© 2001 by The Society for Integrative and Comparative Biology
Serotonergic Neural System not only Activates Swimming but also Inhibits Competing Neural Centers in a Pteropod Mollusc1
1 Department of Biology, Arizona State University, Tempe, Arizona 85287-1501, and Friday Harbor Laboratories, 620 University Road, Friday Harbor, Washington 98250, USA
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Initiation of a particular behavior requires not only activation of the neural center directly involved in its control but also inhibition of the neural networks controlling competing behaviors. In the pteropod mollusc, Clione limacina, many identified serotonergic neurons activate or modulate different elements of the swimming system resulting in the initiation or acceleration of the swimming behavior. Cerebral serotonergic neurons are described here, which produce excitatory inputs to the swimming system as well as inhibitory inputs to the neural centers that control competing behaviors. Whole-body withdrawal behavior is incompatible with swimming activity in Clione. The main characteristic of whole-body withdrawal is complete inhibition of swimming. Cerebral serotonergic neurons were found to produce a prominent inhibition of the pleural neurons that control whole-body withdrawal behavior. By inhibiting pleural withdrawal cells, serotonergic neurons eliminate its inhibitory influence on the swimming system and thus favor increased swimming speed. Serotonergic neurons also produce a prominent inhibition of the Pleural White Cell, which is presumably involved in reproductive or egg-laying behavior. Thus the serotonergic system directly activates swimming system and, at the same time, alters a variety of other neural systems preventing simultaneous initiation of incompatible behaviors.
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All animals can produce a wide variety of behaviors. Many behaviors are mutually exclusive since they follow different goals and might involve incompatible movements of the same body parts. These behaviors can not be performed simultaneously and compete for behavioral output. Therefore, initiation of a particular behavior requires not only activation of the neural circuit directly involved in its control, but also a coordinated inhibition of neural centers underlying incompatible behaviors. In the medicinal leech, swimming and the whole-body shortening are two incompatible behaviors, and swim circuit neurons were found to be inhibited during shortening (Shaw and Kristan, 1997
). In the mollusc Pleurobranchaea, feeding behavior is incompatible with withdrawal behavior, and neurons from the feeding network were found to inhibit the withdrawal command neuron (Kovac and Davis, 1980). In Aplysia, neurons involved in initiation of feeding arousal were found to produce inhibition of defensive withdrawal neurons (Kupfermann et al., 1991). In crayfish, the lateral giant tailflip escape system is inhibited by feeding (Krasne and Lee, 1988) and by the backward walking program (Beall et al., 1990).
In the pteropod mollusc Clione limacina, such mutually incompatible behaviors include swimming and whole-body withdrawal (Norekian and Satterlie, 1996a). Slow swimming in Clione represents a nearly continuous, ‘background’ behavior, which does not require specific sensory inputs for its maintenance. Swimming results from alternate dorsal and ventral flexions of two symmetrical wing-like parapodia. There are two types of escape behaviors. The first is escape swimming, which includes a dramatic increase in swim speed. The second type is a passive avoidance behavior during which the animal ceases swimming, retracts its wings, body-tail and head, and slowly sinks (Sakharov and Kabotyansky, 1986; Norekian and Satterlie, 1996a). The latter occurs when inputs are very strong, applied to the head region, or widespread so that there is no particular direction for escape. We refer to this behavior as the whole-body withdrawal. Unlike escape swimming, whole-body withdrawal behavior is incompatible with swimming and includes its inhibition.
