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Photoresponse from the statocyst hair cell in Lymnaea stagnalis
Neuroscience Letters 337 (2003) 46–50 www.elsevier.com/locate/neulet
Photoresponse from the statocyst hair cell in Lymnaea stagnalis Noriko Tsubata, Akira Iizuka, Tetsuro Horikoshi, Manabu Sakakibara* Laboratory of Neurobiological Engineering, Graduate School of High-Technology for Human Welfare, Tokai University, Numazu 410-0321, Shizuoka, Japan Received 12 September 2002; received in revised form 18 October 2002; accepted 28 October 2002
Abstract Sensory cells for associative learning of light and turbulence were studied in Lymnaea. Intracellular recordings with Lucifer Yellow filled electrodes were made from photoreceptors and statocyst hair cells. Photoreceptors had a long latency, graded depolarizing response to a flash of light; they extended their axon to the cerebral ganglion. The caudal hair cell, one of 12 cells in the statocyst, responded to brief light with a depolarization and superimposed impulse activity. It formed its terminal arborization close to the photoreceptor endings in the cerebral ganglion. Ca 21-free saline reversibly abolished the photoresponse in the hair cell, suggesting the information was conveyed via a chemical synapse. These findings demonstrated that sensory information for associative learning was convergent at the statocyst hair cell. q 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Classical conditioning; Photoreceptor; Statocyst hair cell; Lymnaea; Morphology
Gastropods are well established as models for the study of the molecular and cellular bases of learning and memory. Animals such as Aplysia [5], Limax[6], Hermissenda[1], and Lymnaea[10] exhibit well-characterized associative learning. Lymnaea stagnalis is a well-established animal model exhibiting appetitive classical conditioning [8], we previously demonstrated that Lymnaea are also capable of classical conditioning in response to paired light flashes (conditional stimulus: CS) and orbital rotation (unconditional stimulus: UCS) [12]. Naı¨ve Lymnaea do not have a behavioral response to turning on a light, but do respond to a moving shadow, offset of a long duration light, or orbital rotation with a whole-body withdrawal response. After 30 paired presentations of a light flash and orbital rotation for 3 days, the animals exhibit a robust unconditioned response, namely a whole-body withdrawal, to the CS. Lymnaea to which unpaired light and rotation stimuli are presented exhibit behavior that is significantly different from those receiving paired stimuli. Further, our previous study demonstrates that acquisition and retention of the associative learning depends on developmental stage, and that visual information is conveyed exclusively by the ocular photoreceptors not by the dermal photoreceptors [11]. This form of asso* Corresponding author. Tel: 181-55-968-1211 ext. 4504, fax: 181-55-968-1156. E-mail address: [email protected] (M. Sakakibara).
ciative learning in Hermissenda is dependent on interactions between visual and vestibular sensory neurons [15], and the cellular and molecular mechanism involved in this learning has been characterized [2]. Though the involvement of neuronal circuits in the associative learning of Hermissenda is well-established as to cause long-lasting enhancement of excitability at the type B photoreceptor and the structure of an eye itself is much more complex in Lymnaea [14] than Hermissenda [13], it is not certain whether the neuronal modification in Lymnaea is identical as that in Hermissenda after the same associative learning of visuo-vestibular interaction even in the more complex neuronal system. As a first step we examined the sensory pathways involved in this associative learning. Since the methods were described in detail previously by Kawai et al. [7] and Ono et al. [11] for animal and electrophysiology, respectively, we described the additional methods. Experiments were performed at room temperature controlled at around 20 8C. The circumesophageal ganglion with eyes was removed 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) using the following procedure. Two eyes of Lymnaea, surrounded by a perioptic sinus bounded by the epidermis, were located on the dorsal body surface at the base of tentacle. For intracellular recording from photoreceptors, the part
0304-3940/02/$ - see front matter q 2002 Elsevier Science Ireland Ltd. All rights reserved. doi:10.1016/S0 30 4- 39 40 (0 2) 0 128 9- 2
N. Tsubata et al. / Neuroscience Letters 337 (2003) 46–50
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Fig. 1. The intermediate photoreceptor. (a) A cell located in the central portion of eye was filled with Lucifer Yellow. Its axon extended into the cerebral ganglion. (b) photoresponse to a white light flash of 1 s did not have an off-response and thus it took several seconds to return to the baseline level. Calibration bar: 10 mV and 0.5 s. (c) The enlarged terminal arborization as indicated by the white square in (a).
