Laboratory of Neurobiophysics, School of Pharmaceutical Sciences, The University of Tokyo, Tokyo 113-0033, Japan
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ABSTRACT |
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Inoue, Tsuyoshi, Satoshi Watanabe, and Yutaka Kirino. Serotonin and NO Complementarily Regulate Generation of Oscillatory Activity in the Olfactory CNS of a Terrestrial Mollusk. J. Neurophysiol. 85: 2634-2638, 2001. Synchronous oscillation of membrane potentials, generated by assemblies of neurons, is a prominent feature in the olfactory systems of many vertebrate and invertebrate species. However, its generation mechanism is still controversial. Biogenic amines play important roles for mammalian olfactory learning and are also implicated in molluscan olfactory learning. Here, we investigated the role of serotonin, a biogenic amine, in the oscillatory dynamics in the procerebrum (PC), the molluscan olfactory center. Serotonin receptor blockers inhibited the spontaneous synchronous oscillatory activity of low frequency (approximately 0.5 Hz) in the PC. This was due to diminishing the periodic slow oscillation of membrane potential in bursting (B) neurons, which are essential neuronal elements for the synchronous oscillation in the PC. On the other hand, serotonin enhanced the amplitude of the slow oscillation in B neurons and subsequently increased the number of spikes in each oscillatory cycle. These results show that the extracellular serotonin level regulates the oscillation amplitude in B neurons and thus serotonin may be called an oscillation generator in the PC. Although nitric oxide (NO) is known to also be a crucial factor for generating the PC oscillatory activity and setting the PC oscillation frequency, the present study showed that NO only regulates the oscillation frequency in B neurons but could not increase the spikes in each oscillatory cycle. These results suggest complementary regulation of the PC oscillatory activity: NO determines the probability of occurrence of slow potentials in B neurons, whereas serotonin regulates the amplitude in each cycle of the oscillatory activity in B neurons.
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INTRODUCTION |
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There are many
physiological and morphological resemblances among the olfactory
processing regions in various species, such as the mammalian olfactory
bulb and the procerebrum (PC) of gastropod mollusks (Chase
1986). A most remarkable feature in these olfactory systems is
a coherent electrical oscillation generated by assemblies of neurons
(Adrian 1950
; Gelperin and Tank 1990
).
Additionally, extensive projections of biogenic amine-containing
neurons are widely observed in both the olfactory bulb (McLean
and Shipley 1987
; McLean et al. 1989
) and the PC
(Osborne and Cottrell 1971
). Biogenic amines play
important roles for memory acquisition in mammalian olfaction
(McLean et al. 1993
; Sullivan et al.
1989
). Studies with gastropod mollusks have demonstrated that
serotonin is an essential factor for the associative learning in
Aplysia (Abrams 1985
; Hawkins et
al.1993
), and that serotonin is also essential for
food-aversion learning in Helix (Balaban et al. 1987
), although serotonin is not necessary for a type of
food-attraction learning in Helix (Teyke
1996
).
The PC has experimental advantages over the mammalian olfactory bulb.
Namely, the whole isolated PC survives in vitro after isolation, so
that we can easily study the oscillatory network without disruption of
neuronal circuits (Gelperin and Tank 1990; Kleinfeld et al. 1994
) in contrast to brain slices. To
understand the functions of the oscillation for the memory acquisition,
we have investigated the action of biogenic amines on this coherent oscillation in the PC of the terrestrial mollusk, Limax
marginatus.
Previous reports indicate that biogenic amines modulate the
synchronized oscillatory activity in the PC, recorded as local filed
potential (LFP) oscillation: serotonin, a biogenic amine, increases the
frequency and decreases the amplitude of the LFP oscillation
(Gelperin et al. 1993). However, we have no knowledge about the effect of serotonin at the PC neuron level. The PC contains about 105 neurons, which are classified into only
two types: bursting (B) neurons and nonbursting (NB) neurons
(Kleinfeld et al. 1994
). These two types of neurons both
exhibit synchronous oscillation of their membrane potential
(Kleinfeld et al. 1994
), which has been suggested to be
generated by B neurons (Gelperin and Flores 1997
).
