INTRODUCTION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

A general conclusion derived from studies of motor systems is that the expression of diverse motor acts may be supported by the same neural circuit (for reviews, see Getting 1989; Kupfermann and Weiss 2001; Marder and Calabrese 1996; Pearson 1993). In some examples from invertebrates, multiple behaviors supported by the same neural circuit appear to be quite different. For example, in Tritonia, activation of the swim central pattern generator (CPG) contributes to both escape swimming and ciliary locomotion (Audesirk 1978a; Popescu and Frost 2002). The serotonergic As1-4 neurons in the swim network of Pleurobranchaea also excite pedal G neurons supporting ciliary locomotion (Jing and Gillette 2000). Escape swimming is muscular and rhythmic in contrast to ciliary movement, which is a nonmuscular and nonrhythmic gliding form of locomotion in mollusks (for discussion, see Copeland 1919, 1922; Gainey 1976). In addition to the requirement for selectivity of different motor programs from the same network, both modified and unmodified behaviors must also be expressed in examples where learning alters the activity of a neural circuit supporting different behaviors. As an example in Hermissenda, Pavlovian conditioning results in conditioned stimulus (CS)-elicited inhibition of locomotion (Crow and Alkon 1978, 1980) and CS-elicited foot contraction (Lederhendler et al. 1986). However, these two apparent dissimilar motor acts are not incompatible, and coactivation may help to explain the behavioral consequences of CS presentation in conditioned animals.

The synaptic organization of primary and secondary components of the visual system of Hermissenda has been characterized and described in considerable detail (Akaike and Alkon 1980; Alkon 1973a,b; Alkon and Fuortes 1972; Alkon et al. 1978; Crow and Tian 2000, 2002a; Crow et al. 1979). However, the neural network supporting the generation of visually modulated behaviors such as ciliary locomotion and foot contraction has not been identified. In this report, we have identified and characterized two pedal neurons with different behavioral functions. Pedal neuron VP1 activates cilia on the anterior foot, and pedal neuron VP2 innervates the anterior foot and ventral tentacle. Axonal processes of pedal neurons VP1 and VP2 in pedal nerve P2 were verified by eliciting orthodromic spikes and recording concomitant extracellular spikes in P2 and recording antidromic spikes in VP1 and VP2 elicited by stimulation of nerve P2. We characterized synaptic connections between type I, II, and III interneurons and recently identified pedal neurons VP1 and VP2. Here we show that pedal neurons VP1 and VP2 receive visual input from a polysynaptic pathway consisting of identified photoreceptors, type I_i, II_i, I_e, and II_e interneurons, and recently identified type II_b and III_i interneurons. Inhibitory and excitatory synaptic input from type I and II interneurons were found to project to both VP1 and VP2. Activation of the visual pathway with light results in an increase in the tonic activity of both VP1 and VP2 produced by a combination of disinhibition mediated by type III_i interneurons and excitatory synaptic input from type II_b interneurons. The organization of polysensory interneuronal synaptic projections to neurons in the motor network controlling ciliary locomotion and foot movement may explain how cellular and synaptic modifications associated with Pavlovian conditioning can co-exist with the requirement for normal visual processing expressed by the network.

	METHODS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Adult Hermissenda crassicornis were used in the experiments. The animals were obtained from Sea Life Supply, Sand City, CA, and maintained in closed artificial seawater aquaria at 14 ± 1°C on a 12-h light-dark cycle. All electrophysiological procedures were conducted during the light phase of the light/dark cycle.

Simultaneous intracellular recordings from identified type B photoreceptors, interneurons, and/or pedal neurons were collected from isolated nervous systems. Extracellular recordings were obtained from suction electrodes containing pedal nerve P2. Anatomical and electrophysiological criteria were used to identify type-B photoreceptors within the eyes as described previously (Alkon and Fuortes 1972; Crow and Tian 2000; Frysztak and Crow 1994). The isolated nervous systems were incubated in a protease solution (Sigma Type VIII; 0.67 mg/ml, 5 min) to facilitate microelectrode penetration of photoreceptors. Surgical desheathing of a small area of the cerebropleural and pedal ganglion was conducted to expose the cell bodies of interneurons and pedal neurons. As previously reported (Crow and Tian 2000), the criteria for identifying interneurons consisted of soma size, cell layer, location in the cerebropleural ganglion, electrophysiological responses to light, and extrinsic current stimulation of photoreceptors. In isolated circumesophageal nervous systems, pedal neurons were identified based on size, position along the anterior-ventral edge of the pedal ganglion, and electrophysiological responses to light and extrinsic current stimulation of pedal nerves and interneurons. In semi-intact preparations, pedal neurons VP1 and VP2 were identified by depolarization with extrinsic current and simultaneous video recording of ciliary movement and/or movement of the anterior foot and ventral tentacle.

