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1Laboratory of Neurobiological Engineering, Graduate School of High-Technology for Human Welfare, Tokai University, Shizuoka, Japan; and 2Department of Physiology and Biophysics, Calgary Brain Institute, Faculty of Medicine, University of Calgary, Alberta, Canada
Submitted 6 July 2004; accepted in final form 4 August 2004
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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max of rhodopsin. Additionally they extend their axons in the direction of the other type of photoreceptor neuron, the type T cell. These neurons have a 2-component response to light: a response reversibly reduced in Ca2+-free saline, and a component persisting in Ca2+-free saline. Type T cells send processes into the cerebral ganglion and terminate close to the ending of the statocyst hair cells. Hair cells send their terminal branches to the cerebral ganglia close to the terminations of the type T cells. Caudal hair cells respond to a light flash with a depolarization, whereas the rostral cells respond with a hyperpolarization. The response latency in all hair cells was dependent on the stimulus intensity; the brightest light tested had a latency of 200 ms. The photo-induced response was abolished in Ca2+-free saline, whereas it was still present in high Ca2+–high Mg2+ saline, consistent with the hypothesis that the connection between the photoreceptors and hair cells is monosynaptic. Thus the sensory information necessary for forming an association between photic and rotational stimuli converges on the statocyst neurons. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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). The results from these studies have shown that neuronal changes that are correlated with the behavioral changes involve a number of different mechanisms at multiple loci in the nervous system, that is, sensory neurons, motor neurons, and interneurons, involving both pre- and postsynaptic changes (Antonov et al. 2003; Benjamin et al. 2000; Kimura et al. 1998; Murphy and Glanzman 1999; Sangha et al. 2003; Spencer et al. 2002; Yamada et al. 1992). However, changes in neuronal activity that directly cause associative learning and its consolidation into memory have been difficult to obtain, even in these model systems.
Lymnaea stagnalis is a well-established animal model exhibiting classical and operant conditioning (Benjamin et al. 2000; Kawai et al. 2004b; Lukowiak et al. 2003). Previously we demonstrated that Lymnaea are capable of being classically conditioned by pairing a flash of light (the conditional stimulus [CS]) with orbital rotation (the unconditional stimulus [UCS]) (Sakakibara et al. 1998). After 30-paired presentations of light (CS) and orbital rotation (UCS), the animals exhibit a robust withdrawal response to the CS, whereas initially they did not withdraw to the light flash (i.e., they learned). We further found that the photic information for this learned behavior is mediated exclusively by the ocular photoreceptors and not by the dermal photoreceptors (Ono et al. 2002). We are studying this form of learning in Lymnaea because it occurs with relatively few paired presentations and is behaviorally very robust. However, unlike in Hermissenda, another mollusk that has been extensively used in learning and memory studies (Alkon 1987; Sahley and Crow 1998) using a training procedure similar to the one we used in Lymnaea, little is known about the physiology of either the Lymnaea photoreceptors or the responses of statocyst hair cells to photic stimulation. This information is vital if we are to characterize the neuronal loci and the molecular events that are causal to learning and its consolidation into long-term memory (LTM).
In Hermissenda, synaptic interactions occur between photic and vestibular sensory neurons (Tabata and Alkon 1982). More recent studies have shown that morphological modifications occur at the terminal branches of the type B photoreceptor early on during conditioning (Alkon et al. 1990; Kawai et al. 2002–2004a). Therefore we have to determine whether the more complicated eye of Lymnaea (i.e., 100 s of photoreceptors) works in much the same manner as the relatively simple eye of Hermissenda (i.e., 5 photoreceptors). To accomplish this we must first gain an understanding of how the photoreceptors in the eye of Lymnaea are organized, how they respond to photic stimulation, and additionally how they send afferent information to the rest of the CNS. We then have to determine how photic information can affect information regarding rotational stimuli (i.e., the interaction between the sensory information from the eye and from the statocysts). Only then we can begin to determine the neuronal mechanisms that underlie this form of associative learning.
Previously, based on extracellular recordings from the optic nerve, Stoll and Bijlsma (1973) concluded that there were 2 classes of neurons in the Lymnaea eye: photoreceptors and optic ganglion cells. A more recent morphological study (Bobkova 1998) suggested that there may be 3 types of neurons in the Lymnaea eye. These were termed the type I and type II photoreceptors and the optic ganglion cell. The size and location of each type of photoreceptor in the retina was not distinctive; rather the shape of microvilli differentiated the neurons. With respect to the hair cells in the statocyst, previously published data (Janse et al. 1988) showed that there were 2 populations of statocyst cells: one small (about 30 µm) and one large (about 60 µm). However, how these cells respond to photic stimulation is not certain. The results of our new studies in Lymnaea may enable us in the future to determine where and how the changes that cause learning and its consolidation into memory occur.