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WITHDRAWAL NEURONS |
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A group of neurons that control whole-body withdrawal behavior has been identified in the pleural ganglia (Norekian and Satterlie, 1996a
). Four pleural withdrawal (Pl-W) neurons were found in each ganglion. The largest cell in the group, which was also the largest cell in the pleural ganglia, was designated Pl-W1 neuron. Intracellular stimulation of even one Pl-W neuron that induced a strong burst of spikes in an otherwise silent cell produced a significant withdrawal reaction. This reaction included retraction of the body-tail, symmetrical withdrawal of both wings and retraction of anterior tentacles. Intracellular injection of a fluorescent dye, carboxyfluorescein, revealed that Pl-W neurons had a very unusual morphology. Each cell had three large primary axon branches, which exited the pleural ganglia into all three pleural connectives—cerebral, pedal and intestinal. The axon branches then crossed the cerebral, pedal and intestinal ganglia, where they divided and entered most of the main peripheral nerves that innervate all major regions of the body—head, wings, neck, body wall and the tail. Such a wide innervation field suggested that Pl-W neurons directly activated all major muscle groups involved in producing the whole-body withdrawal response. In addition, Pl-W cells also recruited other central neurons that control local withdrawal networks (Norekian and Satterlie, 1996a). These include, for example, a network of sensory neurons, interneurons and motoneurons in the pedal ganglia, which control local retraction of the wings (Huang and Satterlie, 1990). Pl-W neurons produced strong excitatory inputs to these wing retractor neurons.
Whole-body withdrawal behavior and swimming activity are functionally incompatible in Clione—wings are retracted and swimming stops during withdrawal response. This relationship between the two behaviors was reflected in the activities of their neural centers. The central pattern generator for swimming and swim motoneurons that innervate wings have been previously identified in the pedal ganglia (Arshavsky et al., 1985a, b; Satterlie and Spencer, 1985; Satterlie, 1993). Simultaneous recordings from swim motoneurons and Pl-W cells revealed that spontaneous episodes of increased swimming activity always correlated with inhibitory inputs to Pl-W neurons (Norekian and Satterlie, 1996a). Sensory inputs, such as tactile stimulation of the tail that induced bursts of activity in swim motoneurons produced simultaneous inhibitory inputs to Pl-W neurons (Fig. 1A). Most importantly, direct stimulation of an individual Pl-W neuron produced inhibition of spontaneous swimming activity in swim motoneurons and interneurons (Fig. 1B). Inhibition of swimming activity could last several seconds after the induced spike burst in a Pl-W neurons (Norekian and Satterlie, 1996a). This inhibitory connection was presumably polysynaptic based on experiments with high divalent solution.
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The excitatory effect of serotonin on the locomotory systems has been described in several invertebrate preparations. Serotonin causes activation of crawling in Aplysia californica (Palovcik et al., 1982
; Mackey and Carew, 1983), swimming in Aplysia brasiliana (Parsons and Pinsker, 1989) and Tritonia (Katz et al., 1994), and ciliary locomotion in Tritonia (Audesirk et al., 1979), Lymnaea (Syed et al., 1988), Planorbarius and Helisoma (Sakharov, 1990). Both exogenous serotonin and identified serotonergic neurons produce a dramatic acceleration of swimming in the medicinal leech (Willard, 1981; Nusbaum and Kristan, 1986). Activation of swimming is also induced by application of serotonin in Clione limacina (Arshavsky et al., 1985b; Satterlie, 1989; Kabotyansky and Sakharov, 1990).
Serotonin-immunoreactive neurons in Clione were found only in the pedal and cerebral ganglia (Satterlie et al., 1995). Many of these neurons have been identified, and the majority of identified immunoreactive neurons were involved in different aspects of swim activation (Satterlie and Norekian, 1996). Pedal serotonergic cells, which innervated the wings, produced peripheral modulation of swimming by enhancing the contractility of swim muscles (Satterlie, 1995). Two clusters of small serotonergic neurons, which were found in the anterior and posterior regions of the cerebral ganglia, produced a profound excitatory effect on the central swimming system (Satterlie and Norekian, 1995). These cerebral serotonergic neurons sent their axons to the pedal ganglia and monosynaptically activated interneurons of the swim central pattern generator, swim motoneurons and pedal serotonergic neurons. Their activity initiated swimming behavior or resulted in an increased wing-beat frequency and increased force of wing contractions, and therefore, enhanced ongoing swimming activity. The overall picture was that many central serotonergic neurons directly targeted the swimming system in Clione and either increased swimming speed or activated swimming in quiescent preparations. However, this was not the only swimming-related action produced by neurons of the serotonergic system.