Therefore investigation on the serotonin effects to single B neurons is
the essential step for understanding the action of serotonin on the PC
oscillatory network activity. In the present study, by using serotonin
and serotonin receptor blockers, we first demonstrate that serotonin is
an essential factor for generation of the synchronized oscillatory
activity in the PC, and serotonin regulates the amplitude of the
oscillatory behavior in B neurons.
Previous reports demonstrated that nitric oxide (NO) is also an
essential factor for generation of the synchronized oscillatory activity in the PC, but, unlike serotonin, critically regulates the
oscillation frequency in B neurons: NO depletion slows the oscillation
frequency and finally inhibits the generation (Gelperin 1994; Gelperin et al. 2000
). Therefore we next
investigated the details of NO-induced change of the oscillatory
behavior in B neurons and compared the neuronal kinetics between
serotonin and NO. We demonstrate that NO only increases the oscillation
frequency in B neurons but not the oscillation amplitude.
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METHODS |
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We used slugs, L. marginatus, of 0.3-0.5 g in body
weight. Subjects were immobilized and anesthetized by injecting an
isotonic Mg2+ solution into the body cavity.
After immobilization, the cerebral ganglion of Limax was
quickly removed from the head, and the PC was completely isolated from
the rest of the cerebral ganglion in the dissection saline in which the
MgCl2 concentration was increased to 35 mM and
NaCl was decreased to maintain isotonicity. After that, the PC was
desheathed and transferred to physiological saline containing (in mM)
70.0 NaCl, 2.0 KCl, 4.7 MgCl2, 4.9 CaCl2, 5.0 glucose, and 5.0 HEPES (adjusted to pH
7.6 with NaOH). The physiological saline was continuously perfused at a
rate of 0.8 ml/min. The activity of single PC neurons was recorded in
the current-clamp mode of the perforated patch-clamp recording
configuration (EPC-7, List Electronic, Darmstadt, Germany); the
composition of the pipette solution was (in mM) 35.0 KCl, 35.0 K-gluconate, 5.0 MgCl2, and 5.0 HEPES (adjusted
to pH 7.6 with KOH) plus 250 µg/ml nystatin (Watanabe et al.
1999). On the other hand, synchronous oscillatory activity in
the PC was monitored by recording local field potentials (LFPs) with a
glass electrode whose tip diameter was approximately 20-50 µm. We
used four reagents: serotonin (Sigma, St. Louis, MO), cinanserin and
MDL72222 (Tocris, Bristol, UK) as serotonin receptor antagonists and
(±)-(E)-ethyl-2-[(E)-hydroxyimino]-5-nitro-3-hexeneamide (NOR3; Dojindo, Kumamoto, Japan) as an NO-releasing compound
(Kita et al. 1994
). All reagents were bath-applied using
a perfusion system.
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RESULTS |
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There are extensive serotonergic terminals in the PC
(Osborne and Cottrell 1971). Therefore we first
investigated the effect of serotonin receptor blockers on the PC
oscillatory activities. Synchronous oscillation spontaneously occurs in
the isolated PC and the intrinsic oscillation can be recorded as the
oscillation of the LFP (Gelperin and Tank 1990
).
Cinanserin (500 µM) and MDL72222 (500 µM), which are potent
serotonin receptor antagonists in some molluscan nervous systems
(Vehovszky and Walker 1991
; Yeoman et al.
1994
), suppressed the LFP oscillation in the PC (Fig.
1B; cinanserin: n = 7, MDL72222: n = 3).
The LFP oscillation was recovered by washout of these serotonin
receptor antagonists (Fig. 1B); i.e., the action of the
reagents was reversible. These results indicate that the serotonin
receptor blockers inhibit the synchronous oscillatory activity in the
PC.