We report here, based on both electrophysiological and morphological data, that there are 2 types of photoreceptor cells in the retina of Lymnaea and that one of them, the type T cell, sends an axon out of the eye to the cerebral ganglion close to where the terminal branches from statocyst hair cells terminate. Thus the sensory information necessary for forming an association between photic and rotational stimuli may occur at this putative synaptic contact.
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METHODS |
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Laboratory-reared fresh water pond snails, Lymnaea stagnalis, originally derived from stocks from Vrije Universeit (Amsterdam, The Netherlands) with shell lengths between 5 and 23 mm, were maintained at 20°C in well-aerated water on a 12-h light/12-h dark cycle (on at 0800 h), and fed cabbage and goldfish pellets.
Electrophysiology of the photic receptors
All experiments were performed at room temperature (20°C) during the daylight portion of the diurnal cycle. The circumesophageal ganglia together with the nerves innervating the eyes were first dissected out from Lymnaea with a micro scissors under dim light illumination in Lymnaea saline (51.3 mM NaCl, 1.7 mM KCl, 5.0 mM MgCl2, 1.5 mM CaCl2, and 5.0 mM HEPES, pH 7.9–8.1). This preparation was immobilized on a Silgard-coated culture plate using stainless-steel pins as schematically illustrated in Fig. 1. To facilitate intracellular recordings from photoreceptors in the retina, the epidermis around each eye was carefully removed with fine forceps to better expose the eye. The thin connective tissue sheath surrounding the ganglia was partially digested by incubation in protease (type XIV or type VIII, Sigma Chemical, St. Louis, MO) solution (1 mg/ml) for 5 to 6 min at 20°C.
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(measured with 3 M KCl-filled electrodes) connected by a silver chloride wire to a high-input impedance amplifier (Axoclamp 2B, Axon Instruments, Union City, CA). Voltage responses were recorded on a storage oscilloscope (5111A, Tektronix, Beaverton, OR) and on a chart recorder (Rectihoriz 8, NEC-Sannei, Tokyo, Japan), and analyzed using a microcomputer by an interface board (Digidata 1200, Axon Instruments) with analysis software (p-clamp, Axon Instruments). Electrophysiology of hair cells of the statocysts
The statocysts are located within the paired pedal ganglia of Lymnaea. To facilitate electrode penetration into a statocyst neuron, the commissural fiber between the right and left cerebral ganglion was cut so as to better pin down the preparation. We devised a means of naming each cell from which we recorded. This was based on the position of the neuron within each statocyst (e.g., caudal–rostral; dorsal–ventral; lateral–medial).
To characterize the synaptic input onto the recorded cell, either a Ca2+-free saline (51.3 mM NaCl, 1.7 mM KCl, 6.5 mM MgCl2, and 5.0 mM HEPES, pH 7.9–8.1) or a high-Ca2+ and high-Mg2+ saline (51.3 mM NaCl, 1.7 mM KCl, 24 mM CaCl2, 12 mM MgCl2 and 5.0 mM HEPES, pH 7.9–8.1) was perfused at the rate of 6 ml/min. The high-Ca2+, high-Mg2+ saline raises the action potential threshold, thereby blocking or substantially reducing the efficacy of the polysynaptic pathways (Cohen et al. 1978), whereas the Ca2+-free saline inhibits transmission via chemical synapse.
Photic stimulation
The timing of a 1,000-ms light flash from a 500-W Xenon arc lamp (KSX-25MHMA1, Sanso, Tokyo, Japan) was controlled using a solenoid mechanical shutter (EC-601, Copal, Tokyo, Japan) in the light path and the light directed underneath the preparation using a fiber-optic cable. The light intensity at the preparation was always <3.0 mW/cm2 at 510 nm recorded by a photopower meter (TQ8210, Advantest, Tokyo, Japan). The optical intensity was attenuated with neutral-density filters (Kodak, Hemel Hempstead, UK) covering within 5 log units of intensity to obtain the intensity–response relation. The intensity–response amplitude relation was expressed as follows
 | (1) |
is the half-maximum response intensity. The parameter n was determined by fitting the experimental data to Eq. 1 to obtain the best fit in the sense of the least-mean-square error. The spectral response of the photoreceptors was measured in 20-nm steps from 400 to 700 nm using equal-energized monochromatic light obtained from interference filters. The response to light was recorded every 2 min after 10-min dark adaptation. To characterize the photoreceptor cell light response, Ca2+-free saline (51.3 mM NaCl, 1.7 mM KCl, 6.5 mM MgCl2, and 5.0 mM HEPES, pH 7.9–8.1) was perfused at the rate of 6 ml/min.