of the epidermis around the eye was carefully removed with fine forceps to expose the eyes. In order to facilitate electrode penetration into a statocyst hair cell, the commissural fiber between the right and left cerebral ganglion was cut. The isolated circumesophageal ganglion with eyes was immobilized on a silgard coated culture plate using stainless-steel pins. The thin connective tissue sheath was digested by incubation in protease (type XIV or type VIII, Sigma Chemical Co., St. Louis, MO) solution (0.5 mg/ml) for 8–10 min at 20 8C. To demonstrate the synaptic origin of the hair cell light response, Ca 21-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. Lymnaea is known to have two kinds of light sensitive neuron; dermal photoreceptors and ocular photoreceptors [9]. Thus, to identify the source of visual information to the hair cell, the ipsilateral eye was enucleated mechanically with visual guidance under a binocular dissecting microscope (MZ FLIII, Leica, Heerbrugg, Switzerland) using a quartz-glass rod with a sharp tip. Nomenclature of hair cells was based on their body position (caudal-rostral; abdominal-ventral; lateral-medial). The isolated circumesophageal ganglion was fixed with 4% paraformaldehyde in 0.1 M PO4 buffer. The nucleus of the hair cells was stained with 1 mM of SYTOX Green Nucleic Acid Stain (S-7020, Molecular Probe, Eugene, OR) diluted with Lymnaea saline. Morphological examination of the statocyst to identify hair cells was done using a confocal microscope (TCSNT, Leica, Heerbrugg, Switzerland).
In order to examine the morphological features of neurons, Lucifer Yellow was injected iontophoretically with alternating current of 1 Hz, 20.5 nA in 50% duty cycle for 30 min. 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 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 (Biolet, Oukenn Co., Tokyo). Morphological observation of a photoreceptor together with a hair cell in an isolated circumesophageal ganglion was done on a fluorescent microscope (Microphoto-SA, NIKON, Tokyo). Since the sensory system encoding associative learning of visual and rotational stimuli in Lymnaea has not been examined at the cellular level, we identified the projection of the sensory receptor neurons, both photoreceptors and statocyst hair cells, as a first step. A previous study demonstrated that the optic nerve fiber contained two types of axons; one originating from a photoreceptor and the other from a secondary visual neuron [14]. In that study, extracellular recordings from an optic nerve fiber demonstrated that the response to a 1 s light flash lasted for many tens of seconds. Furthermore, the response was completely abolished in low Ca 21, high Mn 21 saline, suggesting that the light response was from a secondary visual neuron [14]. To evaluate responses in primary sensory receptors, intracellular recordings were made from 21 photoreceptors using
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N. Tsubata et al. / Neuroscience Letters 337 (2003) 46–50
Fig. 2. Caudal hair cell. (a) Photoresponse of the caudal hair cell recorded at different membrane potentials. The membrane potential of the hair cell is changed by constant current injection. Numbers on the left indicate the reading from the amplifier in mV. The top trace shows the timing of the light stimulus. The photoresponse of 3 mV in amplitude at the resting membrane potential of 250 mV decreases under more depolarized membrane potentials (data not shown), while it increases, with action potentials, at more hyperpolarized potentials. We defined the response latency as a time to require to initiate a photoresponse, i.e. initiation of a generator potential, from the onset of illumination. The response latency decreases with hyperpolarization from 420 ms at 250 mV to 280 ms at 2133 mV. Calibrations are 10 mV and 200 ms. (b) Photo-responses recorded from the caudal hair cell reversibly disappear in Ca 21 free saline. This finding indicates that the light response in the hair cell arises from the photoreceptor by way of a chemical synapse. The top trace is the timing of light stimulus. Calibrations are 10 mV and 200 ms. (c) The right caudal hair cell was stained with Lucifer Yellow by intracellular injection. The soma stained bright yellow and is located at the medial aspect of the statocyst. Its axon coursed around the statocyst and the terminal arborization formed at the center of the right cerebral ganglion. This picture is an overlay image of fluorescent microscope. Calibration is 100 mm. (d) Photoresponse from a hair cell abolished after enucleation of the ipsilateral eye without any noticeable membrane potential change. The commissural fiber between the right and left cerebral ganglion was cut beforehand. The hair cell was hyperpolarized due to current injection so as to demonstrate a larger photoresponse. Five minutes was interposed between pre and post enucleation of an eye. Calibration is 5 mV. Light stim represents light stimulus for 1 s.
