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Optic nerve responses to light stimulation in the bioluminescent terrestrial snail, Dyakia (Quantula) striata
Camp. Biochem. Physiof.
Vol. 89A, NO. 3, pp. 391m,
03W9629/88 Sl3.00 + 0.00 Pergamon Press plc
1988
Printed in Great Britain
OPTIC NERVE RESPONSES TO LIGHT STIMULATION IN THE BIOLUMINESCE~ TERRESTRIAL SNAIL, DYAKIA (QVANTULA) STRIATA JONAWN COPELAND Departmentof Biological Sciences, University of Wisconsin-Milwaukee and *Department of Biology, Swarthmore Coll&e, Swarthmore, PA 19081, USA. Telephone: (215) 328-8047 (Received 3 June 1987) Dyukia is the world’s only known bioluminescent terrestrial snail. 2. Since Dyukia’s bioluminescence may be involved in social communication, a study of the visual system was carried out. 3. Extracellular recordings from the optic nerve revealedan on-response. 4. Effects of varying light intensity, duration and waveIen@b were viewed. 5. The on-response of Dyaktamay be considerably more compiex than that of other pulmonate snails.
Abstract-l.
INTRODUCTION A good deal is known about the structure and fun@
tion of the molluscan eye (reviewed in Messenger, 1981). Among the gastropod molluscs, however, the pulmonate eye has received little attention despite its relative simplicity (reviewed in Kerkut and Walker, 1975). The cup-like eye is found on the tip (stylommatophoran} or the base (b~ommatophoran) of the superior tentacles (Runham and Hunter, 1970). In stylommatophorans light passes through a clear carnea and lens and is focused on a structured retina. The structure of the stylommatophoran retina appears to he species specific. It consists of some combination of pigmented and unpigmented sensory receptor cells, pi~ent~ontaining accessory (shielding) cells, and first order ganglion cells (Chetail, 1963; Newell and Newell, 1968; Smith, 1906; Eakin and Brandenburger, 1967, 1975; Kataoka, 1975). Receptor cells are microvillar (Newell and Newell, 1968; Kataoka, 1975; Brandenburger, 1975) and more than one type of receptor cell may be present (Brandenburger, 1975; Kataoka, 1975). No intraretinal synapses have been seen. Both sensory cells and ganglion cells (when present) send processes directly into the optic nerve (Brandenburger, 1975). Visual system responses to stimulation by light have not yet been extensively characterized in stylommatophorans. The ERG has been characterized visa-v& Iight intensity (in Helix pomat~, Berg and Schneider, 1967; Helix aspersa, Gillary, 1970; and Otuiu fuctea, Gillary and Wolbarsht, 1967; Goldman and Hermann, 1967). Also, activity on the optic nerve in response to light has been recorded (in Helix arpersu, Gillary, 1970; 0th lacteu, Goldman and Herman, 1967; Gillary and Wolbarsht, 1967; and Lhax _t%vus, Suzuki et at. 1979). In these stylommatophorans only a simple on-response was found and the delay of the on-response was inversely pro-
*Address to which correspondence should be mailed.
