Laboratory of Neurobiophysics, School of Pharmaceutical Sciences,
The University of Tokyo, Tokyo 113-0033, Japan
 |
INTRODUCTION |
Coherent and periodic oscillations
of electrical activity, generated by a population of interconnected
neurons, have been observed in some mammalian sensory and limbic
systems (Gray and Singer 1989
; Vanderwolf
1969
). The oscillation of membrane potentials in the olfactory
system is a ubiquitous phenomenon, widely observed in many species from
vertebrates to invertebrates (Freeman 1978
; Gelperin and Tank 1990
; Laurent and Naraghi
1994
). Recently, some physiological functions of the electrical
oscillations in information processing and storage have been
demonstrated. For instance, the long-term synaptic modification of the
Schaffer collateral-pyramidal cell pathway in the CA1 region of the
hippocampus is bidirectional and dependent on the phase of the
oscillatory activity (Huerta and Lisman 1995
). In the
present study, we report a new physiological function of the
oscillation, phase-dependent filtering of olfactory information, in the
procerebrum (PC) of the terrestrial mollusk Limax
marginatus. The PC is the olfactory center of
Limax and consists of approximately 105
interneurons that exhibit ongoing synchronous oscillatory activity (Gelperin and Tank 1990
).
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METHODS |
The slugs, L. marginatus, were anesthetized with
Mg2+ buffer injected into the body cavity, and
then the cerebral ganglion with the posterior olfactory nerve attached
was isolated and prepared as previously described (Watanabe et
al. 1998
). The activity of single PC neurons was recorded in
the current-clamp mode of the perforated patch-clamp recording
configuration (List Electronic, EPC-7 or EPC-8); the composition of the
pipette solution was as follows (in mM): 35.0 KCl, 35.0 K-gluconate,
5.0 MgCl2, 5.0 HEPES (adjusted to pH 7.6 with
KOH), and 250 µg/ml nystatin. The bursting (B) and nonbursting (NB)
neurons were identified from their electrical activity (Watanabe
et al. 1998
). On the other hand, synchronous oscillatory
activity in the PC was monitored by recording local field potentials
(LFPs) using a glass electrode whose tip diameter was approximately
20-50 µm. The olfactory nerve was stimulated electrically with a
conventional suction electrode, and the stimulus voltage and duration
were 1-5 V and 1 ms, respectively.
 |
RESULTS |
In Limax and the related species, olfactory information
is received at the olfactory receptors and conveyed to the PC neurons via the olfactory nerve (Fig.
1A; Chase
1986
). Electrical stimulation of the olfactory nerve evoked
transient excitatory postsynaptic potentials (EPSPs), and subsequently
long-lasting inhibitory postsynaptic potentials (IPSPs) in NB neurons
of the PC (Fig. 1Ba). In marked contrast, long-lasting EPSPs
were evoked in B neurons by the olfactory nerve stimulation (Fig.
1Bb), and the rising phase of EPSPs in B neurons consisted
of multiple components (Fig. 1C). The EPSPs evoked in
the NB neurons had their onset approximately 25 ms after the
stimulation of the olfactory nerve (24.8 ± 2.3 ms; mean ± SE, n = 5) and the latency was constant over repeated
stimulation in each preparation (n = 4). Even if the
voltage of stimulation of the olfactory nerve was increased, the input
latency was constant while the amplitude of the EPSP became larger
(Fig. 1Da). On the other hand, the EPSPs in the B neurons
exhibited variable input latencies, which became shorter with a
stronger stimulus; the shortest input-latency was about 50 ms (Fig.
1Db; n = 3). These results show that the
neuronal connection from the olfactory nerve to NB neurons is
monosynaptic excitatory but that to B neurons is polysynaptic
excitatory. Taking into account that PC neurons are classified into
either NB neurons or B neurons (Kleinfeld et al. 1994
;
Watanabe et al. 1998
), the polysynaptic olfactory nerve-B neuron pathway is suggested to be mediated by NB neurons. Additionally, multiple EPSPs observed in B neurons suggest that a large
number of presynaptic NB neurons form convergent synapses onto a single
B neuron.

