Cholinergic Activation of Startle Motoneurons by a Pair of Cerebral Interneurons in the Pteropod Mollusk Clione limacina
Tigran P. Norekian and
Richard A. Satterlie
Department of Zoology, Arizona State University, Tempe, Arizona 85287-1501; and Friday Harbor Laboratories, Friday Harbor, Washington 98250
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ABSTRACT |
Norekian, Tigran P. and Richard A. Satterlie. Cholinergic activation of startle motoneurons by a pair of cerebral interneurons in the pteropod mollusk Clione limacina. J. Neurophysiol. 77: 281-288, 1997. The holoplanktonic pteropod mollusk Clione limacina exhibits an active escape behavior that is characterized by fast swimming away from the source of potentially harmful stimuli. The initial phase of escape behavior is a startle response that is controlled by pedal motoneurons whose activity is independent of the normal swim pattern generator. In this study, a pair of cerebral interneurons is described that produces strong activation of the d-phase startle motoneurons, which control dorsal flexion of the wings. These interneurons were designated cerebral startle (Cr-St) interneurons. Each Cr-St neuron has a small cell body on the dorsal surface of the cerebral ganglia and one large axon that runs into the ipsilateral cerebral-pedal connective and the neuropile of the ipsilateral pedal ganglion. Each spike in a Cr-St neuron produces a fast, high-amplitude (up to 50 mV) excitatory postsynaptic potential (EPSP) in the d-phase startle motoneurons. This 1:1 ratio of spikes to EPSPs and the stable short synaptic latencies (2 ms) persist in high-Mg2+, high-Ca2+ seawater, suggesting monosynaptic connections. Synaptic transmission between Cr-St neurons and startle motoneurons exhibits a very slow synaptic depression, because a number of spikes in Cr-St neurons is required to achieve a noticeable decrease in EPSP amplitude. Synaptic transmission between Cr-St interneurons and startle motoneurons appears to be cholinergic. In startle neurons, 20 µM atropine and 50 µM d-tubocurarine reversibly block EPSPs produced by spike activity in Cr-St interneurons. Hexamethonium only partially blocks EPSPs in startle neurons, and much higher concentrations are required. Exogenous acetylcholine (1 µM) produces a dramatic depolarization of startle motoneurons in high-Mg2+ seawater, and this depolarization is reversibly blocked by atropine. Nicotine also has a depolarizing effect on startle motoneurons, although higher concentrations are required. Cr-St interneurons and startle motoneurons are also electrically coupled; however, the coupling is weak. Stimuli that are known to initiate escape responses in intact animals, such as tactile stimulation of the tail or wings, produce excitatory inputs to Cr-St interneurons. In addition, tactile stimulation of the lips and buccal cones, which is known to trigger prey capture reactions in Clione, also produces excitatory inputs to Cr-St interneurons and startle motoneurons, suggesting involvement of the startle neuronal system in prey capture behavior of Clione.
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INTRODUCTION |
One of the most common and easily identifiable behaviors in the pteropod mollusk Clione limacina is an active avoidance behavior, or escape swimming, which can be induced in intact animals by tactile stimulation of the tail (Sakharov and Kabotyansky 1986
; Satterlie et al. 1985
). Escape swimming is comprised of two phases. The initial phase includes one or two wing beat cycles (total duration <1 s) during which the animal is propelled forward at an extrapolated rate of nearly 18 body lengths per second (Satterlie et al. 1997
). This "startle" response is followed by a period of prolonged fast swimming in which the rate of forward movement is up to six body lengths per second. In the previous paper (Satterlie et al. 1997
) we showed that the startle response is controlled by a group of pedal neurons that is completely separate from the swim pattern generator and swim motoneurons, which control slow and fast swimming (Arshavsky et al. 1985a
,b
; Satterlie 1985
, 1993
; Satterlie and Spencer 1985
). This startle system consists of two pairs of large pedal motoneurons, which are normally silent, with very low membrane potentials and extremely high thresholds for spike generation. Each startle motoneuron has a single large axon that runs into the ipsilateral wing nerve and directly innervates swim musculature in the wings. There are two types of startle motoneurons: d-phase motoneurons, which induce contraction of dorsal swim musculature and produce dorsal flexion of the wings, and v-phase motoneurons, which produce ventral flexion of the wings. The startle motoneurons do not form synaptic connections with the swim pattern generator and swim motoneurons, nor are connections found in the reverse direction. Startle motoneurons can be activated by sensory stimuli that initiate startle responses in intact animals, such as tactile stimulation of the tail. The general properties of startle motoneurons in Clione suggest that they may be functionally analogous to the Mauthner cells in fish and amphibians, which are responsible for the fast escape startle response in these species (for reviews see Eaton and Hackett 1984
; Zottoli et al. 1995
).