Histology
To examine the morphological features of the recorded photoreceptors, Lucifer yellow (LY) was injected iontophoretically with an AC of 1 Hz, –0.5 nA in 50% duty cycle for 30 min after photic characterization. After intracellular dye injection, preparations were placed in the dark in Lymnaea saline for 30 min to allow the dye to diffuse. The nervous tissue with eye was fixed with 4% paraformaldehyde in 0.1 M PO4 buffer at pH 7.4 for 3 h, and then dehydrated through a 70–100% ethanol series. The whole-mount preparation was cleared using xylene on a glass slide embedded with resin (Bioleit, Oukenn, Tokyo, Japan). Morphological observations of LY-filled photoreceptors and its projections into the circumesophageal ganglia were performed using a fluorescent microscope (Microphoto-SA, Nikon, Tokyo, Japan).
For morphological observation on the hair cells the following procedure was followed. After cessation of the physiological experiment the isolated circumesophageal ganglion was fixed with 4% paraformaldehyde in 0.1 M PO4 buffer. The nucleus of the neuron was stained with 1 µM of SYTOX Green Nucleic Acid Stain (S-7020, Molecular Probes) diluted with Lymnaea saline. Morphological examination of the statocyst to identify neurons was done using a confocal microscope (Leica, Heerbrugg, Switzerland).
Statistical test
Statistical comparison of resting membrane potential (RMP) between the type A and T photoreceptors was made with an unpaired t-test and that of response latency and response amplitude among the type A and type T photoreceptors were made with one-way ANOVA (Origin 7.0, Microcal, Northampton, MA).
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RESULTS |
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Two types of photoreceptors, which we call type A and type T, were found and recorded from in the eye of Lymnaea. Although both the A and T photoreceptors were depolarized by photic stimulation, there were clear differences in their response characteristics. Type A photoreceptors had a resting membrane potential of –61.5 ± 26 mV (n = 6), while the type T cells had a resting membrane potential of –65.5 ± 19 mV (n = 11). The resting membrane potential between cells of type A and type T did not differ significantly by an unpaired t-test (t = 0.36557; P = 0.7197). The type A photoreceptor responded to the photic stimulus with a smooth receptor potential without superimposed action potentials. The type T photoreceptors, on the other hand, responded to photic stimulation with a more complex and greater amplitude waveform. Its waveform was composed of a transient shoulder followed by superimposed spike activity (Fig. 2A). The spike amplitude was typically <10 mV and in many instances the response lacked impulses, suggesting that the impulse-generating site was far from soma. The type A and type T photoreceptors in Fig. 2A were recorded from the same eye and both responded to the same range of light intensity. The amplitude of the receptor potential recorded from the type A photoreceptors to photic stimulation was not altered appreciably in Ca2+-free saline, although it tended to be slower (Fig. 2B). In contrast, the response recorded from the type T photoreceptors was significantly affected by the Ca2+-free saline, and the transient shoulder (arrow) was reversibly reduced in Ca2+ free saline while the longer latency hump grew larger.
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Next we determined the photosensitivity of the 2 types of receptors by challenging them with different intensities of white light. Figure 3 shows the response of a typical type T photoreceptor to a range of light intensities from –4 to 0 log units. The response to low light intensities was characterized by a comparatively smooth waveform with an extremely long latency of about 1,000 ms. As the light intensity was increased (in increments of 1 log unit) a transient shoulder became apparent, which saturated at –2 log units of light intensity. The longer latency hump that was seen continued to grow in amplitude up to the maximum light intensity used. In contrast in the type A cell (not shown), the response was characterized as a smooth generator potential without impulse activity and no prominent turning off response. The latency in the type A cells ranged from 300 to 400 ms.
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Spectral response characteristic
The spectral sensitivity (from 400 to 700 nm in 20-nm steps) of the type A (Fig. 5A) and type T (Fig. 5B) photoreceptors in a moderate light intensity (–2 log units) was determined next. Plotted are the spectral sensitivity curves for each individual neuron recorded with either a KCl electrode (open circles) or a Lucifer yellow–filled electrode (filled circles). The type A photoreceptors (n = 9) had a peak response at 480–500 nm corresponding to the absorption maximum of squid rhodopsin (Shichida et al. 1978) or circadian pacemaker of the eye of the mollusk, Bulla gouldiana (Geusz and Page 1991). Type T cells (n = 12), however, had a much broader peak response between 450 and 600 nm, measured at both the shoulder and the longer-latency hump.