The ventral surface of the cerebral ganglia contained a single pair of serotonin-immunoreactive neurons (Fig. 2A). These cells were designated Cerebral Serotonergic Ventral (Cr-SV) neurons (Norekian and Satterlie, 1996b). Their cell body position was bilaterally symmetrical. However, the neuron in the left ganglion was almost twice the size of the contralateral cell (30 µm and 50 µm). The morphological structure of the Cr-SV neurons was studied via intracellular injections with carboxyfluorescein. Each Cr-SV neuron had two primary axon branches (Fig. 2B). One branch ran through the cerebro-pleural connective into the ipsilateral pleural ganglion. The second branch crossed contralateral cerebral ganglion and through cerebro-pleural connective entered the contralateral pleural ganglion. Thus, each Cr-SV neuron innervated both pleural ganglia, with axons branching extensively in their neuropile. Such morphology of the Cr-SV neurons originally suggested that some pleural neurons could receive inputs from these cells. And as previously described, the pleural ganglia contained the central group of neurons, which control whole-body withdrawal behavior.
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SEROTONERGIC NEURONS INHIBIT WITHDRAWAL CELLS |
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Simultaneous recordings from Pl-W cells and Cr-SV neurons demonstrated that Cr-SV neurons produced prominent inhibitory inputs to the Pl-W cells (Norekian and Satterlie, 1996b
). Intracellular stimulation of a Cr-SV neuron, which induced a burst of spikes in the cell, triggered hyperpolarizing inputs in the Pl-W neuron (Fig. 3A). Both left and right Cr-SV neurons produced the same effect—hyperpolarization in all Pl-W neurons, including both ipsilateral and contralateral Pl-W neurons.
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Experiments with high divalent saline revealed two components of the inhibitory responses in Pl-W neurons (Norekian and Satterlie, 1996b
). The first component included fast inhibitory potentials. These IPSPs were blocked in high divalent solution, suggesting that they were polysynaptic in nature. The second component was a slow hyperpolarizing wave of low amplitude (5 mV), which was not blocked by high divalent solution (Fig. 3B). This slow hyperpolarization apparently reflected the direct effect of a Cr-SV neuron on the Pl-W cells. The inhibitory inputs from Cr-SV neurons to Pl-W cells were reversibly blocked by the serotonin antagonist mianserin (Fig. 4). The exogenous serotonin mimicked the effect induced by Cr-SV neurons and produced a 5 mV hyperpolarization of the Pl-W neurons. Both serotonin-induced and Cr-SV neuron-induced hyperpolarizations of Pl-W neurons were found to be associated with a prominent decrease in membrane resistance. The decrease of membrane resistance decreased the excitability of Pl-W neurons, thus providing an additional inhibitory influence on the Pl-W cells (Norekian and Satterlie, 1996b).
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In addition to inhibitory effects on the Pl-W neurons, Cr-SV neurons also produced excitatory inputs to the swimming system (Norekian and Satterlie, 1996b
). However, this excitatory effect was relatively weak—significantly weaker than the effect produced by other cerebral serotonergic neurons. In addition, Cr-SV neurons produced weak excitatory inputs to the serotonergic pedal neurons (Pd-SW), which innervate the wings and enhance the contractility of swim muscles (Fig. 5A). The asymmetrical serotonergic heart excitor neuron (Pd-HE) in the left pedal ganglion also received weak excitatory inputs from Cr-SV neurons (Fig. 5B). The excitatory effects persisted in high divalent saline suggesting that Cr-SV neurons directly activated Pd-SW neurons and the Pd-HE cell.
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The largest neuron in the right pleural ganglion, in addition to the Pl-W1 neuron, is the asymmetrical pleural white (Pl-Wh) cell. This cell was so named because it has a prominent white coloration during the summer reproductive season. The appearance of such white coloration usually indicates an increased production of peptides in the cell body. The Pl-Wh cell became white at the same time as a cluster of small surrounding cells, which showed immunoreactivity to an antibody generated against Aplysia egg-laying hormone. For this reason, and the positive correlation between the white color and maturation of both male and female reproductive structures, we suspect that the Pl-Wh cell may be involved in some aspects of reproductive behavior in Clione.