The partially desheathed circumesophageal nervous systems were pinned to a silicone elastomer (Sylgard; Dow Chemical) stage in a recording chamber filled with artificial seawater (ASW) of the following composition (in mM) 460 NaCl, 10 KCl, 10 CaCl₂, and 55 MgCl₂, buffered with 10 mM HEPES and brought to pH 7.46 with dilute NaOH. The ASW in the recording chamber was monitored by a thermistor and held at 15 ± 0.5°C. Illumination of the eyes was provided by a tungsten halogen incandescent lamp attached to a fiber optic bundle mounted underneath the recording chamber. Maximum light intensity was attenuated with neutral density filters expressed in negative log units. Photoreceptors, interneurons, and pedal neurons were impaled with microelectrodes filled with 4 M KAc. Micoelectrodes were connected to the two headstages of an Axoclamp 2A (Axon Instruments, Foster City, CA). Standard intracellular and extracellular recording and stimulation techniques were employed. Digitized data were analyzed and plotted using Spike 2 software (Cambridge Electronic Design). Single spikes elicited by brief extrinsic current pulses and trains of action potentials elicited by current steps were applied in the dark through a bridge circuit. Depolarizing generator potentials were evoked by light steps of variable duration that followed appropriate periods of dark adaptation. Synaptic connections between photoreceptors, interneurons, and pedal neurons were examined in normal ASW. Evidence for monosynaptic connections between interneurons and pedal neurons was provided by PSPs with relatively short and constant latencies and a one-for-one relationship between interneuron action potentials and PSPs recorded from pedal neurons in normal ASW and in ASW containing high-divalent cations (3× Ca²⁺ and 3× Mg²⁺).

Semi-intact preparations were prepared by cooling the animals to between 0 and 1°C followed by isolation of the circumesophageal nervous system from the buccal crest and body leaving an intact pedal nerve P2. The foot was positioned ventral side up adjacent to the isolated circumesophageal nervous system. The exposed nervous system and anterior foot were visualized in infrared illumination provided by a 45-W tungsten/halogen light source projected by a light guide through an infrared filter (Schott model RG-850). A dissecting microscope formed an image of the nervous system in the infrared light on a Dage MTi video camera connected to a video monitor and video recorder. Ciliary movement was assessed indirectly in infrared illumination by video imaging of the movement of small dried ink particles on the anterior region of the foot during depolarization of VP1 with extrinsic current. Ciliary activity evoked by stimulation of VP1 was quantified by measuring particle movement during the depolarizing current step on a transparency attached to the video monitor. Movement of the anterior foot was videotaped during depolarization of VP2 and quantified by measuring foot displacement from prestimulus baseline positions on the transparency covering the video monitor screen. To rule out the possible contribution of indirect activation of other central neurons to ciliary movement, VP1 and VP2 were depolarized and ciliary movement and anterior foot displacement videotaped with nervous systems exposed to ASW solutions containing high divalent cations (3× Ca²⁺ and 3× Mg²⁺).

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

A neural network diagram showing the synaptic connections among photoreceptors, interneurons, and pedal neurons VP1 and VP2 is shown in Fig. 1. The network represents previously identified and newly identified synaptic connections reported in this paper.

View larger version (25K): [in this window] [in a new window]   Fig. 1. Neural network depicting components of the Hermissenda visual pathway. Diagram of synaptic connections between identified photoreceptors, photoreceptors, and interneurons, and interneurons and pedal neurons VP1, VP2, VP3 and VCMN. Photoreceptors are designated as lateral A (LA), medial A (MA), lateral B (LB), central B (CB) and medial B (MB). Previously identified interneurons designated as type Ie, Ii, type IIe, IIi, and newly identified interneurons designated as type IIb, type IIIi and ventral pedal neurons (VP1, VP2, VP3, and VCMN). , excitatory synapses; , inhibitory synapses. , established monosynaptic connections; - - -, polysynaptic connections with potential interneurons not yet identified.

P2 nerve recordings: projections of VP1 and VP2

Pedal motor neurons that innervate the foot and contribute to locomotion project to postsynaptic targets through identified pedal nerves P1 and P2 (Hodgson and Crow 1991; Richards and Farley 1987). Extracellular recordings from pedal nerve P2 in isolated nervous systems revealed several extracellular spikes whose tonic firing frequency was increased by illumination of the eyes. Previously, backfills of P2 with Ni-Ly labeled a number of candidate pedal motor neurons (data not shown). Two of the labeled pedal neurons designated VP1 and VP2 were examined in the present experiments. Pedal neuron VP1 receives ipsilateral and contralateral polysynaptic input from photoreceptors. As shown in Fig. 2A1, light produced a depolarization and increased tonic firing of pedal neuron VP1 that was associated with an increase in the smaller amplitude extracellular spike activity recorded from nerve P2 (Fig. 2B1). The increase in extracellularly recorded spikes is reflected by an increase in light-evoked baseline amplitude (B1). On a faster time scale shown in Fig. 2, A2 and B2, the VP1 spike is associated one-for-one with an extracellular spike recorded from P2 (; B2). In addition to light-elicited activity of VP1, the largest amplitude extracellular spikes recorded from nerve P2 reflect light-elicited activity generated in pedal neuron VP2. As shown in Fig. 2C, light elicited a depolarization and increase in spike activity in pedal neuron VP2 and a concomitant large-amplitude extracellular spike recorded from pedal nerve P2 (, Fig. 2D). The large-amplitude extracellular spike followed the action potentials recorded from VP2 one-for-one (Fig. 2D).