Startle motoneurons represent the final motor output for the startle neuronal system. The important question of how the excitatory sensory inputs are carried to startle motoneurons and what modifications can occur on the interneuronal level still remains open. In this paper we describe a pair of interneurons in the cerebral ganglia of Clione that receives appropriate excitatory mechanosensory inputs and monosynaptically activates some of the startle motoneurons. The morphological and electrophysiological description of cerebral startle (Cr-St) interneurons is followed by documentation of the interconnections between them and startle motoneurons in the pedal ganglia.
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METHODS |
Adult specimens of C. limacina (body length 3-4 cm) were collected from the breakwater of Friday Harbor Laboratories and held in 1-gallon jars in a refrigerator at 5°C. Animals were anesthetized in a 1:1 mixture of seawater and isotonic MgCl2 and dissected in a petri dish coated with Sylgard (Dow Corning). Electrophysiological experiments were performed on semi-intact preparations consisting of the CNS, head, wings, and body-tail. All digestive and reproductive organs were removed. The body wall, including skin and associated muscle groups, was left intact. All nerves running from the central ganglia to the head, wings, and body-tail also remained intact. Tactile stimulation of the head, tail, and wings was provided by a thin polymeric filament 0.2 mm diam. The filament was attached to a plastic stick that was hand held. The elasticity and length of the filament (4 cm) allowed a relatively consistent strength of the stimulation from trial to trial.
Before recording, ganglia were desheathed by bathing the preparation in a 1-mg/ml solution of protease (Sigma, type XIV) for ~5 min, followed by a 30-min wash. Standard electrophysiological techniques were used for intracellular recording. Glass microelectrodes were filled with 2 M potassium acetate and had resistances of 10-20 M
. Measurments of electrotonic coupling were accomplished by inserting two electrodes into one neuron: one for current injection and one for voltage recording. A third electrode was inserted into the other neuron of the coupled pair. Intracellular staining of neurons was achieved via recording electrodes filled with a 5% solution of carboxyfluorescein (Molecular Probes) that was iontophoresed with the use of negative current pulses. Semi-intact preparations were then immobilized in MgCl2/seawater and photographed live in the recording dish with the use of a fluorescence microscope (Nikon) with a B2 filter cluster. To block chemical synaptic transmission, a high-Mg2+ solution consisting of a 1:3 mix of 0.33 M MgCl2/seawater was superfused into the preparation dish. To test for monosynaptic connections, a high-Mg2+, high-Ca2+ solution (110 mM MgCl2, 25 mM CaCl2) was used.
Acetylcholine chloride (Sigma) and its agonist (
)nicotine (Sigma) and antagonists (atropine sulfate, Sigma; d-tubocurarine chloride, Sigma; hexamethonium chloride, USBC) were applied with the use of a graduated 1-ml pipette. The final concentration was calculated from the volume of injected solution and the volume of the recording dish. To measure the reversal potentials of excitatory postsynaptic potentials (EPSPs) and acetylcholine-induced depolarizations, two microelectrodes were used to penetrate the postsynaptic neuron: one for current injection and the other for voltage recording. Injection of positive current produced a stable depolarization that was long enough to allow recordings of EPSPs or acetylcholine responses. Measurements of EPSPs were performed in high-Mg2+, high-Ca2+ saline to eliminate spontaneous synaptic inputs and decrease the excitability of neurons. Measurements of acetylcholine-induced changes of membrane potential were performed in high-Mg2+ saline.
In all cases in which statistical data are presented, the numbers represent means ± SD.