a Lucifer Yellow filled microelectrode (Fig. 1). The membrane potential in response to a 1.0 s-light flash of 700 mW/cm 2 is illustrated in Fig. 1b. This light response was not suppressed in low Ca 21 high Mg 21 saline (data not shown); thus, we concluded that this response originated from a primary sensory neuron, photoreceptor. The light response of Lymnaeawas almost identical as that of Hermissenda,i.e. a comparatively large generator potential with superimposed impulse activity of around 10–20 mV in amplitude suggestive the impulse generating site was not at soma but along axon. This was previously demonstrated in axotomized preparation in Hermissenda [3] as that the amplitude of an action potential originating from an axon was much more smaller than that of a somatic spike activity. There was no evidence of a prominent off response, i.e., several seconds were required for the light response to return to the resting membrane potential following offset of the light stimulus. Furthermore, the response latency was long in comparison to Hermissenda; the latency shown in Fig. 1b was 210 ms. In contrast to the previous study which demonstrated that numerous sensory cells were located in one eye of Lymnaea [14], we could identify only one type of photoreceptor. This type had a resting membrane potential of 263 ^ 12 mV
(n ¼ 12), and had the characteristic light response as shown in Fig. 1b. After recording the light response and intracellular dye injection, photoreceptor morphology was observed on a fluorescent microscope. The photoreceptor shown in Fig. 1a had a comparatively large cell body located in the central part of the eye. It had an extended axon about 2 mm long which entered the dorsal side of the cerebral ganglion and ran to the abdominal side where it made a terminal branch arborization of several hundred micrometers in diameter (Fig. 1c). Though during the course of penetration we sometime encountered a light response with impulse activity located close to the pigment epithelium, it was not possible to hold the electrode for sufficient time to characterize the response. It is necessary to measure additional details such as light sensitivity, and spectral sensitivity in order to discriminate the photoreceptors in relation to their morphology. Twelve hair cell nuclei stained with SYTOX Green Nucleic Acid Stain were identified from confocal images. Intracellular recordings were made from 25 hair cells of the rostro-caudal plane. The resting membrane potential of caudal hair cells was 256 ^ 10 mV (n ¼ 22). All the caudal hair cell responded to a flash of light with a small amplitude depolarization at the resting membrane potential, and the
N. Tsubata et al. / Neuroscience Letters 337 (2003) 46–50
response amplitude increased or decreased with membrane hyperpolarization or depolarization, respectively, produced by current injection. The cell shown in Fig. 2a responded to a light stimulus with a 3 mV depolarization at a latency of 422 ms at a membrane potential of 250 mV. The response amplitude decreased and the latency increased at more depolarized membrane potentials. At more hyperpolarized membrane potentials, the generator potential had a shorter latency of around 280 ms and was large enough to produce impulse activity. This response latency was a bit longer than observed in the photoreceptor. Since the light response was still depolarizing at the membrane potential of 220 mV (data not shown in Fig. 2a), the equilibrium potential of the channel that generates the light response is presumed to be more positive than 220 mV. Two procedures were performed to estimate the input pathway of the light-response in the hair cell. In the first procedure, the Ca 21 in Lymnaea saline was replaced with Mg 21 to eliminate chemical synaptic transmission. The light response was totally abolished in the Ca 21-free saline at resting membrane potential, and the response recovered when the preparation was returned to normal Lymnaea saline as shown in Fig. 2b. This evidence suggested that the light response observed at the hair cell was mediated by a chemical synapse. Further the impulse initiating 300 ms after the turning off light in Fig. 2b seemed to be off response. The second procedure, to confirm that the hair cell light response originated from ocular photoreceptors and not from dermal photoreceptors, consisted of ipsilateral eye enucleation performed while recording a light response from the caudal hair cell. The photoresponse of 12 mV in amplitude in the hair cell disappeared after enucleation without any noticeable membrane potential change due to the mechanical treatment (Fig. 2d). This was