portional to light intensity (Goldman and Hermann, 1967). The behaviors which are mediated by such visual system responses also have not been extensively studied. However, a number of visually mediated behaviors have been observed in pulmonates: simple orientation responses to light (in Helix pomatia, wheeler, 1921; Agriolimux reticulutus, Crozier and Cole, 1929; Arion uter, Lewis, 1969; and Lima% rnux~~~ Sokolov et ul., 1977) and light triggered circadian activity (in Arion uter, Lewis, 1969; Argiofimux reticulatus, Karlin, 1961; and Limax muximus, Sokolov et al., 1977). Dyukiu (Quuntuiu) striuta is the world’s only known terrestrial mollusc that is bioluminescent. The bioluminescence, yellow-green in color (Haneda, 1981; Parmentier and Barnes, 1975; Copeland and Maneri, 1984), is produced intermittently in flashes (glows) from a luminescent organ located within the foot in the anterior head region. Currently, some information is available concerning Dyukiu’s natural history, the occurrence of the bioluminescence, and certain aspects of its physiological control (Haneda, 1963; Haneda and Tsuji, 1969; Parmentier and Barnes, 1975; Copeland and Maneri, 1984). Some information is also available concerning the structure of the light producing organ, thought to be modified glandular tissue (Bassot and Martoja, 1968; Martoja and Bassot, 1970; Maneri, 1985). However, nothing is known about the visual system of Byakia. Discovered to be biol~ine~nt by Haneda in 1942 (Haneda, 1981), the behavioral function of the flash has remained an enigma. However, recent behavioral work suggests that the flash in Dyakia is involved in a social role (Copeland and Maneri, 1984; Counsilman et al., 1987). Such a social role for bioluminescent behavior has prompted this investigation into the visual system of I3yukiu. MATRRULS AND METHODS Dyakia, cdlwtcd in Singapore, were used. These animals were maintained in 9 x 12 x 3” clear plastic boxes at Z.PC, 391
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12: 12 1ight:dark cycle. Boxes were lined with moist paper towels and changed once a week. Animals were fed a thin pancake of Reechnut Vegetable Chicken Dinner baby food which was spread out across the surface of a Petri dish (for details, see Maneri, 1985). Animals 17-24 mm in shell diameter were used. Each was anesthetized by temperature (3”C, 10min). The shell was removed and the animal was further dissected in a clear Sylgard dish containing chilled saline (3°C). A saline consisting of 58.3 mM Na+, 4.2 mM K+, 7.0 mM Ca2+, 4.6mM Mg2+, 80.3 mM Cl-, 0.4mM H,PO,, 5.0mM CO, and 20mM dextrose was used.
Dissection The retracted superior tentacles were exposed via a dorsal longitudinal incision in the head. Each tentacle was excised and subsequently pinned (eye dorsal) in a fresh dish of chilled saline using 0.001” tungsten pins. Optic nerve and olfactory nerve were then revealed by gentle ~~~is~tion along the midline. Close to the eye the olfactory nerve spread into the digitate ganglion. Here, the optic nerve looped away from the olfactory nerve. The olfactory nerve and digitate ganglion were crushed and the optic nerve cleaned of surrounding connective tissue. The thin layer of skin covering the eye was then removed. Dissection was done at 3°C under white light using a fiber optic. Experiments were initiated only after the completely dissected preparation had been in total darkness for at least 2 hr. Recording Extracelluiar recordings were made from the loop of the optic nerve near the eye. Broken tip glass suction electrodes were used. Optic nerve impulses were amplified by either a capacitatively coupled (low pass, 1OHz) amplifier (DAM 6A, WP Instruments) or a DC coupled amplifier (Pl6 Grass Instruments). Impulses were simultaneously displayed on an oscilloscope (Textronix 5112) and on a storage (Textronix 5113) oscilloscope. The stored display was photographed using a Polaroid oscilloscope camera gextronix). For some of the experiments, amplified optic nerve signals were led through a window discriminator (WP Instruments 121) and the resultant square wave pulse displayed as a dot (RASTER) display via-the Z-axis input of the storage oscilloscope. Thus, depending on the location of the window discriminator, many or few raster dots could be displayed during an optic nerve response to photic stimulation. Stimulation Light was presented via a fiber optic light pipe (DolanJenner model 150). A high intensity light (Sylvania DNE) was used. This was focused on the eye and surrounding tissue via a light pipette (Dolan-Jenner). The maximum intensity of the light (“G” light intensity) was 4.3 x 10’ W/cm measured 3 cm from the end of the liber optic. This value was determined, (and the calibration of neutral density hlters was carried out), using a alibrated IL500 Photometer/Radiometer (International Light). Since the light system was continuously on, a mechanical solanoid armature in the light path acted as a shutter. This shutter was triggered by a laboratory stimulator (Grass S44). Neutral density and colored tilters (Kodak) (blue, green, yellow, orange and red) were inserted in various combinations into a holder in the light path. The occurrence and intensity of the stimulus (light) was monitored at the eye via a photocell (IR 200) whose signal was led to one channel of the oscilloscope. The time between consecutive stimuli was I-2min. All experiments were carried out at room temperature (22°C).