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RESULTS |
A bilaterally symmetrical pair of interneurons was identified in the cerebral ganglia that produced strong excitatory inputs to the startle motoneurons of the pedal ganglia. These interneurons were designated Cr-St interneurons.
Morphology of Cr-St neurons
Each Cr-St interneuron had a small soma, ~30 µm diam, situated in the middle of the dorsal surface of each cerebral ganglion (Fig. 1). The cerebral ganglia can be divided into two regions on the basis of the location and size of somata on the dorsal surface: 1) the central part consists of a number of small neurons 20-40 µm diam, and 2) the anterior-lateral part includes all large dorsal neurons with soma sizes of 70-100 µm. The anterior-lateral region includes such identified neurons as the serotonergic metacerebral cell and Cr-A11 neuron, the only large motoneuron on the dorsal surface from the group of cerebral neurons controlling extrusion of prey capture appendages (Norekian and Satterlie 1993
). The Cr-St interneuron soma was situated in the central region among other small-sized neurons, close to the border of the anterior-lateral region (Fig. 1). Another landmark for localization of the Cr-St neuron is the short nerve N0, which originates from the center of the dorsal surface of each cerebral ganglion and innervates the olfactory ganglion (Wagner 1885
). The Cr-St neuron soma was found near the root of nerve N0.

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| FIG. 1.
Morphology of cerebral startle (Cr-St) interneurons revealed by carboxyfluorescein fills. A: micrograph of the CNS with the left Cr-St neuron filled with dye (arrowhead: position of the cell body). Scale bar: 300 µm. B: schematic representation of the morphological structure of the right Cr-St neuron. Dashed lines: N0 olfactory nerve. N1 and N2, head nerves; L, labial nerve; wing, wing nerve; CER, cerebral ganglion; PED, pedal ganglion; PL, pleural ganglion; INT, intestinal ganglia; ST(d), d-phase startle motoneuron with 1 large axon running into the wing nerve; ST(v), cell body of the v-phase startle motoneuron. Star: identified metacerebral serotonergic cell. Asterisk: identified Cr-A11 neuron.
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Carboxyfluorescein fills (n = 9) revealed that each Cr-St neuron had one large axon that ran laterally and posteriorly inside the cerebral ganglion, entered the ipsilateral cerebral-pedal connective, and ramified within the neuropile of the ipsilateral pedal ganglion (Fig. 1). The initial segment of the Cr-St neuron axon produced several short cerebral branches that spread in an anterior direction and ended near the origin of the cerebral head nerves (Fig. 1).
Physiological properties of Cr-St neurons
Cr-St interneurons did not demonstrate spontaneous spike activity (Fig. 2). They were always silent, with membrane potentials between
60 and
65 mV (
62.1 ± 1.7 mV,n = 15). Thresholds for spike generation were relatively high; however, intracellular depolarization easily produced strong bursts of spikes with frequencies up to 30-50 Hz. A distinguishing characteristic of Cr-St interneurons, which was used for their identification after penetration, was the appearance of rhythmic inhibitory potentials with amplitudes of 3-5 mV and durations of 1-2 s (Fig. 2). These hyperpolarizations appeared regularly, with a frequency of 0.3-0.5 Hz. Left and right Cr-St interneurons did not communicate with each other electrically or chemically (n = 6; Fig. 2). However, changes in their membrane potentials, including subthreshold spontaneous synaptic potentials and induced sensory inputs, were synchronous (Fig. 2).

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| FIG. 2.
Left (L) and right (R) Cr-St interneurons do not have electrical or chemical synaptic connections with each other. Note the regular synchronous inhibitory inputs to both neurons. Scale bars: 15 mV, 1 s.
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Interconnections between Cr-St neurons and startle motoneurons
Induced spike activity in a Cr-St interneuron was found to produce strong activation of ipsilateral pedal startle motoneurons (Fig. 3A). Each Cr-St interneuron activated only d-phase, ipsilateral startle motoneurons (n = 28; Fig. 3, A and C). V-phase Startle motoneurons did not receive excitatory inputs from Cr-St interneurons (n = 4; Fig. 3B). The swim pattern generator and swim motoneurons of the pedal ganglia also were not activated by Cr-St neurons.

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| FIG. 3.