confirmed in another preparations and five out of five ipsilateral eye enucleation always wiped out the light response. This evidence clearly showed that the light response observed in the hair cell arose from the ipsilateral ocular photoreceptor. We could not rule out the possibility that the contralateral eye sends visual information to the caudal hair cell, because the commissural fiber between the right and left cerebral ganglion was cut beforehand in our preparation. Caudal hair cells were stained intracellularly with Lucifer Yellow in order to identify their projection. Fig. 2c displays the one example of the projection of a caudal hair cell which has wide spread axon terminal branches at the center of the cerebral ganglion. Although the whole-body withdrawal response of Lymnaea after classical conditioning is robust [12] in comparison to the behavior of Hermissenda, the neural correlate has not been studied yet because the eyes of Lymnaea are not easy to access for electrophysiology. Our previous study demonstrated that visual information of associative learning in Lymnaea was processed with ocular photoreceptors [11]. The evidence that the light response was observed at the hair cell suggested that the statocyst
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hair cell was one site of convergence of the CS and US signals. The observation that the light response in the caudal hair cell was excitatory strongly suggested that the neural mechanism of the visuo-vestibular-associative learning in Lymnaea is different from that of Hermissenda, because in Hermissenda the light response observed in the caudal hair cell is inhibitory due to inhibitory synaptic input from the type B photoreceptor [15]. The present study demonstrates a characteristic of the Lymnaea photoreceptor response; long response latency. Our long latency light response of 210 ms is consistent with the previous study by Stoll and Bijlsma which measured a response latency of 250 ms [14]. The mechanism for this long latency is the subject of future studies. The long photoresponse-latency of 280 ms observed at the hair cell may simply reflect the latency arising from the photoreceptor mentioned above. From ultrastructural observations of gastropod eyes it is generally accepted that the microvillous receptors in the light sensitive neurons are involved in the ‘on’ response and the ciliary receptors are involved in the ‘off’ response [4,9]. However, the eye of Lymnaea lacks the ciliary type of receptor, and, instead, the light sensitive cells associated with the ‘off’ response are present in the tentacle below the epidermis [16]. These previous findings may explain why there is no obvious ‘off’ response in a photoreceptor to a flash of light as shown in Fig. 1b. However we could observe the ‘off’ photoresponse in the hair cell as shown in Fig. 2, there should be information of ‘turning off a light’ from which we could not yet identify the source. The location of the photoreceptor terminal arborization in the cerebral ganglion was almost identical to that of the hair cell terminal arborization. Though we have not directly identified the synaptic interaction between a photoreceptor and a statocyst hair cell, this overlap is a candidate for the primary loci of associative learning. We thank Drs D.L. Alkon and K.T. Blackwell for critical reading of the manuscript and giving valuable suggestions. We also express our thanks to Ms T. Aritaka for preparing figures. This study was supported by Grant-in-Aids (11168231, 12680783) for Scientific Research, the Ministry of Education, Science, Sports, and Culture of Japan to M.S. and in part by Research and Study Program of Tokai University Educational System General Research Organization to M.S. and T.H., respectively. [1] Alkon, D.L., Associative training of Hermissenda, J. Gen. Physiol., 64 (1974) 70–84. [2] Alkon, D.L., Memory Traces in the Brain, Cambridge University Press, Cambridge, 1987. [3] Alkon, D.L. and Grossman, Y., Evidence for nonsynaptic neuronal interaction, J. Neurophysiol., 41 (1978) 640–653. [4] Barber, V.C. and Wright, D.E., The fine structure of the eye and optic tentacle of the mollusc Cardium edule, J. Ultrastruct. Res., 26 (1969) 515–528. [5] Carew, T.J., Walters, E.T. and Kandel, E.R., Associative
