortex, nonselective cation conductance mediated by
AMPA receptors makes a major contribution to the generation of
traveling waves, because they are strongly suppressed by the AMPA
receptor antagonist CNQX (Golomb and Amitai, 1997). It should be noted
that a network with mutual inhibitory connections could also produce
traveling waves by means of a rebound depolarization, as has been
demonstrated in a modeling study (Rinzel et al., 1998). However, the
propagation speed is much lower than in a network with mutual
excitatory connections, because neurons fire and transmit signals only
after a full recovery from the inhibitory input. Apparently, this mode
of wave propagation is not likely to occur in the PC neurons, because
there is no evidence for prominent hyperpolarizations or rebound
potentials in the bursting PC neurons. The possibility that
Cl conductance exerts its effect on wave
propagation through an inhibitory action is, therefore, unlikely. In
addition, recent work has shown that lowered
[Cl]i may lead
to a tonic influx of cations and to bursting activity (Beck et al.,
2001). This might explain the relatively extended duration of the
[Ca2+]i events
under the Cl-free condition.
Because only bursting neurons have GluClRs in the PC (Watanabe et al.,
1999), the neurons that showed
[Ca2+]i elevations
and triggered the increase in the oscillation frequency in response to
ibotenate should be bursting neurons. In a random sample, ~10% of
the total PC neurons were reported to be bursting neurons (Kleinfeld et
al., 1994). In the present report, we showed that approximately
one-half of the recorded bursting neurons showed a large-amplitude
oscillation using a high Cl electrode,
which was thought to be mediated by Cl
(Fig. 7C). These data are consistent with the result in
isolated PC neurons that ~4% of the total PC neurons showed
excitation after the application of ibotenate (Fig. 3). Thus far,
bursting and nonbursting neurons have been identified in the PC, and
only the former respond to ibotenate (Watanabe et al., 1999);
therefore, the results strongly suggest that the affected neurons in
the culture were bursting neurons. Recently, Wang et al. (2001)
demonstrated the existence of two morphologically distinct subtypes of
bursting neurons. Future studies are needed to relate the physiological and morphological characteristics of bursting neuron subtypes.
The GluClR in the Limax bursting PC neurons has similar
pharmacological and kinetic characteristics with those receptors found in many invertebrate neurons and muscles (Cleland, 1996). However, their roles have been mostly unknown. The ibotenate-induced excitation was thought to occur as a result of a relatively high equilibrium potential for Cl in the bursting
neurons, as has been demonstrated for the mammalian excitatory
GABAA responses (Luhman and Prince, 1991;
Leinekugel et al., 1995; Serafini et al., 1995; Chen et al., 1996;
Owens et al., 1996; Wagner et al., 1997). In mammalian neurons,
[Cl]i is
regulated by
K+/Cl and
Na+/K+/2Cl
cotransporters (Misgeld et al., 1986), and a developmental change in
the
Na+/K+/2Cl
cotransporters may explain the excitatory actions of GABA in specific
stages during development (Marty et al., 2002). Cytoplasmic Cl concentrations are highly variable
between neurons. For example, [Cl]i in two
identified Helix neurons were 11.2 and 24.7 mM (Kerkut and Meech, 1966). The latter value
would give a reversal potential of 42 mV in Helix saline,
and similar values are therefore possible in the Limax
neurons. Although excitatory Cl
conductances have been regarded as exceptional phenomena, our findings
that excitatory Cl conductance regulates
a large-scale network activity suggest a novel role for
Cl conductance in the CNS.
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FOOTNOTES |
Received Aug. 19, 2002; revised Dec. 18, 2002; accepted Jan. 14, 2003.
This work was supported by Grants-in-Aid 12048209, 12307053, 13210036, and 13771353 for Scientific Research from the Ministry of Education,
Culture, Sports, Science, and Technology of Japan.
Correspondence should be addressed to Satoshi Watanabe, Laboratory of
Neurobiophysics, Graduate School of Pharmaceutical Sciences, The
University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
E-mail: satoshi{at}mayqueen.f.u-tokyo.ac.jp.
T. Inoue's present address: Department of Neurosciences, Case Western
Reserve University, Cleveland, OH 44106.
| |
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TOP
ABSTRACT
Introduction