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The PC contains about 105 neurons, which are
classified into only two types: bursting (B) neurons and nonbursting
(NB) neurons (Kleinfeld et al. 1994). These two types of
neurons both exhibit synchronous oscillation of their membrane
potential (Kleinfeld et al. 1994
), which has been
suggested to be generated by B neurons (Gelperin and Flores
1997
). Then, we simultaneously recorded the electrical activity
of a single B neuron and the LFP in the PC in the presence of serotonin
receptor blockers. Periodic oscillatory behavior in B neurons, as well
as LFP oscillations, was inhibited by both serotonin receptor blockers
(Fig. 1, C and D). These results show that
suppression of LFP oscillation, caused by serotonin receptor blockers,
is due to inhibition of the periodic oscillatory activity in B neurons.
As the next step, we investigated the cellular mechanism for the regulation of the oscillatory activity. Periodic activity in B neurons consists of a slow oscillation component phase-locked with the LFP oscillation and a fast spiking component during the depolarizing phase of the slow oscillation (Fig. 2A). Bath-applied serotonin enhanced the amplitude of slow electrical oscillation in B neurons and subsequently elicited more spikes per burst (Fig. 2B; n = 4). On the other hand, the slow wave was weakened and inhibited in the presence of cinanserin (Fig. 2B; n = 3) and MDL 72222 (data not shown). Figure 2C shows the normalized power of the slow oscillation component (0.02-2 Hz) in the B neurons. The power in the slow range was increased by serotonin (245.7 ± 70.8%; mean ± SE), and decreased by cinanserin (12.7 ± 14.3%). These results show that the extracellular serotonin level regulates the amplitude of the slow electrical wave in B neurons.
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Previous reports have demonstrated that exogenously applied NO
increases the PC oscillation frequency, while an NO synthase inhibitor,
which decreases the NO level in the PC, decreases the PC oscillation
frequency (Gelperin 1994). Thus NO is also an essential factor for generating the PC oscillatory activity and plays a crucial
role for setting the PC oscillation frequency. Therefore we compared
the neuronal kinetics in B neurons induced by these two essential
elements, serotonin and NO, for the PC synchronized oscillation (Fig.
3). An NO donor NOR3 (50 µM) increased
the frequency of the LFP oscillation in the PC as previously
demonstrated (Gelperin 1994
). Intracellular recording of
B neurons revealed that NO shifted the bottom of the slow wave in the
depolarizing direction and the top of the slow wave in the
hyperpolarizing direction (Fig. 3Ba; n = 3). As a
result, NO decreased the amplitude of the slow wave and decreased the
number of spikes per burst (Fig. 3, Bb and Bc;
2.24 ± 0.25 per burst in control vs. 1.25 ± 0.25 per burst in NO; P < 0.05 in paired t-test). On the
other hand, serotonin (50 µM) increased the amplitude of the slow
wave as shown in Fig. 2 but retained the bottom of the slow wave or
rather shifted it in hyperpolarizing direction (Fig. 3Aa;
n = 4), resulting in an increase in the number of
spikes per burst (Fig. 3, Ab and Ac; 1.56 ± 0.28 per burst in control vs. 2.52 ± 0.08 per burst in serotonin;
P < 0.05 in paired t-test). This suggests
that serotonin amplifies the slow wave in B neurons without direct
depolarization in B neurons. These results show that NO only regulates
the occurrence probability of the oscillatory events in B neurons but
cannot increase the oscillation amplitude and the number of spikes in B
neurons in each oscillatory events, in quite contrast to serotonin. In
other words, serotonin and NO complementarily regulate the oscillatory
behavior in B neurons and hence the synchronized network oscillation in
the PC.
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DISCUSSION |
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In this paper, we have demonstrated that serotonin is an essential factor for generating the synchronous oscillatory activity. Additionally, we have demonstrated the neuronal mechanism of the serotonin-induced oscillation: namely, the amplitude of the slow wave in B neurons, the critical neuronal element for generating a coherent oscillation in the PC, is controlled by the extracellular serotonin level. This serotonin-induced control of excitability indicates a new physiological function for biogenic amines on the oscillatory dynamics in the olfactory system.