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N. Tsubata et al. / Neuroscience Letters 337 (2003) 46–50 learning in Aplysia: cellular correlates supporting a conditioned fear hypothesis, Science, 211 (1981) 501–504. Culligan, N. and Gelperin, A., One-trial associative learning by an isolated molluscan CNS: use of different chemoreceptors for training and testing, Brain Res., 266 (1983) 319–327. Kawai, R., Horikoshi, T., Yasuoka, T. and Sakakibara, M., In vitro conditioning induces morphological changes in Hermissenda type B photoreceptor, Neurosci. Res., 43 (2002) 363–372. Kemenes, G. and Benjamin, P.R., Appetitive learning in snails shows characteristics of conditioning in vertebrates, Brain Res., 489 (1989) 163–166. Land, M.F., Functional aspects of the optical and retinal organization of the mollusc eye, Symp. Zool. Soc. Lond., 23 (1968) 75–96. Mogilevskii, A. and Verbnyi Ya, I., Influence of different intracellular electrostimulation regimes on the dynamics of the adaptational processes of neurons, Neurosci. Behav. Physiol., 24 (1994) 386–393. Ono, M., Kawai, R., Horikoshi, T., Yasuoka, T. and Sakaki-
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bara, M., Associative learning acquisition and retention depends on developmental stage in Lymnaea stagnalis, Neurobiol. Learn. Mem., 78 (2002) 53–64. Sakakibara, M., Kawai, R., Kobayashi, S. and Horikoshi, T., Associative learning of visual and vestibular stimuli in Lymnaea, Neurobiol. Learn. Mem., 69 (1998) 1–12. Stensaas, L.J., Stensaas, S.S. and Trujillo-Cenoz, O., Some morphological aspects of the visual system of Hermissenda crassicornis (Mollusca: Nudibranchia), J. Ultrastruct. Res., 27 (1969) 510–532. Stoll, C.J. and Bijlsma, A., Optic nerve responses in Lymnaea stagnalis to photic stimulation of the eye, Proc. K. Ned. Akad. Wet., C76 (1973) 406–413. Tabata, M. and Alkon, D.L., Positive synaptic feedback in visual system of nudibranch mollusk Hermissenda crassicornis, J. Neurophysiol., 48 (1982) 174–191. Zylstra, U., Histochemistry and ultrastructure of the epidermis and the subepidermal gland cells of the freshwater snails Lymnaea stagnalis and biomphalaria pfeifferi, Z. Zellforsch. Mikrosk. Anat., 130 (1972) 93–134.
Photoresponse from the statocyst hair cell in Lymnaea stagnalis Noriko Tsubata, Akira Iizuka, Tetsuro Horikoshi, Manabu Sakakibara* Laboratory of Neurobiological Engineering, Graduate School of High-Technology for Human Welfare, Tokai University, Numazu 410-0321, Shizuoka, Japan Received 12 September 2002; received in revised form 18 October 2002; accepted 28 October 2002
Abstract Sensory cells for associative learning of light and turbulence were studied in Lymnaea. Intracellular recordings with Lucifer Yellow filled electrodes were made from photoreceptors and statocyst hair cells. Photoreceptors had a long latency, graded depolarizing response to a flash of light; they extended their axon to the cerebral ganglion. The caudal hair cell, one of 12 cells in the statocyst, responded to brief light with a depolarization and superimposed impulse activity. It formed its terminal arborization close to the photoreceptor endings in the cerebral ganglion. Ca 21-free saline reversibly abolished the photoresponse in the hair cell, suggesting the information was conveyed via a chemical synapse. These findings demonstrated that sensory information for associative learning was convergent at the statocyst hair cell. q 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Classical conditioning; Photoreceptor; Statocyst hair cell; Lymnaea; Morphology
Gastropods are well established as models for the study of the molecular and cellular bases of learning and memory. Animals such as Aplysia [5], Limax[6], Hermissenda[1], and Lymnaea[10] exhibit well-characterized associative learning. Lymnaea stagnalis is a well-established animal model exhibiting appetitive classical conditioning [8], we previously demonstrated that Lymnaea are also capable of classical conditioning in response to paired light flashes (conditional stimulus: CS) and orbital rotation (unconditional stimulus: UCS) [12]. Naı¨ve Lymnaea do not have a behavioral response to turning on a light, but do respond to a moving shadow, offset of a long duration light, or orbital rotation with a whole-body withdrawal response. After 30 paired presentations of a light flash and orbital rotation for 3 days, the animals exhibit a robust unconditioned response, namely a whole-body withdrawal, to the CS. Lymnaea to which unpaired light and rotation stimuli are presented exhibit behavior that is significantly different from those receiving paired stimuli. Further, our previous study demonstrates that acquisition and retention of the associative learning depends on developmental stage, and that visual information is conveyed exclusively by the ocular photoreceptors not by the dermal photoreceptors [11]. This form of asso* Corresponding author. Tel: 181-55-968-1211 ext. 4504, fax: 181-55-968-1156. E-mail address: [email protected] (M. Sakakibara).