A: induced activity in the left Cr-St interneuron produces a strong activation of the ipsilateral, left d-phase startle motoneuron LST(d). B: v-phase startle motoneuron LST(v) does not receive excitatory inputs from the Cr-St neuron. C: contralateral, right d-phase startle motoneuron RST(d) also does not receive inputs from the left Cr-St interneuron. Scale bars: 15 mV, 1 s.
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Each spike in a Cr-St interneuron produced a single EPSP in the ipsilateral, d-phase startle motoneurons (Fig. 4, A and B). These EPSPs were unique in that they were extremely fast and had very high amplitudes. Initially, they were confused with action potentials because their duration and amplitudes were similar to those of regular spikes. The amplitudes varied between 20 and 40 mV, but could be up to 50 mV (Fig. 4, A and B). The duration of each fast EPSP was 8-16 ms (12 ± 3 ms; n = 28; Fig. 4, A and B). EPSPs persisted in high-Mg2+, high-Ca2+ saline and demonstrated the same stable 1:1 ratio of spikes to EPSPs, suggesting a monosynaptic connection (n = 16; Fig. 4B). Monosynapticity was also confirmed by the very short and constant delay between spikes in Cr-St neurons and EPSPs in startle motoneurons (2 ms; n = 26). Both ipsilateral d-phase startle motoneurons simultaneously received a fast monosynaptic EPSP after a single spike in a Cr-St interneuron (n = 5).

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| FIG. 4.
Synaptic connections between Cr-St interneurons and ipsilateral d-phase startle motoneurons (ST). A: each action potential in a Cr-St interneuron produces an individual fast excitatory postsynaptic potential (EPSP) in the startle motoneuron. B: fast EPSPs persist in high-Mg2+, high-Ca2+ seawater with the same 1:1 ratio of spikes to EPSPs. Scale bars: 15 mV, 0.1 s. C: Cr-St interneurons and startle motoneurons are also electrically coupled. Electrical coupling was demonstrated in high-Mg2+ seawater by applying negative or positive current pulses to the startle motoneuron via the 2nd intracellular electrode. Scale bars: 15 mV, 2 s.
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Startle motoneuron EPSPs produced by spike activity in Cr-St interneurons were very stable and slow to show depression. Repetitive spike activity in Cr-St interneurons, such as two to three bursts of spikes with 0.5-s durations and of 40-Hz frequency (total ~ 50 spikes in 2-3 s), had little effect on the amplitudes of startle motoneuron EPSPs (Fig. 5). After 7-10 bursts, however (40-Hz frequency and 0.3- to 0.5-s duration each, presented with 0.5- to 1-s intervals), the amplitudes of startle motoneuron EPSPs were noticeably decreased and could be reduced by ~5 times after 20-30 spike bursts (Fig. 5). After a few minutes of rest, EPSPs showed a dramatic restoration of their amplitude (Fig. 5). In addition to synaptic depression, homosynaptic facilitation was also observed in Cr-St interneuron to startle motoneuron transmission. In each group of startle motoneuron EPSPs, the first three to six EPSPs always showed increases in amplitude before the appearance of synaptic depression (Fig. 5). These experiments were repeated several times on five different preparations with consistent results.

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| FIG. 5.
Repetitive stimulation of a Cr-St interneuron produces a very slow decrease of EPSPs amplitudes in a d-phase startle motoneuron. Intervals between induced bursts of spikes in the Cr-St neuron are 1-2 s. Shown are 1st, 2nd, 3rd, 4th, 7th, 15th, and 25th presentations. Notice that there are no noticeable changes in EPSP amplitudes during the 1st 4 bursts. A 5-min interval partly restores EPSP amplitudes, which were dramatically decreased after 25 bursts. Note the EPSP facilitation within each burst. Scale bars: 15 mV, 0.1 s.
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In addition to the chemical synaptic contacts, Cr-St interneurons were found to be electrically coupled to the ipsilateral, d-phase startle motoneurons (Fig. 4C). These responses persisted in high-Mg2+ solution, and no delay could be recorded between the potential change in the "presynaptic" and "postsynaptic" neurons. The coupling was relatively weak, with coupling coefficients ranging from 0.1 to 0.2(n = 8). This weak electrical coupling was not strong enough to synchronize the activities of Cr-St interneurons and startle motoneurons. Dye coupling was never observed. Thus the main component of the excitatory transmission from Cr-St neurons to startle neurons appeared to be chemical, rather than electrical.