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Figure 7A shows a complete statocyst composed of 13 statocyst neurons stained with SYTOX Green Nucleic Acid. The nuclei of the supporting cell that surround the statocyst neurons stain brightly, whereas the nuclei of the statocyst neurons stain less intensely. Figure 7B shows the results from one experiment in which 13 neurons in a single statocyst were impaled. We found that, although both rostral (1 of 9 cells) and caudal (9 of 15 cells) statocyst neurons responded to the photic stimulus, lateral (7/7 cells), medial (6/6 cells), and centrally (6/6 cells) located neurons did not (Fig. 7B). Moreover, there were regional differences in how the statocyst neurons responded to the photic stimulus. The caudal statocyst neurons responded with a depolarization (Fig. 7B), whereas the rostral cells respond to light with a hyperpolarization. Because it was more frequent to encounter the caudal statocyst neurons (9 out of 15 cells) to respond to light stimulus than the rostral cells (1 out of 9 cells) we therefore directed most of our attention to the caudal statocyst neurons.
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Response latency and light intensity
Both the latency of the response as well as the amplitude of the response in the caudal statocyst neurons were dependent on the intensity of the photic stimulus. As can be seen in Fig. 8A, increasing the intensity of the photic stimulus from –4 log units to 0 log unit results in a shorter response latency and a larger depolarizing response with action potentials superimposed on the depolarizing potential. In Fig. 8B we present data from 5 caudal statocyst neurons plotting the response latency versus stimulus intensity.
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We next ascertained the effect of altering the RMP of the statocyst neuron, by injecting constant current, on the response evoked in it by the photic stimulus. As can be seen in Fig. 9A, the more we hyperpolarized the neuron, the larger the evoked response and vice versa (i.e., the more we depolarized the RMP, the smaller the response). When we plotted the results obtained from 8 caudal statocyst neurons (Fig. 9B), we obtained an apparent reversal potential of the evoked response of approximately 0 mV.
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In our earlier, more preliminary study (Tsubata et al. 2003), we were able to abolish the photo-induced response of the caudal statocyst neurons by perfusing the preparation with a Ca2+-free saline. We repeated these experiments here and found that the response was blocked in the Ca2+-free saline (data not shown). These data are consistent with the hypothesis that the photo-induced response recorded in the statocyst neurons was a result of synaptic input to them from photoreceptor neurons, most likely the type T cells because we proved that the light response in the statocyst neuron disappeared after enucleation of an eye (Tsubata et al. 2003). That is, statocyst neurons were not directly depolarized by the photic stimulus; but rather received synaptic input from photoreceptor cells that were activated by the flash of light. Next we examined whether the response recorded in the caudal hair cell to the photic stimulus was altered in the high Ca2+–high Mg2+ saline. This high divalent cation saline has previously been used by experimenters to increase the action potential threshold and thus provide evidence whether the synaptic pathway is mono- or polysynaptic. We found (Fig. 10) that the photo-induced response in the statocyst neurons was still present. However, there were some changes: the photo-induced response was increased in amplitude and had a longer latency in the high Ca2+–high Mg2+ saline. These data are consistent with the hypothesis that the photic-induced response in the hair cell was mediated by a monosynaptic pathway.
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If the response in the statocyst neurons is the result of monosynaptic interactions between statocyst neurons and photoreceptors, then their respective neuritic terminals should overlap. Therefore we filled lateral, medial, rostral, and caudal statocyst neurons with Lucifer yellow and followed their projections (Fig. 11). All filled neurons (n = 19) projected their terminal branches into the cerebral ganglion by coursing along the commissure at the edge of the pleural ganglion. Once they entered into the cerebral ganglion, they branch out into what we call terminal branches in the central neuropile (Fig. 11, A and B). Because we had to cut the commissure between the cerebral ganglia, we could not determine whether they would course into the contralateral ganglion.
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DISCUSSION |
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; Sakakibara et al. 1998). However, because little is known about the anatomy and physiology of the Lymnaea visual and vestibular system, we could not begin to determine the underlying neuronal mechanisms of this associative learning and its consolidation into memory. Thus we undertook this morphological and physiological study of the eye and statocyst of Lymnaea as an initial step in attempting to elucidate the neuronal mechanisms of learning and memory. The initial short study we conducted demonstrated that there was a physiological interaction between the visual system and the vestibular system at the level of the statocyst hair cell (Tsubata et al. 2003). However, we did not know how visual information was processed in the retina nor did we know how it was sent to the vestibular system. Thus our understanding in Lymnaea of how the visual system interacts with the vestibular system was at best poorly understood. Classification of the photoreceptors
Here we have been able to show, using both morphological and physiological data, that there are 2 types of photoreceptors: type A and type T cells. These neurons possess distinctive differences in photosensitivity, spectral sensitivity, and morphological features. A previous morphological study by Stoll and Bijlsma (1973) also concluded that there were 2 classes of neurons in the Lymnaea eye: photoreceptors and optic ganglion cells. Bobkova (1998), on the basis of morphological analyses, suggested that there were 3 types of neurons in the Lymnaea eye, termed type I and type II photoreceptors and the optic ganglion cell. The defining morphological characteristic in that study was the shape of the microvilli. Morphologically, as shown in Fig. 6, type A and type T cells also differ. The type A photoreceptors have axons that terminate only in the eye, whereas type T cells have long axons that extend out of the retina to the cerebral ganglion. In the cerebral ganglion type T cells appear to make a terminal arborization close to the site of the hair cell terminals that exit from the statocyst. It is quite likely that what we refer to as type A photoreceptor corresponds to the photoreceptors of Bobkova (1998), whereas our type T photoreceptor corresponds to an "optic ganglion cell" in Bobkova's study.