ciative learning in Hermissenda is dependent on interactions between visual and vestibular sensory neurons [15], and the cellular and molecular mechanism involved in this learning has been characterized [2]. Though the involvement of neuronal circuits in the associative learning of Hermissenda is well-established as to cause long-lasting enhancement of excitability at the type B photoreceptor and the structure of an eye itself is much more complex in Lymnaea [14] than Hermissenda [13], it is not certain whether the neuronal modification in Lymnaea is identical as that in Hermissenda after the same associative learning of visuo-vestibular interaction even in the more complex neuronal system. As a first step we examined the sensory pathways involved in this associative learning. Since the methods were described in detail previously by Kawai et al. [7] and Ono et al. [11] for animal and electrophysiology, respectively, we described the additional methods. Experiments were performed at room temperature controlled at around 20 8C. The circumesophageal ganglion with eyes was removed 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) using the following procedure. Two eyes of Lymnaea, surrounded by a perioptic sinus bounded by the epidermis, were located on the dorsal body surface at the base of tentacle. For intracellular recording from photoreceptors, the part
0304-3940/02/$ - see front matter q 2002 Elsevier Science Ireland Ltd. All rights reserved. doi:10.1016/S0 30 4- 39 40 (0 2) 0 128 9- 2
N. Tsubata et al. / Neuroscience Letters 337 (2003) 46–50
47
Fig. 1. The intermediate photoreceptor. (a) A cell located in the central portion of eye was filled with Lucifer Yellow. Its axon extended into the cerebral ganglion. (b) photoresponse to a white light flash of 1 s did not have an off-response and thus it took several seconds to return to the baseline level. Calibration bar: 10 mV and 0.5 s. (c) The enlarged terminal arborization as indicated by the white square in (a).
of the epidermis around the eye was carefully removed with fine forceps to expose the eyes. In order to facilitate electrode penetration into a statocyst hair cell, the commissural fiber between the right and left cerebral ganglion was cut. The isolated circumesophageal ganglion with eyes was immobilized on a silgard coated culture plate using stainless-steel pins. The thin connective tissue sheath was digested by incubation in protease (type XIV or type VIII, Sigma Chemical Co., St. Louis, MO) solution (0.5 mg/ml) for 8–10 min at 20 8C. To demonstrate the synaptic origin of the hair cell light response, Ca 21-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. Lymnaea is known to have two kinds of light sensitive neuron; dermal photoreceptors and ocular photoreceptors [9]. Thus, to identify the source of visual information to the hair cell, the ipsilateral eye was enucleated mechanically with visual guidance under a binocular dissecting microscope (MZ FLIII, Leica, Heerbrugg, Switzerland) using a quartz-glass rod with a sharp tip. Nomenclature of hair cells was based on their body position (caudal-rostral; abdominal-ventral; lateral-medial). The isolated circumesophageal ganglion was fixed with 4% paraformaldehyde in 0.1 M PO4 buffer. The nucleus of the hair cells was stained with 1 mM of SYTOX Green Nucleic Acid Stain (S-7020, Molecular Probe, Eugene, OR) diluted with Lymnaea saline. Morphological examination of the statocyst to identify hair cells was done using a confocal microscope (TCSNT, Leica, Heerbrugg, Switzerland).
In order to examine the morphological features of neurons, Lucifer Yellow was injected iontophoretically with alternating current of 1 Hz, 20.5 nA in 50% duty cycle for 30 min. 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 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 (Biolet, Oukenn Co., Tokyo). Morphological observation of a photoreceptor together with a hair cell in an isolated circumesophageal ganglion was done on a fluorescent microscope (Microphoto-SA, NIKON, Tokyo). Since the sensory system encoding associative learning of visual and rotational stimuli in Lymnaea has not been examined at the cellular level, we identified the projection of the sensory receptor neurons, both photoreceptors and statocyst hair cells, as a first step. A previous study demonstrated that the optic nerve fiber contained two types of axons; one originating from a photoreceptor and the other from a secondary visual neuron [14]. In that study, extracellular recordings from an optic nerve fiber demonstrated that the response to a 1 s light flash lasted for many tens of seconds. Furthermore, the response was completely abolished in low Ca 21, high Mn 21 saline, suggesting that the light response was from a secondary visual neuron [14]. To evaluate responses in primary sensory receptors, intracellular recordings were made from 21 photoreceptors using
48
N. Tsubata et al. / Neuroscience Letters 337 (2003) 46–50
Fig. 2. Caudal hair cell. (a) Photoresponse of the caudal hair cell recorded at different membrane potentials. The membrane potential of the hair cell is changed by constant current injection. Numbers on the left indicate the reading from the amplifier in mV. The top trace shows the timing of the light stimulus. The photoresponse of 3 mV in amplitude at the resting membrane potential of 250 mV decreases under more depolarized membrane potentials (data not shown), while it increases, with action potentials, at more hyperpolarized potentials. We defined the response latency as a time to require to initiate a photoresponse, i.e. initiation of a generator potential, from the onset of illumination. The response latency decreases with hyperpolarization from 420 ms at 250 mV to 280 ms at 2133 mV. Calibrations are 10 mV and 200 ms. (b) Photo-responses recorded from the caudal hair cell reversibly disappear in Ca 21 free saline. This finding indicates that the light response in the hair cell arises from the photoreceptor by way of a chemical synapse. The top trace is the timing of light stimulus. Calibrations are 10 mV and 200 ms. (c) The right caudal hair cell was stained with Lucifer Yellow by intracellular injection. The soma stained bright yellow and is located at the medial aspect of the statocyst. Its axon coursed around the statocyst and the terminal arborization formed at the center of the right cerebral ganglion. This picture is an overlay image of fluorescent microscope. Calibration is 100 mm. (d) Photoresponse from a hair cell abolished after enucleation of the ipsilateral eye without any noticeable membrane potential change. The commissural fiber between the right and left cerebral ganglion was cut beforehand. The hair cell was hyperpolarized due to current injection so as to demonstrate a larger photoresponse. Five minutes was interposed between pre and post enucleation of an eye. Calibration is 5 mV. Light stim represents light stimulus for 1 s.