Cholinergic nature of the synaptic inputs from Cr-St neurons to startle motoneurons
The cholinergic antagonist atropine was found to reversibly block transmission from Cr-St interneurons to startle motoneurons (n = 11; Fig. 6A). The effective concentration of atropine for the complete blockade of EPSPs in startle neurons was
20 µM. EPSPs were usually blocked 3-5 min after atropine application and were restored after 10 min of wash in seawater (Fig. 6A). d-Tubocurarine was also effective and reversibly blocked startle motoneuron EPSPs induced by spike activity in Cr-St interneurons at concentrations of
50 µM (n = 5; Fig. 6B). Hexamethonium produced partial, reversible blocking of synaptic transmission from Cr-St neurons to startle neurons (n = 4). The required concentrations were much higher than those for atropine and d-tubocurarine, because 500 µM hexamethonium reduced EPSP amplitude by 50% (Fig. 6C).

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| FIG. 6.
Blocking effect of cholinergic antagonists on the excitatory synaptic transmission between Cr-St interneurons and d-phase startle motoneurons. A: in startle motoneurons, 50 µM atropine completely blocks EPSPs induced by spike activity in Cr-St interneurons. The remainder is due to electrical coupling. The transmission is restored after a 10-min wash in seawater. B: 100 µM d-tubocurarine blocks EPSPs in the startle neurons. The effect is washed out after 15 min in seawater. C: 500 µM hexamethonium partially blocks startle neuron EPSPs. The amplitude of EPSPs is restored after a 15-min wash. Scale bars: 15 mV, 0.2 s.
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Exogenous acetylcholine had a dramatic effect on d-phase startle neuron membrane potentials. To establish the direct sensitivity of startle motoneurons to acetylcholine, the experiments were conducted in high-Mg2+ saline, which completely blocks chemical transmission in the CNS, thus producing a chemical isolation of neurons. Acetylcholine in concentrations as low as 1 µM produced depolarization of the d-phase startle neurons with an amplitude up to 70 mV (n = 12; Fig. 7A). Startle neurons responded to acetylcholine concentrations down to 0.2 µM. Atropine at 50 µM reversibly blocked depolarization produced by 10 µM acetylcholine (n = 4; Fig. 8). Nicotine also produced depolarization of startle neurons, although it appeared to be less potent than acetylcholine because it required a concentration of
5 µM to induce a noticeable depolarization (n = 5; Fig. 7B).

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| FIG. 7.
A: bath application of 5 µM acetylcholine (ACh) produces depolarization of a d-phase startle motoneuron. B: bath application of 20 µM nicotine also produces depolarization of a startle motoneuron. Experiments are performed in high-Mg2+ seawater, which blocks all chemical synaptic transmission in the CNS. Scale bars: 20 mV, 3 s.
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| FIG. 8.
Atropine at 50 µM reversibly blocks the depolarization produced by bath-applied 10 µM acetylcholine in high-Mg2+ seawater. Scale bars: 20 mV, 3 s.
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Additional confirmation of the cholinergic nature of synaptic transmission between Cr-St interneurons and startle motoneurons came from measurements of the reversal potentials for EPSPs and for depolarizations produced by exogenous acetylcholine. The amplitudes of the EPSPs induced in startle neurons by Cr-St interneurons decreased to the zero level when the startle neurons were depolarized to membrane potentials between +10 and +15 mV (n = 5). Similarly, the amplitude of the depolarization induced by application of exogenous acetylcholine decreased to the zero level when the membrane potentials of startle neurons were between +10 and +15 mV (n = 3).