View larger version (22K): [in this window] [in a new window]   Fig. 2. Simultaneous recordings from pedal neurons VP1 and VP2 and nerve P2 during illumination of the eyes. Light elicited an increase in tonic firing of VP1 (A1) and a corresponding increase in extracellular spike activity recorded in P2 (B1). The same recording on a faster time scale showed that the action potential recorded in VP1 (A2) is associated with an extracellular spike () 1-for-1 in the recording from nerve P2 (B2). The large amplitude extracellular spike in B2 is associated with spike activity in pedal neuron VP1. Action potentials recorded in VP2 (C) are associated 1-for-1 () with the large amplitude extracellular spike recorded from verve P2 (D).

Extracellular spike activity recorded in nerve P2 followed VP1 and VP2 spike activity with a brief latency as shown in Fig. 3. A current-elicited spike generated in VP1 (Fig. 3A) resulted in a short-latency extracellular spike recorded from nerve P2 (Fig. 3B). In the same preparation, stimulation of nerve P2 with extrinsic current elicited a short-latency antidromic spike recorded in VP1 (Fig. 3C). Antidromic spikes exhibited a short and constant latency after stimulation of nerve P2 as shown by the three superimposed spikes in Fig. 3D. Similar results were obtained from recordings of pedal neuron VP2 as shown by the examples of orthodromic and antidromic spikes in Fig. 3, E-G. A spike in VP2 elicited by extrinsic current was associated with a brief latency extracellular spike recorded from nerve P2 (Fig. 3F). A short-latency antidromic spike recorded in VP2 followed stimulation of nerve P2 (Fig. 3G).

View larger version (13K): [in this window] [in a new window]   Fig. 3. Pedal neurons VP1 and VP2 project axonal processes in pedal nerve P2. A single spike elicited by an extrinsic current pulse in VP1 (A) is followed by a brief and constant latency extracellular spike recorded in nerve P2 (B). An antidromic spike recorded in VP1 (C) followed stimulation of nerve P2. Antidromic spikes recorded in VP1 exhibited a brief and constant latency following stimulation of nerve P2 as shown by the 3 superimposed VP1 antidromic spikes in D. A single spike elicited by an extrinsic current pulse in VP2 (E) is followed by a brief and constant latency extracellular spike recorded in nerve P2 (F). Stimulation of nerve P2 elicited an antidromic spike recorded from VP2 (G).

VP1 stimulation activates cilia on the anterior foot and stimulation of VP2 elicits movement of the anterior foot and ventral tentacle in semi-intact preparations

Depolarization of VP1 with a 10-s extrinsic current pulse elicited ciliary movement on the anterior foot in a semi-intact preparation. Figure 4A shows an example of increased tonic firing of VP1 elicited by extrinsic current and the concomitant movement of small dried ink particles on the anterior foot analyzed from videotape data (B). The foot was imaged in infrared illumination during activation of VP1 in normal ASW (Fig. 4A) and in a preparation whose nervous system was exposed to a high-divalent cation ASW solution (Fig. 4C). The high-divalent cation ASW solution did not block ciliary activation during depolarization of VP1 (Fig. 4D), indicating that VP1 does not indirectly activate cilia through polysynaptic connections with central neurons. As shown in Fig. 5 depolarization of VP2 in semi-intact preparations produced movement of the anterior foot measured from videotape recordings. Circumesophageal nervous systems exposed to the high-divalent cation ASW solution could still express anterior foot movement elicited by depolarization of VP2 (Fig. 5, B and C), indicating that indirect activation of central neurons was not contributing to anterior foot movements elicited by stimulation of VP2.

View larger version (26K): [in this window] [in a new window]   Fig. 4. Depolarization of VP1 evoked activation of cilia on the anterior foot in semi-intact preparations. A: intracellular recording from VP1 during depolarization with a 10-s 0.8-nA extrinsic current pulse. Movement of dried ink particles on the anterior foot in arbitrary units during the 10-s depolarization of VP1 in normal artificial seawater (ASW; B). C: depolarization of VP1 (1 nA) in a circumesophageal nervous system exposed to a high-divalent cation solution (3× Ca2+ and 3× Mg2+). Movement of dried ink particles on the anterior foot during the 10-s depolarization of VP1 in a high-divalent cation solution (D).

View larger version (9K): [in this window] [in a new window]   Fig. 5. Depolarization of VP2 evoked movement of the anterior foot. A: intracellular recording from VP2 in normal ASW during a 10-s depolarization (2 nA). B: depolarization of VP2 (5 nA) in a circumesophageal nervous system exposed to a high-divalent cation solution. C: group data (mean ± SE) of anterior foot movement evoked during the 10-s depolarization in normal ASW and nervous systems exposed to a high-divalent cation solution (n = 4).

Synaptic projections from photoreceptors and interneurons

We next examined synaptic input to VP1 and VP2 from previously identified components of the visual pathway. Evidence showing polysynaptic input to VP1 from an identified type-B photoreceptor is shown in Fig. 6. Stimulation of a medial type-B photoreceptor with extrinsic current (Fig. 6A) resulted in an increase in tonic firing of pedal neuron VP1 (Fig. 6B) and an increase in extracellular spike activity recorded in nerve P2 as indicated by an increase in prestimulus baseline amplitude (Fig. 6C). The increase in the discharge frequency of the largest amplitude extracellular spike recorded in nerve P2 suggests that the medial type-B photoreceptor also projects to both VP1 and VP2 (see preceding text).