By recording extracellularly from the optic nerve, Stoll and Bijlsma (1973) further concluded that, based on their ability to reversibly abolish photic-evoked neural activity in a low-Ca2+ and high-Mg2+ saline, the afferent photoresponse originated from the optic ganglion cells. We showed here that the type A cell was characterized by its smooth photo-induced waveform, whereas the response of type T cells to photic stimulation was more complex. There was a transient "shoulder" followed by a longer-latency "hump." This 2-component response of the type T cell agrees with the findings of a previous study (Sharko and Osipov 1981). However, we extended this earlier study by demonstrating that the transient shoulder was reversibly reduced in a Ca2+-free saline. In the Ca2+-free solution, the amplitude of the longer-latency hump was also increased. Because we were able to record simultaneously from both a type A and a type T cell (Fig. 2A) and continued to see a response to photic stimulation in the type T photoreceptor in the Ca2+-free solution, we are confident that the type T cell responds directly to the photic stimulus. Together, our data and the previous data show that there are 2 classes of photoreceptor neurons in the eye, one of which, the type T cell, sends a process to the cerebral ganglion.
It appears, based on the data reported here, that the type T cell is more than just a relay cell. In the Ca2+-free saline, which would block synaptic input to it from the type A neurons, the type T cell still responds to photic stimulation. Thus the T cell is both a photoreceptor and, because it extends a neurite to the cerebral ganglion, a neuron that potentially sends afferent information to neurons in other ganglia. The type T cell may also possess a distinctive visual pigment from that in the type A neuron because the type T cell has a much broader peak spectral response between 450 and 550 nm. The type A neuron does not have this broad spectral response. Whether the broader response peak of the type T neurons plays a role such as helping to set the circadian rhythms of Lymnaea as the optic ganglion cell in the related mollusc, Onchidium (Katagiri et al. 1995), remains to be determined.
The impulse activity recorded in the type T photoreceptor was characterized by a comparatively small amplitude spike (<10 mV). The amplitudes of these responses, however, were identical to spike activity in Hermissenda B type photoreceptors, which originate in the axon far from the soma (Tabata and Alkon 1982). This may also be the reason that we encountered less-frequent spike activity than that observed in Hermissenda photoreceptors as we mechanically manipulated the axons in the optic nerve in making our preparations.
We have shown that the visual information for this conditioning paradigm (i.e., paired light and rotation) in Lymnaea is mediated exclusively with the ocular photoreceptive system (Ono et al. 2002), not with a nonocular photoreceptive system (Chono et al. 2002; Ovchinnikov 1986). A fundamental interpretation of this result is possible because it has been clearly shown that the eyes are essential for positive phototaxic behavior (Stoll 1973). The inference is that the eyes continuously inform the CNS about intensity and direction of light stimuli. The present result supports the idea that in molluscs behavioral response to sustained illumination and shadow-response are mediated by different type of photoreceptors that can be distinguished by both physiological and morphological criteria (Land 1968; Tsubata et al. 2003).
Input to statocyst neurons
It has been well established that statocyst neurons receive afferent innervation from a number of different sensory systems including tactile and chemosensory neurons (i.e., pO2; Janse et al. 1988). Our data also confirm our earlier findings (Tsubata et al. 2003) that certain statocyst neurons receive sensory input from photoreceptors. Thus the statocyst neurons that receive photic input may thus play a major role in mediating classical conditioning of the withdrawal response brought about by pairing a photic stimulus with a rotational stimulus (Ono et al. 2002).