RESULTS
Optic nerve response to light stimulation
A response occurred on the optic nerve subsequent to photic (light) stimulation (Fig. I A). This response occurred each time the eye was stimulated and appeared as a complex burst of activity. Using a short duration (100-500 msec) stimulus pulse, the burst consisted of an initial phasic (first) response of relatively large amplitude potentials followed by lower amplitude tonic potentials. First responses were similar, though not identical, each time the eye was stimulated (Fig. 1B). Sometimes a small amplitude DC shift was seen at the beginning of the response (Fig. 1A). Eflect of decreasing stimulus intensity
The first response’s delay (measured from stimulus onset to the first spike on the optic nerve trace or to the first dot on the RASTER trace), was relatively constant for almost any given stimulus intensity (Figs 1B and 2). As light intensity decreaed, response delay increased (Fig. 2) and the number of action potentials (indicated by the number of RASTER dots) decreased (Fig. 2). When optic nerve responses were compared over an intensity range of 4-6 log units, delay varied linearly with log stimulus intensity (Fig. 3). Other parameters, such as number of RASTER dots (spike frequency), RASTER burst duration ( = duration of RASTER phasic and tonic response), and the average RASTER frequency (number of RASTER dots/burst duration = average spike frequency of the initial phase and subsequent tonic response) showed no consistent relationships over this same intensity range. However, if the extremes were compared for any of these parameters, the expected trend was seen, so with decreasing light intensity, the number of dots decreased, RASTER burst duration decreased, and average RASTER frequency decreased. The delay at 0 intensity was 260-640 msec, depending on the animal. This delay increased to over 2 set at very low light intensities in one animal (Fig. 3). One eye showed saturation at high intensities (Fig. 3, dashed line). EfSect of stimulus duration
The effect of stimulus duration was studied by keeping the stimulus intensity consta.nt and increasing the stimulus duration to almost 7 sec. By using the window discriminator and photographing the storage oscilloscope at successively slower sweep speeds (5sec/cm in Fig. 6), a second feature of the optic nerve response was revealed. When the stimulus duration was varied between 100 and 6700 msec, both response delay and initial response duration were relatively constant (Fig. 4). The initial phasic (first) response was followed by lower amplitude tonic potentials, as before (in Figs 1 and 2) and was displayed as a continuous stream of RASTER dots). However, by using the slower sweep speed the first response, with its phasic and tonic component, was seen to be followed by a period of decreased activity (no RASTER dots). Then, a second response occurred (Fig. 4).
Optic nerve responses in Dyakia sfriata
Fig. 1. Response of Dyakia optic nerve to photic stimulation. In B, three responses separated by 1 min are shown. Stimulus duration in A and B, light = 450 msec. Intensity = 0.8 O.D. units. The second response consisted exclusively of low amplitude tonic potentials. (The optic nerve showed activity during the period of decreased activity, but this activity was below the window of the window discriminator.) The second response increased in duration as the duration of the flashes increased (Figs 5 and 6). The period of decreased activity showed no consistent relationship to the stimulus duration.