View larger version (28K): [in this window] [in a new window]   Fig. 6. Identified type B photoreceptors exhibit polysynaptic projections to pedal neuron VP1. Simultaneous recordings from a medial type B photoreceptor, pedal neuron VP1 and pedal nerve P2. A depolarizing extrinsic current step applied to a medial type B photoreceptor (A) elicited an increase in tonic firing of pedal neuron VP1 (B) and an increase in extracellularly recorded spikes of different amplitudes recorded from pedal nerve P2 (C).

We previously showed that identified photoreceptors exhibited monosynaptic projections to two aggregates of interneurons designated as type I_e and type I_i, and polysynaptic connections to type II_e and type II_i interneurons (Crow and Tian 2000, 2002a). Here we show that synaptic projections from photoreceptors to pedal neurons VP1 and VP2 involve previously identified type I and type II interneurons. An example of a light-elicited depolarization and increase in tonic spike frequency in an identified type I_e interneuron is shown in Fig. 7A1. Depolarization of the same type I_e interneuron with extrinsic current (Fig. 7A2) elicited an increase in extracellular spike activity recorded from nerve P2 (Fig. 7B). The increase in the discharge frequency of the large-amplitude extracellular spike suggests that the type I_e interneuron also projected by polysynaptic pathways to pedal neuron VP2. As shown previously, the increase in light-elicited extracellular spike activity recorded from nerve P2 is associated with increased tonic firing of both pedal neurons VP1 and VP2.

View larger version (39K): [in this window] [in a new window]   Fig. 7. Identified type Ie interneurons project to pedal neurons with axonal processes in nerve P2. The type Ie interneuron was identified by a characteristic light-elicited depolarization and increase in tonic firing (A1). Stimulation of the same type Ie interneuron shown in A1 with an extrinsic depolarizing current pulse (A2) resulted in an increase in the firing of extracellularly recorded spikes of different amplitudes in nerve P2.

Convergence of type I and II interneuron synaptic input

A characteristic of the synaptic activity recorded in pedal neuron VP1 is the generation of inhibitory postsynaptic potentials (IPSPs) recorded in the dark and a decrease in IPSP frequency during illumination. The average IPSP frequency of VP1 pedal neurons in the dark (n = 20) was 3.0 ± 0.17 and 1.9 ± 0.19 during the light step. The decrease in IPSP frequency of VP1 neurons evoked by light was statistically significant (t₁₉ = 5.6; P < 0.001). An example of a simultaneous recording from a type I_i interneuron and pedal neuron VP1 in the dark and during a light step is shown in Fig. 8, A and B. The light step elicited a characteristic complex IPSP recorded from the type I_i interneuron (Fig. 8A). At the onset of light and during the light step, IPSP frequency decreased in pedal neuron VP1 as shown in Fig. 8B. During the period of illumination, the presentation of an extrinsic depolarizing current pulse in the type I_i interneuron increased the frequency of IPSPs recorded in VP1 (Fig. 8B). After the termination of the light step, IPSP frequency increased in VP1 to prelight baseline levels. Light and extrinsic current stimulation of type I_i interneurons produced similar effects on IPSP frequency recorded from VP2 pedal neurons (Fig. 8, C and D). The average IPSP frequency of VP2 pedal neurons recorded in the dark (n = 10) was 3.2 ± 0.34 and 2.1 ± 0.27 during the light step. The decrease in IPSP frequency of VP2 neurons evoked by light was statistically significant (t₉ = 6.1; P < 0.001). Light hyperpolarized and decreased activity in the type I_i interneuron and the depolarizing extrinsic current pulse applied during the light step increased IPSP frequency recorded in VP2 (Fig. 8D). These results indicate that type I_i interneurons project to interneurons that inhibit VP1 and VP2 pedal neurons, and light results in a disinhibition of VP1 and VP2 pedal neurons as a result of light-elicited inhibition of spike activity in type I_i interneurons. The effect of light on changes in IPSP frequency recorded from VP1 and VP2 pedal neurons and extrinsic current depolarization on IPSP frequency recorded from VP1 and VP2 neurons is summarized in the group data shown in Fig. 8, E and F. The analysis of the group data revealed that light produced a significant decrease in IPSP frequency in VP1 (t₁₉ = 6.0; P < 0.001) and VP2 (t₉ = 6.2; P < 0.001) as compared with prelight baseline activity. In addition, the depolarizing current pulse applied to the I_i interneurons produced a significant increase in IPSP frequency recorded in VP1 (t₆ = 4.1; P < 0.006) and VP2 (t₅ = 2.4, P < 0.05) during the light step (Fig. 8F). A similar finding was detected in type II_i interneurons as shown in Fig. 9. A light step elicited a decrease in type II_i spike activity (Fig. 9A1) and a decrease in IPSP frequency recorded in VP1 (Fig. 9B1). Stimulation of the same type II_i interneuron with depolarizing extrinsic current (Fig. 9A2) elicited an increase in IPSP frequency recorded in VP1 during the current pulse (Fig. 9B2).