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Fig. 1.
A membrane potential in bursting (B) neurons and nonbursting (NB)
neurons evoked by electrical stimulation of the posterior olfactory
nerve (ON). A: a schematic drawing of the olfactory
system of Limax, based on the previous morphological
observations. The PC is clearly divided into the somatic region (SR)
and neuropil region (NR). The PC neurons consist of B neurons (filled
circle) and NB neurons (empty circles) and only NB neurons extend their
neurites to the NR. B: representative responses of NB
neurons (Ba) and B neurons (Bb). Dashed
lines indicate the membrane potentials just before the ON stimulus is
applied. Arrowheads indicate the stimulus artifact. The horizontal
scale bar is 200 ms, and the vertical bar is 20 mV for NB neuron and
12.5 mV for B neuron. C: expansion of the rising phase
of the evoked excitatory postsynaptic potential (EPSPs) in a B neuron.
The time range indicated by the horizontal bar at the bottom of Fig.
1Bb is expanded and the action potential was truncated.
Arrows indicate the onset of individual EPSP. Scale bars are 25 ms and
5 mV. D: EPSPs in an NB neuron (Da) and a
B neuron (Db) evoked by an ON stimulation. In both
neurons, the intensity of the ON stimulus was varied from 1.0 to 3.0 V
at the phase range of 1.0-1.4 . Since the phase-dependency of the
transmissions is not observed in this phase range (see Fig.
2C), we can examine the effects of the stimulus
intensity. In these experiments, hyperpolarizing DC currents were
injected to keep the membrane potential at about 70 mV to prevent
discharges. The horizontal scale bar is 50 ms, and the vertical bar is
3 mV for NB neurons and 8 mV for B neurons.
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Polysynaptic transmission from the olfactory nerve to B neurons was
further characterized. Electrical activities of a B neuron and an NB
neuron were recorded simultaneously (Fig.
2A). The neural activities of
both types of neurons were spontaneously oscillating and phase-locked
to the LFP of the PC and to each other (Kleinfeld et al.
1994
; see Fig. 2A). When the phase of the periodic
activities of the PC is defined as shown at the bottom of Fig.
2A, the input latency of evoked EPSPs in B neurons was
longer when the stimulus was applied at an earlier phase, i.e., just
after the peak of depolarization (Fig. 2B). In Fig.
2C, input latencies and averaged membrane potentials of NB
neurons were both indicated as a function of the phase of the PC
oscillation. Recovery of the IPSP in NB neurons and input latencies in
B neurons were resolved into fast and slow components. The phase at the
boundary between the two components was approximately
. The input
latencies of B neurons were well correlated with the membrane
potentials of NB neurons (Fig. 2D), but not with those of B
neurons (data not shown). These results indicate that the response of B
neurons to a stimulus depends on the phase of the oscillatory activity
when the stimulus was applied, and the phase-dependency of latency of
the evoked response in B neurons may arise from the phasic oscillation
of the intervening NB neurons. Thus, electrical oscillation of NB neurons operates as a neuronal filter in the polysynaptic pathway from
the olfactory nerve to B neurons.

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Fig. 2.
Relationship between the membrane potentials of NB neurons and input
latencies of B neurons in response to ON stimulation. A:
simultaneous recording from an NB neuron (upper) and an
adjacent B neuron (lower). The phase of oscillation was
defined based on the interval between adjacent spikes of B neurons, as
shown at the bottom. Scale bars are 1 s and 10 mV.