Response to light of dyerent wavelengths Dyakia optic nerve responded to all wavelengths of
light presented (Fig. 7). As colored light intensity decreased, delay increased (data not presented here). Although the delay to blue light was sometimes relatively less than the delay to green and yellow light (Fig. 7), this was not always the case. However, the delay to blue, green or yellow light was always much less than the delay to orange or red (Fig. 7). There were aiso fewer RASTER dots (i.e. less nerve activity) with orange and red light than there were with blue, green or yellow light (Fig. 7). Because of the complexity of the optic nerve
JONAI-HAN C~PELAND
Fig. 2. Response of optic nerve to three different intensities of light: A, full intensity (O.D. = 0); B, reduced by 90% from full intensity (O.D. = - 1) and C, reduced by 99% from full intensity (O.D. = -2). Calibration: upper voltage values indicate light trace calibration for A, B and C respectively; lower voltage value indicates voltage value of optic nerve trace. The Brst three traces in A, B and C were recorded simultaneously. Thereafter,with subsequent stimulations, the first two traces were eliminated and only the RASTER display shown.
rise, it was not possible to measure spectral sensitiivity of single units at high stimulus intensities. (Tests at low intensities, where single units were seen, gave inconsistent results.) However, threshold sensi-
tivity for the entire optic nerve response could be estimated by defining threshold as the value of the light intensity that was just capable of producin ga weak (2-4 dots) first response whose delay varied by
Optic nerve responses in D$cia striata
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less than 100 msec. Using these criteria for threshold, the eyes responded to blue, green and yellow light at very weak intensities (between - 2.5 and - 4.4 O.D. units) and to orange and red light between 0 (full intensity) and - 1.OO.D. units (Fig. 8).
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DISCUSSION
Optic nerve response
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Fig. 3. Effect of decrease in light intensity on delay. Five different preparations were used. Mean and 1 SD shown at each light intensity for each eye. Dashed line indicates eye which showed saturation at high intensities of light.
When stimulated with light, Dyakiu’s optic nerve shows a complex burst of activity. A phasic first response is followed by tonic activity (Figs 1, 2 and 4-6). Additionally, when stimulus durations are increased to behaviorally relevant ranges (Copeland and Maneri, 1984), beyond the usual physiological range normally employed in gastropod vision studies, and furthermore, when a window discriminator circuit is used to simplify the recorded response, the optic nerve response is seen to be even more complex than a phasic on-response followed by tonic activity. The response consists of first response, period of decreased activity, and second response (Figs 4-6). Either because a different type of analysis was attempted, or because longer (behaviorally relevant) stimulus durations were used, or because Dyakiu’s optic nerve response is, indeed, more complex than that of other stylommatophorans, Dyukia’s optic nerve response to light seems to be different from other pulmonates.
Fig. 4. Effect of stimulus duration on optic nerve response. As in Fig. 2, first three traces were recorded simultaneously. Thereafter, the first and second traces were eliminated and only the RASTER display shown. Optic nerve response consists of first response, period of decreased activity (no RASTER dots), and second response. Note time calibration equals 1 sec.
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Fig. !i. Effect of stimulus duration on optic nerve response. As in Fig. 4. Note time calibration 5 sec.
Because the response delay is constant from stimulus onset (Figs 1, 2 and 4) when stimulus duration is increased over a very large range (Figs 5 and 6), this response may be considered as an on-response. On-responses have also been found on the optic nerve of the terrestrial pulmonates Helix aspersa
(Gillary, 1970) Otala fatea (Gillary and Wolbarsht, 1967; Goldman and Hermann, 1967) and Limax jlaous (Suzuki et al., 1979) and the aquatic pulmonates Helisoma trivolvis (Patton and Kater, 1972) and Lymnea stagnalis (St011and Bijlsma, 1973). However, these on-responses appear as a compound
Optic nerve responses in Dyakia striata
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LimaxJlaous (Kataoka, 1975) and Ariolimax californicus (Eaken and Brandenburger, 1975) and, using a
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Fig. 6. Effect of stimulus duration on optic nerve response. Left vertical axis for second response duration; right vertical axis for first response duration and for period of decreased activity. Same data as Fig. 5. Mean and 1SD shown. Similar results were found in two additional preparations.