View larger version (34K): [in this window] [in a new window]   Fig. 8. Type Ii interneurons excite interneurons that are a source for inhibitory postsynaptic potentials (IPSPs) recorded in pedal neurons VP1 and VP2. Light elicited a complex IPSP recorded from interneuron Ii (A) and a concomitant decrease in IPSP frequency recorded from pedal neuron VP1 (B). During the light step, an extrinsic current step depolarized Ii (- - -, A) and produced an increase in the number of IPSPs recorded from VP1 (B). Similar effects of light- and current-elicited depolarization were detected in simultaneous recordings from a type Ii interneuron and pedal neuron VP2. Depolarization (- - -) of the type Ii interneuron during the light step (C) in a different preparation produced an increase in IPSPs recorded in VP2 (D). IPSP frequency decreased during the light step and increased after the termination of light (D). Group data showing the mean decrease in IPSPs from baseline recorded in VP1 and VP2 elicited by the light step (E). *VP1, P < 0.001; *VP2, P < 0.001. Group data showing the significant increase in IPSP frequency elicited by the extrinsic current pulse presented during light in VP1 and VP2 (F). VP1, *P < 0.006; VP2, P < 0.05

View larger version (17K): [in this window] [in a new window]   Fig. 9. Type IIi interneurons excite interneurons that inhibit VP1 pedal neurons. Light elicited a complex IPSP and decrease in tonic firing recorded in a type IIi interneuron (A1) and a concomitant decrease in IPSP frequency recorded in pedal neuron VP1 (B1). Stimulation of the same type IIi interneuron with a depolarizing extrinsic current pulse (- - -, A2) produced an increase in IPSP frequency recorded from VP1 (B2).

Similar synaptic connections were found between type II_e interneurons and VP1 and VP2 pedal neurons. As shown in Fig. 10, a light step resulted in a characteristic depolarization of a type II_e interneuron, the generation of a spike, an increase in excitatory postsynaptic potential (EPSP) frequency (Fig. 10A1), and a concomitant decrease in IPSP frequency recorded in pedal neuron VP1 (Fig. 10B1). The decrease in IPSP frequency recorded in VP1 could be the result of light-elicited inhibition of type I_i and II_i interneurons. However, stimulation of the type II_e interneuron with depolarizing extrinsic current (Fig. 10A2) produced a decrease in IPSP frequency recorded in VP1 (Fig. 10B2). These results suggest that excitation of type II_e interneurons inhibit the activity of the inhibitory interneurons that are the source of IPSPs recorded from VP1. In addition, extrinsic depolarizing current applied to a type II_e interneuron (Fig. 10C) decreased IPSPs recorded from a VP2 pedal neuron (Fig. 10D). In summary, light affects two pathways projecting to VP1 and VP2. Light decreased inhibition of VP1 operating through both type I_i and type II_i interneurons by inhibiting the activity of interneurons that normally inhibit VP1 and VP2. The second pathway involves excitation of type II_e interneurons that inhibit the activity of inhibitory interneurons.

View larger version (26K): [in this window] [in a new window]   Fig. 10. Type IIe interneurons inhibit the source of IPSPs recorded in pedal neurons VP1 and VP2. Light elicited a depolarization, a spike, and an increase in excitatory postsynaptic potentials (EPSPs) recorded from a type IIe interneuron (A1) and a concomitant decrease in IPSP frequency during the light recorded in VP1 (B1). Depolarization of the IIe interneuron (A2) with extrinsic current resulted in a disinhibition (decrease in IPSPs) of pedal neuron VP1 (B2). Depolarization of a different type IIe interneuron (C) produced a decrease in IPSP frequency and a disinhibition of pedal neuron VP2 (D).

Identification of type III inhibitory interneurons

Simultaneous recordings from VP1 and VP2 pedal neurons suggested that IPSPs may be generated by a common presynaptic source. As shown in Fig. 11, spontaneous IPSPs occurred synchronously in both VP1 and VP2. We next examined the source of the IPSPs recorded from pedal neurons VP1 and VP2. Simultaneous recordings from an interneuron designated as a type III_i and pedal neuron VP1 in normal ASW and after exposing the nervous system to a high-divalent cation solution (3× Ca²⁺ and 3× Mg²⁺) are shown in Fig. 12, A and B. A spike generated in the type III_i interneuron (Fig. 12A1) elicited a short-latency monosynaptic IPSP recorded in VP1 (Fig. 12B1). The high-divalent cation ASW did not block the IPSP recorded in VP1 (Fig. 12B2) elicited by the spike in the type III_i interneuron (Fig. 12A2). The IPSPs recorded from VP1 followed spikes in the type III_i interneuron one-for-one with a short and relative constant latency. As shown in Fig. 12, C and D, three consecutive superimposed IPSPs with a relatively constant latency recorded from VP1 followed three superimposed single spikes in the type III_i interneuron elicited by a brief extrinsic current pulse (Fig. 12D).

View larger version (9K): [in this window] [in a new window]   Fig. 11. IPSPs recorded from pedal neurons VP1 and VP2 may be from a common presynaptic source. Simultaneous intracellular recordings from pedal neuron VP1 (A) and VP2 (B) show that IPSPs occur synchronously in each pedal neuron.