a Lucifer Yellow filled microelectrode (Fig. 1). The membrane potential in response to a 1.0 s-light flash of 700 mW/cm 2 is illustrated in Fig. 1b. This light response was not suppressed in low Ca 21 high Mg 21 saline (data not shown); thus, we concluded that this response originated from a primary sensory neuron, photoreceptor. The light response of Lymnaeawas almost identical as that of Hermissenda,i.e. a comparatively large generator potential with superimposed impulse activity of around 10–20 mV in amplitude suggestive the impulse generating site was not at soma but along axon. This was previously demonstrated in axotomized preparation in Hermissenda [3] as that the amplitude of an action potential originating from an axon was much more smaller than that of a somatic spike activity. There was no evidence of a prominent off response, i.e., several seconds were required for the light response to return to the resting membrane potential following offset of the light stimulus. Furthermore, the response latency was long in comparison to Hermissenda; the latency shown in Fig. 1b was 210 ms. In contrast to the previous study which demonstrated that numerous sensory cells were located in one eye of Lymnaea [14], we could identify only one type of photoreceptor. This type had a resting membrane potential of 263 ^ 12 mV
(n ¼ 12), and had the characteristic light response as shown in Fig. 1b. After recording the light response and intracellular dye injection, photoreceptor morphology was observed on a fluorescent microscope. The photoreceptor shown in Fig. 1a had a comparatively large cell body located in the central part of the eye. It had an extended axon about 2 mm long which entered the dorsal side of the cerebral ganglion and ran to the abdominal side where it made a terminal branch arborization of several hundred micrometers in diameter (Fig. 1c). Though during the course of penetration we sometime encountered a light response with impulse activity located close to the pigment epithelium, it was not possible to hold the electrode for sufficient time to characterize the response. It is necessary to measure additional details such as light sensitivity, and spectral sensitivity in order to discriminate the photoreceptors in relation to their morphology. Twelve hair cell nuclei stained with SYTOX Green Nucleic Acid Stain were identified from confocal images. Intracellular recordings were made from 25 hair cells of the rostro-caudal plane. The resting membrane potential of caudal hair cells was 256 ^ 10 mV (n ¼ 22). All the caudal hair cell responded to a flash of light with a small amplitude depolarization at the resting membrane potential, and the
N. Tsubata et al. / Neuroscience Letters 337 (2003) 46–50
response amplitude increased or decreased with membrane hyperpolarization or depolarization, respectively, produced by current injection. The cell shown in Fig. 2a responded to a light stimulus with a 3 mV depolarization at a latency of 422 ms at a membrane potential of 250 mV. The response amplitude decreased and the latency increased at more depolarized membrane potentials. At more hyperpolarized membrane potentials, the generator potential had a shorter latency of around 280 ms and was large enough to produce impulse activity. This response latency was a bit longer than observed in the photoreceptor. Since the light response was still depolarizing at the membrane potential of 220 mV (data not shown in Fig. 2a), the equilibrium potential of the channel that generates the light response is presumed to be more positive than 220 mV. Two procedures were performed to estimate the input pathway of the light-response in the hair cell. In the first procedure, the Ca 21 in Lymnaea saline was replaced with Mg 21 to eliminate chemical synaptic transmission. The light response was totally abolished in the Ca 21-free saline at resting membrane potential, and the response recovered when the preparation was returned to normal Lymnaea saline as shown in Fig. 2b. This evidence suggested that the light response observed at the hair cell was mediated by a chemical synapse. Further the impulse initiating 300 ms after the turning off light in Fig. 2b seemed to be off response. The second procedure, to confirm that the hair cell light response originated from ocular photoreceptors and not from dermal photoreceptors, consisted of ipsilateral eye enucleation performed while recording a light response from the caudal hair cell. The photoresponse of 12 mV in