action potential in Helix (Gillary, 1970) or as a slow wave form with a spike discharge riding on it in Helisoma (Patton and Kater, 1972). In the stylommathophorans Limax (Suzuki et al., 1979), Lymanea @toll and Bijlsma, 1973) and Otulu (Gillary and Wolbarsht, 1967; Goldman and Hermann, 1967), the on-response appears as a complex spike discharge. In these animals, however, only short duration flashes of light were used for stimuli. The physiological or anatomical causes of this response complexity in Dyakia are unknown, as is also the case for the somewhat simpler responses recorded in other pulmonates. Part of the response complexity could be due to the presence of both compound action potentials and single unit discharges in the recordings. Such potentials could be produced by the presence of different types of receptors or secondary cells. These receptors or secondary cells could adapt at different rates to continual or repeated stimulation. Although neither anatomical cross section of the optic nerve or structure of the retinal receptors and second order neurons are known for Dyakia, clues may be obtained from other pulmonates. With respect to optic nerve, both Helix aspersa (Eaken and Brandenburger, 1967) and Limax maximus (Gelperin, unpublished observations) possess several hundred small axons in the optic nerve, making the recording of compound action potentials a possibility. In the eye itself, two different photoreceptor types have been found in Helix aspersa (Brandenburger, 1975),
slightly larger phylogenetic perspective, five different photoreceptor types have been found in the eye of the opisthobranch gastropod Aplysia californica (Herman and Strumwasser, 1984). Additionally, second order cells, called ganglion cells, have been reported in the periphery of the retina in Helix aspersa (Brandenburger, 1975). Also, the eye of Agriolimax reticulatus contains an “accessory retina”, possibly infra-red sensitive (Newell and Newell, 1968) and the Aplysia californica eye, in addition to having five different receptor types, also possesses many afferent neurosecretory neurons (Herman and Strumwasser, 1984). Thus, the response complexity may be due to a heterogeneity of receptor and second order cell types, their different response properties, and different rates of adaptation. Using white light, the intensity-delay function for the first response of the on-response in Dyakia (Fig. 3) shows an inverse relationship between intensity and delay over 6 log units. A similar relationship has also been found for all wavelengths tested. Similar intensity-delay functions for white light have been shown over 6 log units in Lhux (Suzuki et al., 1979) and 4 log units in Otula (Goldman and Hermann, 1967). The threshold response, -6.0 log units in one individual (Fig. 3), to white light in Dyukiu, may actually be l-2 orders of magnitude lower than that of Limaxflavus (Suzuki et al., 1979). This is difficult to compare, however, because in Limax flavus a brighter stimulus was used (different 0 log intensity) and threshold was defined differently (Suzuki et al., 1979). phe very low number of preparations showing any response at all in Otala (Goldman and Hermann, 1967) can be explained by Goldman and Hermann’s methods, i.e. dissection in white light and immediate testing versus 3 hr dark adaptation time in Limax (Suzuki et al., 1979) and 2 hr here.] This 6 log unit response range encompasses much of the range of light intensities that Dyakia might experience in the field. Direct sunlight at sea level at noon at mid-latitudes has a radiant flux density of5 x 104~Wcm-2,moonlight is3 x lO-‘~W~rn-~, and the energy of starlight is 3 x 10-4pW cm-2 (reviewed in Nichol, 1978). The dynamic range of stimulus intensities that could evoke responses on the optic nerve in Dyakiu was from 4.3 x 1ticrnm2 (0 intensity) to 1.2 x 10m2PW cmw2 ( - 6 intensity) measured at 3 cm from the source. The in vitro preparation, then, is sensitive enough to be stimulated by moonlight. This means that the in viuo preparation, probably more sensitive, would be likely to be stimulated by the weak flash of a conspecific snail. What these sensitivities and background luminances translate into in terms of range and form vision in Dyakia cannot be estimated until the radiant energy emitted by the luminescent organ is measured, the optical characteristics of the eye are assessed, and appropriate psychophysical experiments are done. Second response