B: phase-dependence of input latencies of the EPSPs in B
neurons. The arrowhead indicates the stimulus artifact and the arrows
indicate the onset of the first EPSP. Input latency is defined as the
time between the arrowhead and the arrow. Input latency (68 ms) evoked
by the stimulus at phase was shorter than that (152 ms) evoked at
phase 0.5 . Scale bars are 150 ms and 10 mV. C: input
latencies of the first EPSPs in a B neuron (empty marks) and membrane
potentials of averaged NB neuron (solid thick lines) as a function of
the phase when the stimulus was applied. Since the phase is determined
on the basis of the spike intervals in B neurons, the averaged membrane
potential of NB neuron as a function of the phase was estimated from
the data of paired B and NB neuron recordings (n = 3), while the latencies of the first EPSPs in the B neuron were
estimated from recordings of B neurons alone (n = 5). In Fig. 2, Ca and Cb, representative
data of the input latency in a B neuron and the individual data of the
normalized input latencies in all preparations (indicated by the 5 different marks) are indicated, respectively. In Fig.
2Cb, normalized input latency in B neurons were
calculated from the following equation: normalized IL = (IL ILmin)/(ILmax ILmin),
where IL is input latency, ILmin or ILmax are
minimum or maximum input latency in each preparation, respectively. The
input latencies at phase 0.4-1.0 and phase 1.0-1.6 were fitted
using the least-squares approximation, which are indicated by the 2 thin straight lines. D: correlation between the input
latencies of B neurons and the membrane potential of NB neurons. Figure
2, Da and Db, are derived from Fig. 2,
Ca and Cb, respectively. In the inset of
Fig. 2Db, the correlation coefficient in each
preparation is indicated.
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Then, what will be the consequence of the phase-dependent response
evoked in B neurons? The B neurons are an essential factor for
frequency modulation of the ongoing synchronous oscillation (Gelperin 1994
; S. Watanabe, T. Inoue, M. Murakami,
Y. Inokuma, S. Kawahara, and Y. Kirino, unpublished
observations). Frequency modification is known to be evoked also by
odor application to the olfactory receptors (Gelperin and Tank
1990
). When a single electrical stimulus was applied to the
olfactory nerve, it induced modulation in the oscillation frequency of
the PC depending on the phase of the PC oscillation (Fig.
3). The PC oscillation frequency was more
strongly modulated when the stimulus was applied at a later phase,
while stimulation of the olfactory nerve affected only slightly the
oscillation frequency when the stimulus was given at an earlier phase
(Fig. 3). These results clearly indicate that a modulation of the
frequency of the PC oscillation is also phase-dependent and neuronal
spikes in the olfactory nerve have different effects on the PC
oscillation frequency, depending on the phase at their arrival. In
other words, only selected spikes can modulate the oscillation
frequency.

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Fig. 3.
Phase-dependent modulation of the LFP oscillation frequency in the PC.
A: representative data of frequency modulation of LFP
oscillation in the PC by a single stimulus to the ON. Arrowhead
indicates the stimulus artifact. The olfactory nerves were stimulated
at a later phase in lower traces of the LFP. Scale bar is 2 s.
B: summary of the data (mean ± SE,
n = 4). The phase was determined on the basis of
the onset of local field potential (LFP) events, which approximately
coincided with the spike in B neurons. The vertical axis indicates the
normalized frequency change, (fpost fpre)/fpre, where fpre refers to
instantaneous LFP frequency, which is the inverse of the interval
between the two LFP events, just before the ON stimulus, and
fpost is average of instantaneous LFP frequency for 3 cycles just after the ON stimulus.
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 |
DISCUSSION |
In this paper, we have addressed mainly two points: 1)
the synaptic connection from the olfactory nerve to the PC neurons, and
2) the neuronal mechanism of phase-dependent synaptic
transmission of sensory information, which results in phase-dependent
modulation of the PC oscillatory activity. This neuronal mechanism
suggests that the PC oscillation functions as a phase-dependent filter of sensory information. This paper is the first report that described phase-dependent transmission, elucidated its neuronal mechanism, and
proposed the physiological function as a neuronal filter of electrical
oscillation in the olfactory system, although phase-dependent modification of PC activity has been previously reported
(Gelperin and Tank 1990
).
Figure 1 shows that olfactory information conveyed from the olfactory
nerve first excites NB neurons in the PC, and subsequently B neurons.