amplitude in the hair cell disappeared after enucleation without any noticeable membrane potential change due to the mechanical treatment (Fig. 2d). This was confirmed in another preparations and five out of five ipsilateral eye enucleation always wiped out the light response. This evidence clearly showed that the light response observed in the hair cell arose from the ipsilateral ocular photoreceptor. We could not rule out the possibility that the contralateral eye sends visual information to the caudal hair cell, because the commissural fiber between the right and left cerebral ganglion was cut beforehand in our preparation. Caudal hair cells were stained intracellularly with Lucifer Yellow in order to identify their projection. Fig. 2c displays the one example of the projection of a caudal hair cell which has wide spread axon terminal branches at the center of the cerebral ganglion. Although the whole-body withdrawal response of Lymnaea after classical conditioning is robust [12] in comparison to the behavior of Hermissenda, the neural correlate has not been studied yet because the eyes of Lymnaea are not easy to access for electrophysiology. Our previous study demonstrated that visual information of associative learning in Lymnaea was processed with ocular photoreceptors [11]. The evidence that the light response was observed at the hair cell suggested that the statocyst
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hair cell was one site of convergence of the CS and US signals. The observation that the light response in the caudal hair cell was excitatory strongly suggested that the neural mechanism of the visuo-vestibular-associative learning in Lymnaea is different from that of Hermissenda, because in Hermissenda the light response observed in the caudal hair cell is inhibitory due to inhibitory synaptic input from the type B photoreceptor [15]. The present study demonstrates a characteristic of the Lymnaea photoreceptor response; long response latency. Our long latency light response of 210 ms is consistent with the previous study by Stoll and Bijlsma which measured a response latency of 250 ms [14]. The mechanism for this long latency is the subject of future studies. The long photoresponse-latency of 280 ms observed at the hair cell may simply reflect the latency arising from the photoreceptor mentioned above. From ultrastructural observations of gastropod eyes it is generally accepted that the microvillous receptors in the light sensitive neurons are involved in the ‘on’ response and the ciliary receptors are involved in the ‘off’ response [4,9]. However, the eye of Lymnaea lacks the ciliary type of receptor, and, instead, the light sensitive cells associated with the ‘off’ response are present in the tentacle below the epidermis [16]. These previous findings may explain why there is no obvious ‘off’ response in a photoreceptor to a flash of light as shown in Fig. 1b. However we could observe the ‘off’ photoresponse in the hair cell as shown in Fig. 2, there should be information of ‘turning off a light’ from which we could not yet identify the source. The location of the photoreceptor terminal arborization in the cerebral ganglion was almost identical to that of the hair cell terminal arborization. Though we have not directly identified the synaptic interaction between a photoreceptor and a statocyst hair cell, this overlap is a candidate for the primary loci of associative learning. We thank Drs D.L. Alkon and K.T. Blackwell for critical reading of the manuscript and giving valuable suggestions. We also express our thanks to Ms T. Aritaka for preparing figures. This study was supported by Grant-in-Aids (11168231, 12680783) for Scientific Research, the Ministry of Education, Science, Sports, and Culture of Japan to M.S. and in part by Research and Study Program of Tokai University Educational System General Research Organization to M.S. and T.H., respectively. [1] Alkon, D.L., Associative training of Hermissenda, J. Gen. Physiol., 64 (1974) 70–84. [2] Alkon, D.L., Memory Traces in the Brain, Cambridge University Press, Cambridge, 1987. [3] Alkon, D.L. and Grossman, Y., Evidence for nonsynaptic neuronal interaction, J. Neurophysiol., 41 (1978) 640–653. [4] Barber, V.C. and Wright, D.E., The fine structure of the eye and optic tentacle of the mollusc Cardium edule, J. Ultrastruct. Res., 26 (1969) 515–528. [5] Carew, T.J., Walters, E.T. and Kandel, E.R., Associative
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