Using a stimulus of several seconds, only the second response period’s duration was directly pro-
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COPELAND
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Optic nerve responses in Dyakiu striutu
the results in fireflies see, for example, Biggley et al., 1967; La11 et al., 1980). Visually guided behavior in snails
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Fig. 8. Threshold sensitivity of four eyes to colored light of
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portional to stimulus duration (Fig. 6). Thus, at behaviorally significant durations, the flash duration of a Dyakia could be encoded by the time between the beginning of the first response and end of the second response. Behavioral tlashes produced by Dyakia at 28°C are 0.5-6.0 set duration at 28°C (Copeland and Maneri, 1984). Depending upon the Q10 of the physiological processes associated with the optic response, the behavioral data here suggests an even broader range of stimulus durations might have been tested physiologically. (However, some visual responses measures, such as average ERG amplitude, increase from 10 to 20°C and decrease from 20 to 30°C (Gillary and Wolbarsht, 1967; Gillary, 1970) whereas others, such as ERG delay, decrease between 10 and 20°C (Gillary and Wolbarsht, 1967). Effect of colored light
The optic nerve response is most sensitive to blue, green and yellow light (Figs 7 and 8). These results are similar to those of Gillary and Wolbarsht (1967). where the dark adapted ERG’s constant response action spectrum was most sensitive in the blue-green (490 nm), and those of Suzuki et al. (1979), where the dark adapted optic nerve response showed a peak sensitivity to blue (480nm). (In this latter study, the light adapted response showed a shift in peak sensitivity to 460 nm if the amount of light adaptation exceeded 3 log units. Suzuki et al. (1979) interpreted this as providing neurophysiological evidence for two kinds of photosensory cells in the retina of Climax flavus.)
The color of Dyukiu’s in oioo bioluminescence is yellow-green (Copeland and Maneri, 1984). This lies well within the very broadly defined range of greatest spectral sensitivity found here (Figs 7 and 8). This observation, that the spectral sensitivity maximum of the bioluminescence and the sensitivity maximum of the eye are similar, has been found in other bioluminescent systems (reviewed in Nichol, 1978) (for
Slug and snail behavior has usually been thought to be largely guided by mechano- and chemoreception (Runham and Hunter, 1970). Only simple behaviors (tropisms, taxes, locomatory rhythms) were considered to be photically guided (Kerkut and Walker, 1975) and true visual behavior was not known. However, Otala lactea shows a body orientation guidance system to a slot of darkness (22.5”C x 45” at 25 cm) in a band of uniform brightness (Hermann, 1968). Additionally, Helix uspersu shows some preference for vertical bars subtending a visual field of 14.5-24.6” (versus horizontal or diagonal bars) (Hamilton and Winter, 1982). Also, Euparina pisuriu, closely related to Helix, may return to vegetation from which it has been displaced via visual orientation (Zanforlin, 1976). Additionally, prosobranch snails like the intertidal Littorina irrorata and Techtariurn muricatus and the subtidal Turbo castanea show l’esponse thresholds to 0.3-6.5” dark vertical bars (Hamilton and Winter, 1982, 1984). Thus, the visual world of snails may be considerably more complex than originally thought and simple form vision may be possible. Dyakia, being a bioluminescent terrestrial snail, is probably a reasonable candidate for possessing an unusual visual system. It might be appropriate that, if Dyukia’s bioluminescence is involved in a social role (Copeland and Maneri, 1984; Counsilman et al., 1987), the visual system should be