As shown here the type T cell sent its axon into the cerebral ganglion. There it could synapse with neuritic projections of statocyst neurons (or other neurons). Our Lucifer yellow fills of statocyst neurons are consistent with this hypothesis. They extend into the same area of the cerebral ganglion that the type T photoreceptors do. It is thus not inconceivable that the synaptic interactions that occur between retinal and statocyst neurons resulting from photic stimulation of the eyes may occur here. That the synaptic interaction occurs here is supported, but not proved, by our Lucifer yellow fills. However, in conjunction with the data from the high Ca 2+–high Mg 2+ saline experiments (which suggest, but do not prove a monosynaptic connection) and the lack of any other site of possible overlap of neuritic processes by the Lucifer yellow fills, we conclude that the cerebral ganglion is the most probable site of the monosynaptic interaction. Definite proof that a monosynaptic connection can occur between the type T photoreceptor and caudal statocyst neuron could be demonstrated in culture as has been done previously with other Lymnaea neurons (Inoue et al. 1996; Syed et al. 1990). These experiments will be performed in the future.
Characterization of statocyst hair cells
We found that not all statocyst neurons responded to photic stimulation and those which did responded in different ways. Thus 9 of 15 caudal statocyst neurons (60%) responded to the photic stimulus with a depolarization, whereas only 1 of 9 rostral cells responded with a hyperpolarization to light. In a previous study on Helix hair cells (Ovchinnikov 1986) the response to the onset of a light flash was a membrane depolarization and an increase in the superimposed firing frequency of impulse activity with a latency ranging from 0.3 to 2 s, which was dependent on the light intensity, similar to what we observed in Fig. 8. Ovchinnikov further demonstrated that in some instance adequate stimulation of photoreceptors caused inhibitory response in 2 of 50 hair cells as shown in Fig. 7B (Ovchinnikov 1986). We are uncertain whether one reason more caudal and rostral neurons did not respond to photic stimulation was attributable to the surgical manipulations (e.g., removal of the connective tissue surrounding the photoreceptor axons) necessary to make recordings from statocyst neurons. These surgical procedures may compromise photoreceptor input to many of the caudal and rostral statocyst neurons. We are uncertain whether cell size determines if a statocyst neuron will respond with a depolarizing or a hyperpolarizing response. We are also uncertain whether the same type T photoreceptor mediates both excitation and inhibition to the different statocyst neurons. Future experiments will attempt to answer these questions.
Comparison of photic responses in Hermissenda and Lymnaea statocyst neurons
Our results regarding photic input to statocyst neurons differ from the data obtained in Hermissenda and suggest that the visual–vestibular interactions in Lymnaea are organized differently. We found that photic stimulation resulted in depolarizing input to caudal statocyst neurons, whereas in Hermissenda photic stimulation resulted only in hyperpolarizing input to statocyst neurons. Thus although both Hermissenda and Lymnaea can be classically conditioned by pairing a photic stimulus (the CS) with a rotational stimulus (the UCS), the underlying synaptic interactions between the photoreceptors and the statocyst neurons may be fundamentally different. This suggests that the underlying neuronal mechanisms subserving learning and memory formation in these 2 gastropod molluscs may also be different. Alternatively, it is possible that only the statocyst neurons that receive the inhibitory input mediate learning. The eyes of these 2 molluscs are organized differently. In Hermissenda there are only 5 photoreceptor cells: 3 type B photoreceptors and 2 type A cells, whereas in Lymnaea there are a few hundred photoreceptors. There is a larger number of sensory receptor cells in each eye of Lymnaea compared with that of Hermissenda (Stoll and Bijlsma 1973) and thus Lymnaea may view the outer world much more precisely than Hermissenda, as suggested by behavioral analysis (Andrew and Savage 2000). In Hermissenda the A and B photoreceptors not only differ in their photosensitivity but differ in the roles they play in mediating phototaxic behavior. Type B cells play an inhibitory role in the mediation of phototactic behavior, whereas the A cells play an excitatory role (Akaike and Alkon 1980; Alkon 1987). In Hermissenda the B cells tend to be more excitable after conditioning, thus making it more likely that the animal will not move toward the light (Alkon et al. 1985; West et al. 1982). This increased excitability is thought to result from the synaptic interaction that occurs between the visual and vestibular sensory cells.
In Lymnaea, the behavioral consequences of pairing the photic stimulus with the rotational stimulus are different from in Hermissenda. Pairing a photic stimulus (light on) with a rotational stimulus (which causes the withdrawal response) in Lymnaea results in learning, which is expressed behaviorally as an increase in the probability that the photic stimulus will cause withdrawal. Thus although the sensory modalities used in the Hermissenda and Lymnaea studies are the same, the conditioned behaviors are different. Moreover, because the synaptic interactions between these 2 sensory modalities are also different, we expect that the neuronal mechanisms underlying learning and memory are also different. These analyses can begin to be undertaken because we now know much more about the photic input from the eye to statocyst neurons.