The Pl-Wh cell was also a target for Cr-SV neurons and was very sensitive to Cr-SV neuron activities. An induced burst of spikes in a Cr-SV neuron produced a prominent hyperpolarization of the Pl-Wh cell (Fig. 6). The response represented a slow hyperpolarizing wave of high amplitude without any rapid inflections that could be interpreted as fast synaptic potentials (n = 24; with latencies of 100–200 msec, time-to-peak of 1–1.5 sec, relaxation time of 1–1.5 sec and amplitudes up to 15 mV). This hyperpolarization was not blocked by high divalent solution, suggesting a direct connection between neurons (Fig. 6A). Inputs were sufficiently strong to completely inhibit the spontaneous spike activity in the Pl-Wh cell. The serotonin antagonist, mianserin, completely and reversibly blocked the Cr-SV neuron-induced inhibition of the Pl-Wh cell (Fig. 6B).
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Exogenous serotonin produced a prominent hyperpolarization of the Pl-Wh cell terminating its spontaneous spike activity. This high amplitude hyperpolarization persisted in high Mg++ solution (Fig. 7A). The Pl-Wh cell was very sensitive to serotonin responding with hyperpolarization to exogenously applied transmitter in concentrations as low as 0.5 µM. Thus, exogenous serotonin mimicked the effect produced by Cr-SV neurons on the Pl-Wh cell. The Cr-SV neurons themselves were sensitive to serotonin and responded to its application with strong activation. In high Mg++ solution, serotonin produced a prominent depolarization of Cr-SV neurons (Fig. 7B).
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Cerebral serotonergic Cr-SV neurons produce excitatory inputs to swim neurons, Pd-SW neurons that facilitate wing muscle contractions, and the Heart Excitor neuron (Fig. 8). However, their excitatory influence is relatively weak. The main action of the Cr-SV neurons is in the pleural ganglia. Each Cr-SV neuron extensively innervates the pleural ganglia and produces strong inhibitory inputs to the pleural withdrawal neurons, which control the whole-body withdrawal behavior (Fig. 8). Withdrawal behavior is incompatible with swimming activity. Pleural withdrawal neurons are normally silent, but when active produce inhibitory inputs to swim neurons. Thus, Cr-SV neurons inhibit pleural withdrawal neurons, decreasing the probability of initiation of the whole-body withdrawal behavior and therefore indirectly facilitating the performance of swimming behavior. In addition, Cr-SV neurons inhibit a pleural White cell, which is presumably involved in the control of a reproductive behavior (Fig. 8).
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The main conclusion of the present story is the notion that the swimming system is not an isolated system. There are numerous connections and influences coming from the neural centers that control other behaviors. Some of these behaviors, such as the whole-body withdrawal, are incompatible with swimming. And their neural centers produce a prominent inhibitory influence on the swimming system. The serotonergic system plays a very important role in initiation or reinforcement of swimming activity in Clione. However, it does not only directly activate the swimming system. It also indirectly facilitates the performance of swimming behavior through inhibition of the competing neural systems, such as the whole-body withdrawal. The emerging picture is of a multi-neuronal serotonergic system that alters a variety of behavioral activities, including whole-body withdrawal and reproductive behavior, all in a way directed at the increased swimming activity, while simultaneously decreasing the probability of activation of mutually exclusive behaviors.
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ACKNOWLEDGMENTS |
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This work was supported by NSF grant IBN-9630805, NINDS grant NS-34662 and NIH FIRCA grant TW00935 to T.P.N. and NSF grant IBN-9319927 to R.A.S. The Symposium was supported by National Science Foundation grant IBN 9905990.
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1 From the Symposium Swimming in Opisthobranch Mollusks: Contributions to Control of Motor Behavior presented at the Annual Meeting of the Society for Integrative and Comparative Biology, 4–8 January 2000, at Atlanta, Georgia.
2 To whom correspondence should be addressed. E-mail: Tigran.Norekian{at}asu.edu
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