specialized when compared to other terrestrial pulmonates. At the level of recording used here, the visual system of Dyukiu seems similar to that of other terrestrial pulmonates. However, the level of analysis used here suggests a complexity of response to photic stimulation (first response, period of decreased activity, second response) not found in other pulmonates. Furthermore, Dyukiu’s eye seems more sensitive than other pulmonate eyes. Thus, this visual system may, indeed, be unusually adapted to receive bioluminescently produced light. Acknowledgements-This work was supported in part by a grant from National Geographic Society (2716-83). The author thanks Dr George Daston and MS Maryellen Daston for their criticisms of the manuscript, Dr. James Counsilman for collection and shipment of the snails, and Mr David Lichtman for the loan of the Photometer/ Radiometer. REFERENCES
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Kerkut, G. A. and Walker R. J. (1975) Nervous system, eye, and statocyst. In Pulmonates. I. Eunctional Aruztomv and Physiology (Edited by Fretter V. and Peake J). Academic Press. New York. La11A. D., Seliger, H. H., Biggley W. H. and Lloyd J. E. (1980) Ecology of colors of 6refly bioluminescence. Science 210, 560-562. Lewis R. D. (1969) Studies on the locomotor activity of the slug, Arion ater. I. Humidity, temperature, and light reactions. Mulucologiu 7, 295-306. Maneri M. (1985) Bioluminescence and sexual maturity in the terrestrial snail Dyakiu striuta. Master’s Thesis, University of Wisconsin-Milwaukee. Martoja MI and Bassot J. M. (1970) Etude histologique du complexe glandulaire pedieux de Dyakia striate, Godwin et Austen, gasteropode pulmone don&s sur lorgane hunineux. Vie et milieu. Ser. A: Biol. Mar. Tome XXI-Fast. 2-A, 395452. Messenger I. (1981) Vision in mollusks. In Har&ook of Sensory Physiology (Edited by Autrum H.). Springer, Berlin. Newell P. F. and Newell G. E. (1968) The eye of the slug, Agriolimax reticulutus. Symp. 2001. Sot. (Land.) 23, 97-111. Nichol J. A. C. (1978) Bioluminescence and vision. In Bioluminescence in Action (Edited by Herring P. J.). Academic Press, New York. Patton M. L. and Kater S. B. (1972) J. exp. Biol. 56, 695-702. Parmentier J. and Barnes A. (1975) Observations on the luminescence production by the Malayan gastropod Dyakiu striata. Maluvan Nature J. 28. 173-180. R&ham N. and Hunter P. (1970) Terrestrial Slugs. Hutchinson, London. Smith G. (1906) The eyes of certain pulmonate gastropods, with special reference to the neurofibrillae in Lima-x maximus. Bull. Mus. Comp. 2001. (Harvard) 48,233283. Sokolov P. G., Beiswanger C. M., Prior D. J. and Gelperin A. (1977) A cirdadian Rhythm in the locomotory behavior of the giant garden slug Limax maximus. J. exp. Biol. as, 47-64. Stoll C. J. and Bijlsma A. (1973) Optic nerve response in Lymnea stugnalis (L) (Pulmonata, Basonatophora) to photic stimulation of the eye. Prod. K. Ned. Akad. We. Ser. C. 76, 406413. Suzuki H., Watanabe M., Tsukahara Y. and Tasaki K. (1979) Duplex systems in the simple retina of a gastropod mollusc, LimaxJlaous L. J. camp. Physiol. 133, 125130. Wheeler G. C. (1921) The phototropism of land snails. J. camp. Physiol. 1, 149-154. Zanforlin M. (1976) Observations on the visual perception of the snail Euparipha pisana (Muller). Boll. Zool. 43, 303-31s.
Vol. 89A, NO. 3, pp. 391m,
03W9629/88 Sl3.00 + 0.00 Pergamon Press plc
1988
Printed in Great Britain
OPTIC NERVE RESPONSES TO LIGHT STIMULATION IN THE BIOLUMINESCE~ TERRESTRIAL SNAIL, DYAKIA (QVANTULA) STRIATA JONAWN COPELAND Departmentof Biological Sciences, University of Wisconsin-Milwaukee and *Department of Biology, Swarthmore Coll&e, Swarthmore, PA 19081, USA. Telephone: (215) 328-8047 (Received 3 June 1987) Dyukia is the world’s only known bioluminescent terrestrial snail. 2. Since Dyukia’s bioluminescence may be involved in social communication, a study of the visual system was carried out. 3. Extracellular recordings from the optic nerve revealedan on-response. 4. Effects of varying light intensity, duration and waveIen@b were viewed. 5. The on-response of Dyaktamay be considerably more compiex than that of other pulmonate snails.