Although conditioning of Lymnaea to a photic stimulus is robust (Sakakibara et al. 1998) little is known of the neural correlates underlying this learning in Lymnaea. The data presented here form a foundation on which we can begin to elucidate the neuronal mechanisms that underlie this form of classical conditioning in Lymnaea. However, the larger number of receptor units in the Lymnaea retina may complicate the unraveling of the synaptic interactions that occur as a result of pairing of the photic with rotational stimuli (Sakakibara et al. 1998).
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Address for reprint requests and other correspondence: M. Sakakibara, Laboratory of Neurobiological Engineering, Department of Biological Science and Technology, School of High-Technology for Human Welfare, Tokai University, 317 Nishino, Numazu 410-0321, Shizuoka, Japan (E-mail:manabu{at}tokai.ac.jp)
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REFERENCES |
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Alkon DL. Memory Traces in the Brain. New York: Cambridge Univ. Press, 1987.
Alkon DL, Ikeno H, Dworkin J, McPhie DL, Olds JL, Lederhendler I, Matzel L, Schreurs BG, Kuzirian A, Collin C, and Yamoah E. Contraction of neuronal branching volume: an anatomic correlate of Pavlovian conditioning. Proc Natl Acad Sci USA 87: 1611–1614, 1990.
Alkon DL, Sakakibara M, Forman R, Harrigan J, Lederhendler I, and Farley J. Reduction of two voltage-dependent K+ currents mediates retention of a learned association. Behav Neural Biol 44: 278–300, 1985.[CrossRef][ISI][Medline]
Andrew RJ and Savage H. Appetitive learning using visual conditioned stimuli in the pond snail, Lymnaea. Neurobiol Learn Mem 73: 258–273, 2000.[CrossRef][ISI][Medline]
Antonov I, Antonova I, Kandel ER, and Hawkins RD. Activity-dependent presynaptic facilitation and Hebbian LTP are both required and interact during classical conditioning in Aplysia. Neuron 37: 135–147, 2003.[CrossRef][ISI][Medline]
Benjamin PR, Staras K, and Kemenes G. A systems approach to the cellular analysis of associative learning in the pond snail Lymnaea. Learn Mem 7: 124–131, 2000.
Bobkova MV. Structural and functional organization of the optic system's peripheral area in the pond snail Lymnaea stagnalis. Zh Evol Biokhim Fiziol 34: 716–731, 1998.
Chono K, Fujito Y, and Ito E. Non-ocular dermal photoreception in the pond snail Lymnaea stagnalis. Brain Res 951: 107–112, 2002.[CrossRef][ISI][Medline]
Cohen JL, Weiss KR, and Kupfermann I. Motor control of buccal muscles in Aplysia. J Neurophysiol 41: 157–180, 1978.
Geusz ME and Page TL. An opsin-based photopigment mediates phase shifts of the Bulla circadian pacemaker. J Comp Physiol A Sens Neural Behav Physiol 168: 565–570, 1991.[CrossRef][Medline]
Inoue T, Takasaki M, Lukowiak K, and Syed NI. Identification of a putative mechanosensory neuron in Lymnaea: characterization of its synaptic and functional connections with the whole-body withdrawal interneuron. J Neurophysiol 76: 3230–3238, 1996.
Janse C, van der Wilt GJ, van der Roest M, and Pieneman AW. Modulation of primary sensory neurons and its relevance to behaviour in the pond snail Lymnaea stagnalis. Symp Biol Hungarica 36: 559–568, 1988.
Katagiri N, Katagiri Y, Shimatani Y, and Hashimoto Y. Cell type and fine structure of the retina of Onchidium stalk-eye. J Electron Microsc 44: 219–230, 1995.
Kawai R, Horikoshi T, and Sakakibara M. Involvement of the ryanodine receptor in morphologic modification of Hermissenda type B photoreceptors after in vitro conditioning. J Neurophysiol 91: 728–735, 2004a.
Kawai R, Horikoshi T, Yasuoka T, and Sakakibara M. In vitro conditioning induces morphological changes in Hermissenda type B photoreceptor. Neurosci Res 43: 363–372, 2002.[CrossRef][ISI][Medline]
Kawai R, Sunada H, Horikoshi T, and Sakakibara M. Conditioned taste aversion with sucrose and tactile stimuli in the pond snail Lymnaea stagnalis. Neurobiol Learn Mem 82: 164–168, 2004b.[CrossRef][ISI][Medline]
Kawai R, Yasuoka T, and Sakakibara M. Dynamical aspects of in vitro conditioning in Hermissenda type B photoreceptor. Zoolog Sci 20: 1–6, 2003.[CrossRef][ISI][Medline]
Kimura T, Suzuki H, Kono E, and Sekiguchi T. Mapping of interneurons that contribute to food aversive conditioning in the slug brain. Learn Mem 4: 376–388, 1998.