View larger version (12K): [in this window] [in a new window]   Fig. 12. Type IIIi interneurons directly inhibit VP1 pedal neurons. Simultaneous recordings from a type IIIi interneuron and pedal neuron VP1 in normal ASW and in a high-divalent cation solution (3× Ca2+ and 3× Mg2+). A single spike generated in the type IIIi interneuron by a current pulse (A1) elicited a monosynaptic IPSP recorded from VP1 (B1). Exposure of the nervous system to the high-divalent cation ASW did not block the IPSP recorded in VP1 (B2) evoked by the spike in the type IIIi interneuron (A2). IPSPs recorded from VP1 followed spikes in the type IIIi interneuron 1-for-1 with a brief and constant latency as shown by 3 superimposed spikes generated from a type IIIi interneuron (C) and superimposed IPSPs recorded from pedal neuron VP1 (D).

Type II_b projections to pedal neurons VP1 and VP2

In addition to the disinhibition of VP1 and VP2 produced by type I_i, II_i, and II_e interneurons, newly identified type II_b interneurons also contributed to light-elicited depolarization of VP1 and VP2 pedal neurons. Figure 13 shows a light-evoked generator potential recorded from a central type-B-photoreceptor (Fig. 13A1) and a depolarization and increase in EPSP frequency recorded from a type II_b interneuron (Fig. 13B1). Depolarization of the B-photoreceptor with extrinsic current elicited spikes in the type-B photoreceptor (Fig. 13A2) and EPSPs recorded from the II_b interneuron (Fig. 13B2), indicating the central type-B photoreceptor projected to the type II_b interneuron. Because light evoked more EPSPs than stimulation of a single type-B photoreceptor, it is likely that multiple photoreceptors project to a single type II_b interneuron. As shown in Fig. 14A1, light elicited an increase in the tonic spike activity of a type II_b interneuron and a concomitant increase in the spike activity recorded in VP1 (Fig. 14B1). Hyperpolarizing VP1 to 68 mV to block spike activity (Fig. 14B2) showed that the light step elicited a complex EPSP and depolarization of VP1. Stimulation of the same type II_b interneuron with a depolarizing extrinsic current pulse (Fig. 14A3) produced an increase in spike activity recorded from VP1 (Fig. 14B3). Hyperpolarizing VP1 to 67 mV blocked spike activity and revealed a complex EPSP and a depolarization of VP1 (Fig. 14B4) elicited by the depolarizing extrinsic current pulse applied to the type II_b interneuron (Fig. 14A4).

View larger version (11K): [in this window] [in a new window]   Fig. 13. Synaptic connections between identified photoreceptors and type IIb interneurons are polysynaptic. A light step elicited a depolarizing generator potential in a central B photoreceptor (A1) and EPSPs recorded from a type IIb interneuron hyperpolarized to block spike activity (B1). Stimulation of the same central type B photoreceptor with extrinsic current (A2) evoked EPSPs in the type IIb interneuron (B2).

View larger version (23K): [in this window] [in a new window]   Fig. 14. Newly identified type IIb interneurons project to VP1 pedal neurons. Light elicited an increase in tonic firing of the IIb interneuron (A1) and a concomitant increase in firing of pedal neuron VP1 (B1). Hyperpolarizing VP1 to 68 mV to block spike activity revealed that the light-elicited increase in firing of the IIb interneuron (A2) resulted in a depolarization of pedal neuron VP1 (B2). Depolarization of the same type IIb interneuron with an extrinsic current pulse (A3) produced an increase in tonic firing of VP1 (B3). Depolarization of the same type IIb interneuron with extrinsic current revealed a complex EPSP recorded from pedal neuron VP1 (B4) hyperpolarized to 67 mV to block spike generation.

We next examined synaptic input to pedal neuron VP2 from type II_b interneurons. As shown in Fig. 15, A1 and B1, light produced a depolarization of the type II_b interneuron (Fig. 15A1) and a slow depolarization and increased spike activity of pedal neuron VP2 (Fig. 15B1). A depolarizing extrinsic current pulse elicited spikes in the type II_b interneuron (Fig. 15A2) and a slow depolarization and single action potential recorded from VP2 (Fig. 15B2). Hyperpolarizing VP2 to block spike activity showed that the spikes elicited by the current step in the same type II_b interneuron (Fig. 15A3) produced EPSPs recorded in VP2 (Fig. 15B3). Taken collectively, the results provide evidence for convergence of type II_e, II_b, and II_i synaptic input to VP1 and VP2 pedal neurons.

View larger version (21K): [in this window] [in a new window]   Fig. 15. Newly identified type IIb interneurons project to pedal neuron VP2. Light elicited an increase in tonic firing of the type IIb interneuron (A1) and a concomitant depolarization and increased spike activity recorded from pedal neuron VP2 (B1). Depolarization of the type IIb interneuron with extrinsic current (A2) produced a slow depolarization and spike in VP2 (B2). Depolarization of the same type IIb interneuron with extrinsic current (A3) produced a depolarization and complex EPSP recorded from pedal neuron VP2 (B3) hyperpolarized to 59 mV to block spike generation.