Escape reaction and mechanosensory inputs from the tail
Pedal startle motoneurons have been found to be responsible for the initial phase of escape behavior, and stimuli that initiate this behavior in intact animals, such as tactile stimulation of the tail, were found to produce excitatory inputs to them (Satterlie et al. 1997
). Experiments with semi-intact preparations demonstrated that tactile stimulation of the tail produced excitatory inputs to the Cr-St interneurons as well as to the startle motoneurons (n = 14; Fig. 9A). The body-tail of Clione is innervated primarily by intestinal nerves N10 and N11 (Wagner 1885
; Zakharov and Ierusalimsky 1992
). Cobalt backfilling of nerves N10 and N11 showed stained processes that ran through the pleural ganglia into the cerebral ganglia, in addition to a number of axons originating from stained neurons in intestinal ganglia (n = 4). These experiments demonstrated the morphological basis for connections between the cerebral ganglia and tail mechanoreceptors.

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| FIG. 9.
Mechanosensory inputs to Cr-St interneurons and d-phase startle motoneurons related to escape behavior. Tactile stimulation of the tail (A) and the wing (B) produces excitatory inputs to both Cr-St and startle neurons. Arrowheads: tactile stimuli. Scale bars: 15 mV, 1 s.
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Tactile stimulation of the wings, in addition to the tactile stimulation of the tail, can trigger activity in startle motoneurons and initiate escape reactions in Clione (Satterlie et al. 1997
). Experiments with semi-intact preparations demonstrated that tactile stimulation of the wings also produced excitatory inputs to the Cr-St interneurons, as well as to the startle neurons (n = 9; Fig. 9B). Although the identified Cr-St interneurons participated in transferring information from the tail and wing mechanoreceptors to the startle motoneurons, they were not the only source of excitatory inputs to the startle neurons. In the majority of all simultaneous recordings of Cr-St neuron and startle neuron activities, some EPSPs recorded in startle neurons were not related to Cr-St neuron spikes (Fig. 9).
Prey capture and mechanosensory inputs from the lips and buccal cones
Dramatic acceleration of swimming is characteristic of behavioral activities other than escape responses in Clione. The main example is prey capture behavior (Litvinova and Orlovsky 1985
). Is feeding-related acceleration of swimming based only on the activation of the swim central pattern generator, or can it also involve activation of startle neurons? It has been previously shown that mechanoreceptive input from prey is a very important factor in triggering the prey capture reaction in Clione (Conover and Lalli 1972
; Lalli 1970
). Tactile stimulation of the lips or buccal cones in semi-intact preparations has been shown to produce strong excitatory inputs to all identified neuronal systems involved in the control of feeding behavior (Norekian 1995
). We have found in the present study that tactile stimulation of the buccal cones or lips produced, in addition to the acceleration of swimming activity in the swim central pattern generator and swim motoneurons, excitatory inputs to the startle motoneurons (n = 16; Fig. 10A) and Cr-St interneurons (n = 10; Fig. 10, B and C). In addition, the system of cerebral neurons controlling extrusion of buccal cones (Cr-A neurons) was found to produce weak subthreshold, polysynaptic excitatory inputs to Cr-St interneurons and startle motoneurons (n = 6; Fig. 10D).

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| FIG. 10.
Excitatory inputs to startle interneurons and motoneurons related to prey capture behavior. A: tactile stimulation of the buccal cones (arrowhead) produces activation of the swim motor program, as shown by the general excitor (GE) swim motoneuron activity, and at the same time produces excitatory inputs to the d-phase startle motoneuron. B and C: tactile stimulation (arrowhead) of the buccal cones (B) and lips (C) produces excitatory inputs to Cr-St interneurons. D: strong induced bursts of spikes in a Cr-A3 neuron, which controls extrusion of the buccal cones, produces stable subthreshold excitatory inputs to a d-phase startle motoneuron. Scale bars: 10 mV, 1 s.
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DISCUSSION |
Cr-St interneurons
Startle motoneurons in the pteropod mollusk C. limacina have been shown to be responsible for the startle response (Satterlie et al. 1997
), which appears to be analogous to those in many other animals, including fish, amphibians, and some other invertebrates. A startle response is an abrupt strong reaction with very short latency to a sudden stimulus that is unexpected and alarming. Such characteristics mean that the underlying neuronal system is supposed to be normally silent, unless activated by appropriate sensory inputs, should transfer the signal very fast with minimum synaptic delay, and should be very reliable.
