Serotonin and NO Complementarily Regulate Generation of Oscillatory Activity in the Olfactory CNS of a Terrestrial Mollusk

Tsuyoshi Inoue, Satoshi Watanabe, and Yutaka Kirino

Laboratory of Neurobiophysics, School of Pharmaceutical Sciences, The University of Tokyo, Tokyo 113-0033, Japan


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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.


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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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Fig. 1. Serotonin is an essential factor for generation of synchronous oscillatory activity in the procerebrum (PC). A: schematic drawing of the hemispherical cerebral ganglion of Limax and recording sites. The PC (shaded area) was dissected from the rest of the cerebral ganglion (outlined by dotted lines). PC neurons are classified into 2 types: bursting (B) neurons (filled circles) and nonbursting (NB) neurons (open circles). B: modulation of the local field potential (LFP) oscillation in the PC, elicited by serotonin receptor blockers, cinanserin and MDL72222. These serotonin receptor blockers inhibited the LFP oscillation in the PC about 10 min after the application, and the washout for about 1 h fully recovered the LFP oscillation. Times after the application of these antagonists are indicated in the parentheses. Scale bars are 5 s, 10 µV in cinanserin and 5 s, 7 µV in MDL72222. C and D: simultaneous recording of the LFP (top) and the membrane potentials of B neurons (bottom). In the control, the membrane potentials in B neurons and the LFP in the PC revealed a phase-locked oscillation. Serotonin receptor blockers, cinanserin (C) and MDL72222 (D), inhibited both the LFP oscillation and the oscillatory activity in B neurons. Scale bars are 4 s, 25 µV (top) and 25 mV (bottom) in C and 4 s, 12.5 µV (top) and 25 mV (bottom) in D.

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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Fig. 2. Serotonin regulates the amplitude of the slow electrical oscillation in B neurons. A: filtered electrical activity in a B neuron. Electrical activity in a B neuron (top) was band-pass filtered at 0.02-2 Hz (middle) and 2-3,000 Hz (bottom). This activity consists of 2 components: slow electrical oscillation (middle) and periodic spiking activity (bottom). Scale bars are 1 s and 5 mV. B: modulation of the oscillatory behavior in B neurons induced by serotonin or a serotonin receptor antagonist. Serotonin or cinanserin, a serotonin receptor antagonist, enhanced or reduced the amplitudes of the slow electrical oscillation in the B neurons, respectively. We could not determine whether or not the effects are reversible because perforated patch-recording from small B neurons for a period as long as 1 h was too difficult. Scale bars are 500 ms and 7.5 mV. C: normalized power of the slow (0.02-2 Hz) signal component. Values are given as the means ± SE percentage change.

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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Fig. 3. Modulation of oscillatory behavior in B neurons, induced by serotonin (A) and nitric oxide (NO, B). Both NOR3 (50 µM) and serotonin (50 µM) increased the oscillation frequency in B neurons. NOR3 shifted the bottom of slow wave in B neurons in the depolarizing direction and shifted the top of the slow wave in the hyperpolarizing direction, which results in decrease in the number of the spikes per burst in B neurons (Ba). On the other hand, serotonin enhanced the amplitude of the slow wave, and consequently increased the number of spikes per burst in B neurons (Aa). Scale bar is 1 s. A, b and c: changes of the spike number in B neurons, induced by serotonin (n = 4; Ab shows individual data; Ac shows averaged data; P < 0.05 in paired t-test). B, b and c: changes of the spike number in B neurons, induced by NO (n = 3; Bb shows individual data; Bc shows averaged data; P < 0.05 in paired t-test).


    DISCUSSION
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ABSTRACT
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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.


    ACKNOWLEDGMENTS

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.


    FOOTNOTES

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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0022-3077/01 $5.00 Copyright © 2001 The American Physiological Society