Taking into account the previously suggested inhibitory connection from
B neurons to NB neurons (Kleinfeld et al. 1994
; Watanabe
et al., unpublished observations), the PC can be viewed as a
feedback inhibitory circuit; activity levels of NB neurons evoked by
olfactory input depend both on the direct depolarization and on the
recurrent hyperpolarization via B neurons. This synaptic circuit is
similar to that in the olfactory bulb, the vertebrate olfactory center,
where olfactory information is received by mitral/tufted cells, which
form an inhibitory feedback loop via the granule cells (Isaacson
and Strowbridge 1998
; Jahr and Nicoll 1980
). In addition to the similarity in the morphology of the neurons
(Scott 1986
; Watanabe et al. 1998
) and
the existence of synchronous oscillations (Freeman 1978
;
Gelperin and Tank 1990
), the synaptic connections revealed in the present study indicate a novel type of conserved function between the vertebrates and invertebrates.
As shown in Fig. 2, B and C, the efficacy of
synaptic transmission from the olfactory nerve to B neurons was
modified phase-dependently, presumably because of the phasic
oscillation of the intervening NB neurons. Namely, the fact that a
500-ms difference in stimulus timing (i.e., approximately 0.5
phase
difference) leads to a 110-ms difference in EPSP latency (Fig.
2B) is presumably due to the depth of the hyperpolarizing
IPSP in the NB neurons and the steep nonlinear depolarizing ramp during
recovery from the IPSP. Additionally, more hyperpolarizing membrane
potentials of the NB neurons not only would produce a longer interval
between the onset of the EPSP and the spike generation, but also might inhibit the EPSP from reaching the threshold in the extreme case. On
the other hand, more depolarizing membrane potentials would elicit
spike generation more easily and the interval is shorter. Since the
excitability in B neurons determines oscillation frequency in the PC
(Gelperin 1994
; Watanabe et al., unpublished
observations), the phase-dependency of evoked responses in B neurons in
turn results in the phase-dependent modulation of synchronous
oscillatory activity in the PC by the olfactory nerve stimulus (Fig.
3).
Then, how does this phase-dependent filtering affect the physiological
operation of olfactory information processing? In Limax and
the related species, tentacle ganglion (TG) is located upstream of the
PC in information flow (Chase 1986
) and some of the
neurons in the TG project their axons to the olfactory nerve
(Chase and Kamil 1983
). In recent studies, synchronous
oscillation in the TG and the olfactory nerve, which are noncoherent
with spontaneous oscillation in the PC, have been observed and spiking
activities in the olfactory nerve were shown to be phase-locked with
the oscillation in the TG (Kimura et al., personal communication). Thus, spiking activities in the olfactory nerve are noncoherent with
the spontaneous oscillation in the PC, and filtering effect of
olfactory information in the PC might be determined by the phase-relationship between the two synchronous oscillatory networks: the TG and the PC. In other words, phase-dependent filtering system in
the PC might transmit olfactory information to the further information
processing unit in the PC only when the TG and PC oscillations are in a
certain phase relationship. Odor-induced dynamic interactions between
the primary and secondary olfactory processing regions will be a key
mechanism for understanding physiological function of the newly
proposed mechanism in olfactory information processing.
We are grateful to Dr. Hiroo Ooya for supplying the slugs.
This study was supported by Grants-in-Aid for Scientific Research
11771408, 11168212, and 10480176 from the Ministry of Education, Science, Sports and Culture, Japan, and by a grant from the Program for
Promotion of Basic Research Activities for Innovative Biosciences, Japan.
Address for reprint requests: Y. Kirino, Laboratory of
Neurobiophysics, School of Pharmaceutical Sciences, The University of
Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan (E-mail: kirino{at}mayqueen.f.u-tokyo.ac.jp).
The costs of publication of this article were defrayed in
part by the payment of page charges. The article must
therefore be hereby marked "advertisement"
in accordance with 18 U.S.C. Section 1734 solely to indicate
this fact.