In the PC, extensive serotonergic terminals (Osborne and
Cottrell 1971) and NO-containing neurons (Gelperin
1994
) are both observed. As shown in Figs. 1 and 2 (serotonin)
and a previous paper (Gelperin 1994
) (NO), serotonin and
NO are both essential factors for generation of synchronous oscillatory
activity in the PC, and the extracellular serotonin and NO levels
regulate the PC oscillatory activity. In other words, the PC contains a potential oscillatory circuit, but exogenous serotonergic inputs and
endogenous NO activation are necessary to actually generate a coherent
oscillation in this oscillatory circuit. However, as described in the
present paper, serotonin and NO regulate the PC oscillatory activity in
different manners. The main effect of serotonin is to increase the
amplitude of the oscillatory activity in B neurons (Fig. 2), resulting
in the increase in the number of spikes per oscillatory cycle in B
neurons (Fig. 3A). In contrast, the main effect of NO is to
increase the oscillation frequency in B neurons (Gelperin
1994
), but NO cannot increase the oscillation amplitude and the
spikes in B neurons (Fig. 3B). These indicate that NO is an
essential factor for regulation of occurrence probability of
oscillation in B neurons, while serotonin is an essential factor for
regulation of the oscillation amplitude in B neurons. Thus serotonin
and NO act as an amplifier and frequency determinant of the oscillatory
event in B neurons, respectively, and these two transmitter systems are
proposed to complimentarily regulate the PC oscillatory activity.
In the present study, we used the in vitro PC preparation, which was
completely isolated from the rest of the cerebral ganglion. A previous
report indicated that there are no somata of serotonergic neurons in
the PC, but there are only extensive serotonergic terminals in the PC
(Osborne and Cotterell 1971). However, the results in Figs. 1 and 2 indicate that endogenous serotonin does elicit the periodic bursting activity in B neurons and synchronized oscillatory activity in the PC, even if the PC was completely isolated. Some reports in other molluscan systems indicate that serotonergic terminals
severed from their somata of origin remain alive as a lot of smaller
neurites (Chiasson et al. 1994
; Murphy et al. 1985
). Therefore the spontaneous oscillation in the isolated PC must be driven by the endogenous serotonin that is released from severed terminals of the serotonergic neurons yet to be identified. It
is of no doubt that identification of such serotonergic neurons makes a
great contribution to elucidation of the generation mechanism of the
synchronized oscillatory activity in the PC.
It has been demonstrated in several neural systems that a
neurotransmitter could have two types of functions, i.e., memory acquisition and generation of a synchronous oscillation. For example, the cholinergic innervation from the medial septum and diagonal band of
Broca (Wainer et al. 1985) generates synchronous
oscillatory activity at the theta frequency in the mammalian
hippocampus (Konopacki et al. 1987
), and this
cholinergic system is important for memory acquisition during spatial
learning (Blokland et al. 1992
) and hippocampal
long-term synaptic plasticity (Huerta and Lisman 1993
). In the mammalian olfactory bulb, serotonergic (McLean and
Shipley 1987
) and noradrenergic terminals (McLean et al.
1989
) have been observed, and these biogenic amines have been
demonstrated to be involved in olfactory learning (McLean et al.
1993
; Sullivan et al. 1989
) and learning-induced
changes of bulbar activity (Gray et al. 1986
). However,
the role of biogenic amines in the oscillatory activity in the
mammalian olfactory bulb is poorly understood. In the present study, we
demonstrated that serotonin, a biogenic amine, acts as an oscillation
generator in the PC of the molluscan olfactory center. Taking into
account of high resemblance between the mammalian olfactory bulb and
the PC (Chase 1986
), biogenic amines may also act as an
oscillation-generator in the mammalian olfactory bulb in a manner
similar to that in the PC.
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ACKNOWLEDGMENTS |
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We are grateful to Dr. Hiroo Ooya for the supply of the slugs.
This study was supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Science, Sports, and Culture, Japan (Grant Nos. 11771408, 11168212, and 10480176) and by a grant from the Program for Promotion of Basic Research Activities for Innovative Biosciences, Japan.
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FOOTNOTES |
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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).
Received 24 April 2000; accepted in final form 14 March 2001.
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REFERENCES |
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