In this study we describe a pair of Cr-St interneurons that monosynaptically activate pedal startle motoneurons and transfer to them at least part of the specific sensory information. Cr-St interneurons were normally silent and could be activated by sensory stimuli that initiate startle responses in intact animals, such as tactile stimulation of the tail or wings. Action potentials in Cr-St neurons produced monosynaptic EPSPs in ipsilateral d-phase startle motoneurons. These EPSPs had uniquely high amplitudes and short durations. High-amplitude EPSPs, which could reach up to 50 mV, appeared to be essential because startle motoneurons have very high thresholds for spike generation. In addition, such high-amplitude EPSPs presumably increased the reliability of signal transmission via the synaptic connection between startle interneurons and motoneurons, which presumably works as a relay rather than a modulatory or integrative unit. The necessity of transferring sensory signals to the motor system as fast as possible explains the very short latencies of EPSPs (2 ms) and their short durations (10 ms).
The synaptic connection between Cr-St and startle neurons was found to be somewhat resistant to synaptic depression, because a number of spikes had to be fired in a Cr-St interneuron in a short period of time before a noticeable decrease in startle motoneuron EPSP amplitude would occur. The behavioral startle response in Clione is known to be depressed very quickly and requires 10-15 min of rest before the reaction can be triggered again (Satterlie et al. 1997
). In the previous report on startle motoneurons it was demonstrated that the neuromuscular junction, from startle motoneurons to the swim musculature, is very susceptible to depression, because a dramatic decrease of muscle contractility occurred with repetitive stimulation of startle motoneurons (Satterlie et al. 1997
). The synaptic connection from Cr-St interneurons to startle motoneurons, on the other hand, is stable and reliable, with the ability to transfer signals to startle motoneurons without significant loss. Thus it appears that synaptic depression of the startle response in Clione occurs primarily at the neuromuscular and possibly the sensory levels, rather than at the level of transmission between interneurons and motoneurons. The peripheral location of response depression in Clione appears to be quite unique because in other startle-escape systems that show habituation, the site of plasticity is central rather than peripheral. For example, startle response habituation in crayfish has been found to be a central phenomenon that occurs at the sensory afferent-primary interneuron synapse (Krasne 1969
; Miller et al. 1992
). We have not yet examined the sensory afferent-to-startle interneuron synapses for such plasticity in Clione.
Cholinergic nature of excitatory inputs to startle motoneurons
Excitatory inputs to the startle motoneurons, including monosynaptic EPSPs produced by action potentials in the Cr-St interneurons, appeared to be cholinergic. They were blocked by cholinergic antagonists and mimicked by exogenous acetylcholine. The pharmacology of cholinoreceptors in gastropod mollusks is complicated and does not reflect the original classification of muscarinic and nicotinic types based on the use of selective vertebrate antagonists and agonists (for review on cholinergic transmission in gastropod mollusks see Walker 1986
). A similar situation was found for cholinoreceptors on the startle motoneurons of Clione.
The most effective blocker of startle motoneuron EPSPs (and acetylcholine-induced depolarization) was found to be atropine, a known selective antagonist of muscarinic receptors. However, EPSPs were also effectively blocked by d-tubocurarine, which is a nicotinic receptor antagonist. The effect of acetylcholine was mimicked by nicotine, although it appeared to be less effective than acetylcholine. In addition, EPSPs in startle motoneurons resembled nicotinic-type junctional potentials in that they were very fast, with short latencies (2 ms), short durations (10 ms), very high amplitudes (up to 50 mV), and reversal potentials around +10 mV. Muscarinic responses are typically much slower than these EPSPs, because muscarinic receptors are normally coupled to ion channels by second-messenger systems, whereas nicotinic receptors are directly associated with ion channels (for reviews see Monferini 1995
; Skok et al. 1989
; Wastek and Yamamura 1981
).
It is thus difficult to pharmacologically define the postsynaptic receptors as nicotinic or muscarinic. Regardless, the synaptic connection between Cr-St interneurons and startle motoneurons appears to be cholinergic, because the transmission was blocked by cholinergic antagonists and was mimicked by exogenous acetylcholine in very low concentrations, and reversal potentials were the same for EPSPs and depolarizations induced by exogenous acetylcholine.