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Characteristics of VP1 and VP2

In this report we have identified two pedal neurons (VP1 and VP2), characterized their electrophysiological activity, and documented the increase in tonic-firing of the neurons by illumination of the eyes and the role of VP1 in ciliary movement and VP2 in foot movement. Both VP1 and VP2 have axonal projections to the anterior foot through pedal nerve P2 as verified by recordings of orthodromic and antidromic spikes generated by stimulation of VP1 and VP2 and stimulation of nerve P2. Lucifer yellow labeling of VP1 and VP2 confirmed the electrophysiological results (unpublished results). Activation of a single photoreceptor with extrinsic current is sufficient to increase tonic firing of VP1. Stimulation of a medial type-B photoreceptor with extrinsic current was shown to produce an increase in tonic spike activity recorded from VP1 with concomitant changes in extracellular activity recorded from pedal nerve P2. The increase in the discharge rate of several extracellular spikes with different amplitudes suggests that a single type-B-photoreceptor projects polysynaptically to multiple pedal neurons. Synaptic divergence of the visual system is further supported by the observation that depolarization of an identified type I_e interneuron elicited an increase in the activity of several extracellularly recorded spikes from nerve P2. This suggests that the same components of the neural network mediate both ciliary movement and foot movement. While different, these two behaviors are not incompatible, and coactivation may facilitate ciliary movement by increasing the contact area between the foot and substrate. The control over different behaviors by the same network has been shown in Tritonia (Popescu and Frost 2002), Pleurobranchaea (Jing and Gillette 2000), and Lymnaea (Syed and Winlow 1989).

Synaptic inhibition of VP1 and VP2

Previous research has shown that light inhibits type I_i and II_i interneurons and excitates type I_e and II_e interneurons (Crow and Tian 2000, 2002a). Here we show that light evoked a complex IPSP in type I_i interneurons and a concominant decrease in IPSP frequency recorded from VP1 and VP2. Our evidence suggests that the source of the IPSPs in VP1 and VP2 is the type III_i interneuron. Depolarization of type I_i interneurons during the light step resulted in an increase in IPSP frequency recorded in both VP1 and VP2. Therefore light-evoked synaptic inhibition of type I_i interneurons results in a disinhibition of VP1 and VP2 due to a decrease in normal synaptic excitation of type III_i interneurons. A similar finding was observed for both light and extrinsic current depolarization of type II_i interneurons. Light decreased spike activity recorded in type II_i interneurons and decreased IPSP frequency recorded in both VP1 and VP2. Depolarization of type II_i interneurons in the dark increased IPSP frequency recorded in both VP1 and VP2 pedal neurons. In contrast, the effects of depolarization of type I_e and type II_e interneurons were less clear. Light-evoked depolarization of type II_e interneurons also decreased IPSP frequency recorded from VP1 pedal neurons, and depolarization with extrinsic current of type II_e interneurons produced disinhibition of VP1, e.g., decrease in IPSPs during the current pulse. The effect of current depolarization on type I_e interneurons was more variable. In some experiments, current depolarization of type I_e interneurons produced inhibition of VP1, and in other examples, no effect on PSP frequency or changes in tonic firing could be detected in either VP1 or VP2. The variability of type I_e depolarization on changes in tonic firing of VP1 and VP2 may be due in part to the labeled-line organization between photoreceptors and type I interneurons (Crow and Tian 2000). It is suggested that because lateral type-B photoreceptors project to the first layer of type I_e interneurons and other B photoreceptors project to type I_e interneurons located in deeper layers, not all type-B photoreceptors may have polysynaptic connections with VP1 and VP2 pedal neurons through type I_e and II_e interneurons. Our results show that convergence of type I_i, II_i, I_e, and II_e interneuron synaptic input is to type III_i interneurons, and type III_i interneurons form monosynaptic connections with VP1 and VP2 pedal neurons.

Type II_b interneuron synaptic projections to VP1 and VP2

In contrast to the decrease in IPSP frequency recorded in VP1 and VP2 elicited by light, light and extrinsic current depolarization of newly identified type II_b interneurons produced excitation of pedal neurons VP1 and VP2. However, light-elicited increased tonic firing of II_b interneurons was more variable than observed in type II_e interneurons. Taken collectively, the neural network mediating ciliary movement consists of two independent pathways from the photoreceptors to identified interneurons that converge on VP1. The pathway from type I_i, II_i, I_e, and II_e interneurons is through the type III_i inhibitory interneurons and, when activated by light, produces a disinhibition and increased tonic firing of VP1. The excitatory pathway from type II_b interneurons produces a depolarization and increase in the tonic firing of VP1 when activated by light.

Pedal neuron control of ciliary locomotion

Many gastropod mollusks locomote by the movement of pedal cilia on the sole of the foot (for examples see Audesirk 1978a,b; Copeland 1919; Popescu and Frost 2002; Willows et al. 1997). Ciliary locomotion is a nonrhythmic, nonmuscular, smooth gliding form of movement. The neural analysis of ciliary locomotion has been conducted in Tritonia diomedea (Audesirk 1978a,b; Popescu and Frost 2002; Popescu and Willows 1999), Pleurobranchaea (Jing and Gillette 2000), and Lymnaea (Syed and Winlow 1989). Interestingly, the swim CPG network contributes to both escape swimming and ciliary locomotion in Tritonia (Popsecu and Frost 2002). Because the foot is not in contact with the substrate during swimming, ciliary activation and swimming are not incompatible behavioral acts. Indeed, coactivation may be beneficial because after the swim ciliary locomotion is expressed as soon as the foot is in contact with the substrate (Popescu and Frost 2002). In Pleurobranchaea, the As1-4 interneurons provide both excitation to the swim CPG and locomotor G neurons that are homologs of ciliary activating neurons in other species (Jing and Gillette 2000).