Land MF. Functional aspects of the optical and retinal organization of the mollusc eye. Symp Zool Soc Lond 23: 75–96, 1968.
Lukowiak K, Sangha S, MacCom C, Varshney N, Rossengger D, Sadamoto H, and Scheibenstock A. Associative learning and memory in Lymnaea stagnalis: how well do they remember? J Exp Biol 206: 2097–2103, 2003.
Murphy GG and Glanzman DL. Cellular analog of differential classical conditioning in Aplysia: disruption by the NMDA receptor antagonist DL-2-amino-5-phosphonovalerate. J Neurosci 19: 10595–10602, 1999.
Ono M, Kawai R, Horikoshi T, Yasuoka T, and Sakakibara M. Associative learning acquisition and retention depends on developmental stage in Lymnaea stagnalis. Neurobiol Learn Mem 78: 53–64, 2002.[CrossRef][ISI][Medline]
Ovchinnikov AV. Intersensory interactions in the CNS of Helix lucorum. Neirofiziologiia 18: 17–26, 1986.[Medline]
Sahley C and Crow T. Invertebrate learning: current perspectives. In: Neurobiology of Learning and Memory. San Diego, CA: Academic Press, 1998.
Sakakibara M, Kawai R, Kobayashi S, and Horikoshi T. Associative learning of visual and vestibular stimuli in Lymnaea. Neurobiol Learn Mem 69: 1–12, 1998.[CrossRef][ISI][Medline]
Sangha S, Scheibenstock A, and Lukowiak K. Reconsolidation of a long-term memory in Lymnaea requires new protein and RNA synthesis and the soma of right pedal dorsal 1. J Neurosci 23: 8034–8040, 2003.
Sharko NV and Osipov BS. Electrical responses of the eye and optic nerve of the pond snail Lymnaea stagnalis. Neirofiziologiia 13: 652–654, 1981.[Medline]
Shichida Y, Tokunaga F, and Yoshizawa T. Circular dichroism of squid rhodopsin and its intermediates. Biochim Biophys Acta 504: 413–430, 1978.[Medline]
Spencer GE, Kazmi MH, Syed NI, and Lukowiak K. Changes in the activity of a CpG neuron after the reinforcement of an operantly conditioned behavior in Lymnaea. J Neurophysiol 88: 1915–1923, 2002.
Stoll CJ. On the role of eyes and non-ocular light receptors in orientational behaviour of Lymnaea stagnalis (L.). Proc K Ned Akad Wet C 76: 203–214, 1973.[Medline]
Stoll CJ and Bijlsma A. Optic nerve responses in Lymnaea stagnalis to photic stimulation of the eye. Proc K Ned Akad Wet C 76: 406–413, 1973.
Syed NI, Bulloch AG, and Lukowiak K. In vitro reconstruction of the respiratory central pattern generator of the mollusk Lymnaea. Science 250: 282–285, 1990.
Tabata M and Alkon DL. Positive synaptic feedback in visual system of nudibranch mollusk Hermissenda crassicornis. J Neurophysiol 48: 174–191, 1982.
Tsubata N, Iizuka A, Horikoshi T, and Sakakibara M. Photoresponse from the statocyst hair cell in Lymnaea stagnalis. Neurosci Lett 337: 46–50, 2003.[CrossRef][ISI][Medline]
West A, Barnes E, and Alkon DL. Primary changes of voltage responses during retention of associative learning. J Neurophysiol 48: 1243–1255, 1982.
Yamada A, Sekiguchi T, Suzuki H, and Mizukami A. Behavioral analysis of internal memory states using cooling-induced retrograde amnesia in Limax flavus. J Neurosci 12: 729–735, 1992.[Abstract]
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max) peaked between 460 and 540 nm and this characteristic was maintained regardless of the measuring point on either the shoulder or the hump. In both A and B the open circles are the responses recorded from cells using KCl-filled electrodes, whereas the filled circles are the responses recorded using Lucifer yellow–filled electrodes.
) can be seen. B: statocyst neurons at different locations show different responses to photic stimulation (–3 log units of the maximum intensity) of the eye. Lateral–caudal cells respond with a depolarization, lateral–rostral cells respond with a hyperpolarization, whereas the remaining cells do not respond to light. Particular laterorostral hair cell was hyperactive with spontaneous impulse activity. Light stimulus was indicated with bars below each recoding trace. C: response of a caudal statocyst neuron to photic stimulation of the eye. Three successive recordings of photoresponse from a caudal neuron whose resting membrane potential (RMP) was –58 mV. Eye was stimulated with a 1-s flash of light (–2 log units of the maximum intensity) every 90 s. With this intensity of light the statocyst neuron responded with an initial generator potential with superimposed spike activity. Bars under the trace indicate the light stimulus.