Involvement of startle neurons in the feeding behavior
Our original focus was on the role of startle motoneurons and Cr-St interneurons in initiating the startle response, which represents the initial phase of escape behavior in Clione. It appears, however, that startle motoneurons and Cr-St interneurons may be active during another behavior, feeding, which is also characterized by a dramatic acceleration of swimming. Clione is an aggressive carnivore that feeds on the actively swimming prey, shelled pteropods from the genus Limacina. Swim acceleration is important for successful prey capture, providing a forward lunge toward the prey. We have found in the present study that sensory inputs such as tactile stimulation of the lips and buccal cones, which are known to initiate prey capture reactions in intact animals and to activate neurons involved in the control of this behavior (Norekian 1995
), produced excitatory inputs to Cr-St interneurons and startle motoneurons. The system of startle neurons thus may be involved in triggering a forward lunge during prey capture in Clione, thus increasing the success of prey capture.
A similar dual function has recently been suggested for Mauthner cells, which may represent an analogous neuronal system in fish and amphibians. Physiological and behavioral studies originally indicated that Mauthner cells initiate the characteristic fast start escape response of fish (for reviews see Eaton and Hackett 1984
; Zottoli et al. 1995
). However, it was also demonstrated that Mauthner cells may be active in behaviors other than fast startle responses. The activation of Mauthner cells during prey capture has recently been shown in goldfish where, the cells fire when the fish performs a C-shaped flexion in association with the terminal phase of prey capture (Canfield and Rose 1993
). Mauthner-initiated flexions during feeding rapidly remove the prey from the water surface, thus increasing both the success of prey capture and minimizing the fish's own susceptibility to surface predation (Canfield and Rose 1993
).
Functioning of the startle neuronal system
This and the previous paper (Satterlie et al. 1997
) are the first publications describing a unique gastropod neuronal system that underlies a startle response. Although the basic arrangement of the neuronal organization has been described, there are still questions to be answered. First is the question of how the coordination between d-phase and v-phase neurons is organized during the startle response. Obviously, startle neurons from d-phase and v-phase cannot be active at the same time. However, no reciprocal inhibitory connections were found between d-phase and v-phase motoneurons, suggesting that coordination occurs at higher levels in the circuit (Satterlie et al. 1997
). The Cr-St interneurons described here were found to activate only ipsilateral d-phase startle motoneurons. This is logical, because the cerebral interneurons cannot simultaneously activate both d-phase and v-phase motoneurons. However, Cr-St interneuron connections do not explain how coordination between d-phase and v-phase neurons is organized. We have not found cerebral interneurons that activate v-phase startle motoneurons. This is a critical question because v-phase startle motoneurons are always activated first, and behavioral startle responses always begin with a ventral wing movement (Satterlie et al. 1997
). One possible scenario is that sensory inputs from the tail are conducted directly to the v-phase startle motoneurons in the pedal ganglia, whereas d-phase startle motoneurons receive delayed inputs that are routed through Cr-St interneurons in the cerebral ganglia.
The next question is how the coordination is organized between left and right startle neurons. Contralateral startle motoneurons do not communicate with each other (Satterlie et al. 1997
). Cr-St interneurons from the left and right cerebral ganglia also do not show interconnections. It appears that bilateral coordination is mainly due to common sensory inputs. The option of independent firing of left and right startle neurons may also have an important implication. At times it might be necessary to produce a stronger contraction in one wing to turn the animal away from the source of an alarming and unexpected stimulus. Investigation of the startle neuronal system and mechanisms of coordination, as well as a search for additional neuronal elements, primarily on the level of interneurons and sensory cells, represent the main target for our future experiments.
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ACKNOWLEDGEMENTS |
We express our gratitude to Professor A. O. Dennis Willows and the staff of the Friday Harbor Laboratories for kind cooperation, Dr. C. Mills for help in collecting animals, and Dr. G. Mackie for providing additional micromanipulators.
This study was supported by National Institute of Neurological Disorders and Stroke Grant R01 NS-27951 and National Science Foundation Grant IBN-9319927.
 |
FOOTNOTES |
Address reprint requests to T. P. Norekian.
Received 22 April 1996; accepted in final form 18 September 1996.
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