Previous work has shown that pedal nerves P1 and P2 contain the axons of neurons responsible for generating locomotion in Hermissenda (Hodgson and Crow 1991; Richards and Farley 1987). Richards and Farley (1987) showed that the posterior three-fourths of the foot is innervated by nerve P1, while the remaining anterior portion is innervated by nerve P2. Consistent with their findings, here we report that extracellular recordings from pedal nerve P2 revealed light-elicited increases in tonic firing reflected by extracellular spikes with different amplitudes. We have identified VP1 and VP2 as two pedal neurons contributing to the spike activity in nerve P2. In addition, experiments conducted with semi-intact preparations showed that depolarization of VP1 produced ciliary movement and depolarization of VP2 produced anterior foot and ventral tentacle movement. The visual network consisting of photoreceptors, type I, II, and III interneurons is presynaptic to both VP1 and VP2, thus the same circuit elements can support both light-elicited ciliary movement and light-evoked muscular foot movements in Hermissenda. Muscular and ciliary effector systems are different; however, these two behavioral acts are not necessarily incompatible because the lateral foot movements elicited by stimulation of VP2 may increase the contact area of the anterior foot and thus facilitate locomotion in conjunction with activation of cilia on the sole of the anterior foot. Interestingly, a recently identified pedal neuron that produces a rapid contraction of the anterior foot does not receive synaptic input from type II_e or II_i interneurons (unpublished observation). This observation suggests that Pavlovian conditioning may modify different components of the circuit supporting ciliary locomotion and foot shortening (contraction).

Conditioned modification of locomotion and foot contraction

Previous studies have examined light-responsive putative motor neurons in the pedal ganglia, although progress has not been remarkable (Crow 1981; Goh and Alkon 1984; Goh et al. 1985; Hodgson and Crow 1991, 1992; Jerussi and Alkon 1981; Richards and Farley 1987). The most extensive analysis of a possible link between the visual system and motor behavior was reported by Goh and Alkon (1984). They identified a putative motor neuron in the pedal ganglion, classified as motor neuron one (MN1), that produced turning of the posterior half of the foot when the MN1 was stimulated with extrinsic depolarizing current. They reported that MN1 received indirect synaptic input from medial type A photoreceptors via a group of identified interneurons similar to type I and II interneurons. In response to light, MN1 exhibited an increase in EPSP frequency and an increase in the spike discharge frequency. Interneurons in the cerebropleural ganglion that received excitatory synaptic input from the medial type A photoreceptor produced excitation of MN1 when activated by light or extrinsic depolarizing current. Previous research has shown that type B excitability is enhanced by conditioning (Crow and Alkon 1978). Therefore it was proposed that in conditioned animals, the enhanced inhibition of the medial type A produced by type-B-enhanced excitability in response to the CS would result in a diminished direct excitatory synaptic input to the interneurons and decreased indirect excitation of MN1. Consistent with this hypothesis, they found that in conditioned animals, light elicited a significantly smaller increase in the spike frequency of MN1 as compared with normal controls or random control groups (Goh et al. 1985). Goh and Alkon (1984) further proposed that MN1 was involved in mediating the animal's turning behavior toward a source of illumination, and thus decreased activity of MN1 may lead to reduced motor activity and diminished phototactic behavior. However, it is clear that inhibition of type A photoreceptor activity cannot account for the neural correlates of Pavlovian conditioning identified from pedal neuron recordings and extracellular activity recorded from pedal nerves. These studies showed light-elicited decreases in spike frequency below baseline activity that cannot be related to type A activity because type A photoreceptors are not spontaneously active in the dark. Therefore inhibiting their activity during the presentation of the CS would not be expected to produce inhibition of the activity of pedal neurons below their baseline levels (Hodgson and Crow 1992; Richards and Farley 1987).

Visually mediated behaviors that have been examined in Hermissenda include the effects of light gradients on locomotion (Lederhendler et al. 1980), positive phototactic responses (Alkon 1974), foot shortening (Lederhendler et al. 1986), and the initiation of locomotion (Crow and Offenbach 1983). These examples of behaviors influenced by light and their modification by Pavlovian conditioning involve, either directly or indirectly, locomotion. The network mediating ciliary locomotion in Hermissenda expresses enhanced cellular excitability (Sahley and Crow 1998), facilitation of monosynaptic PSPs between identified photoreceptors (Frysztak and Crow 1994; Gandhi and Matzel 2000), and facilitation of PSPs between photoreceptors and type I interneurons produced by Pavlovian conditioning (Crow and Tian 2000, 2002b). The previously identified facilitation of type I interneuron monosynaptic and complex PSPs detected after conditioning would be expected to decrease the tonic activity of VP1 during the presentation of the CS. Neural correlates of Pavlovian conditioning in VP1 and VP2 pedal neurons are currently under investigation.