Abstract-l.
INTRODUCTION A good deal is known about the structure and fun@
tion of the molluscan eye (reviewed in Messenger, 1981). Among the gastropod molluscs, however, the pulmonate eye has received little attention despite its relative simplicity (reviewed in Kerkut and Walker, 1975). The cup-like eye is found on the tip (stylommatophoran} or the base (b~ommatophoran) of the superior tentacles (Runham and Hunter, 1970). In stylommatophorans light passes through a clear carnea and lens and is focused on a structured retina. The structure of the stylommatophoran retina appears to he species specific. It consists of some combination of pigmented and unpigmented sensory receptor cells, pi~ent~ontaining accessory (shielding) cells, and first order ganglion cells (Chetail, 1963; Newell and Newell, 1968; Smith, 1906; Eakin and Brandenburger, 1967, 1975; Kataoka, 1975). Receptor cells are microvillar (Newell and Newell, 1968; Kataoka, 1975; Brandenburger, 1975) and more than one type of receptor cell may be present (Brandenburger, 1975; Kataoka, 1975). No intraretinal synapses have been seen. Both sensory cells and ganglion cells (when present) send processes directly into the optic nerve (Brandenburger, 1975). Visual system responses to stimulation by light have not yet been extensively characterized in stylommatophorans. The ERG has been characterized visa-v& Iight intensity (in Helix pomat~, Berg and Schneider, 1967; Helix aspersa, Gillary, 1970; and Otuiu fuctea, Gillary and Wolbarsht, 1967; Goldman and Hermann, 1967). Also, activity on the optic nerve in response to light has been recorded (in Helix arpersu, Gillary, 1970; 0th lacteu, Goldman and Herman, 1967; Gillary and Wolbarsht, 1967; and Lhax _t%vus, Suzuki et at. 1979). In these stylommatophorans only a simple on-response was found and the delay of the on-response was inversely pro-
*Address to which correspondence should be mailed.
portional to light intensity (Goldman and Hermann, 1967). The behaviors which are mediated by such visual system responses also have not been extensively studied. However, a number of visually mediated behaviors have been observed in pulmonates: simple orientation responses to light (in Helix pomatia, wheeler, 1921; Agriolimux reticulutus, Crozier and Cole, 1929; Arion uter, Lewis, 1969; and Lima% rnux~~~ Sokolov et ul., 1977) and light triggered circadian activity (in Arion uter, Lewis, 1969; Argiofimux reticulatus, Karlin, 1961; and Limax muximus, Sokolov et al., 1977). Dyukiu (Quuntuiu) striuta is the world’s only known terrestrial mollusc that is bioluminescent. The bioluminescence, yellow-green in color (Haneda, 1981; Parmentier and Barnes, 1975; Copeland and Maneri, 1984), is produced intermittently in flashes (glows) from a luminescent organ located within the foot in the anterior head region. Currently, some information is available concerning Dyukiu’s natural history, the occurrence of the bioluminescence, and certain aspects of its physiological control (Haneda, 1963; Haneda and Tsuji, 1969; Parmentier and Barnes, 1975; Copeland and Maneri, 1984). Some information is also available concerning the structure of the light producing organ, thought to be modified glandular tissue (Bassot and Martoja, 1968; Martoja and Bassot, 1970; Maneri, 1985). However, nothing is known about the visual system of Byakia. Discovered to be biol~ine~nt by Haneda in 1942 (Haneda, 1981), the behavioral function of the flash has remained an enigma. However, recent behavioral work suggests that the flash in Dyakia is involved in a social role (Copeland and Maneri, 1984; Counsilman et al., 1987). Such a social role for bioluminescent behavior has prompted this investigation into the visual system of I3yukiu. MATRRULS AND METHODS Dyakia, cdlwtcd in Singapore, were used. These animals were maintained in 9 x 12 x 3” clear plastic boxes at Z.PC, 391
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12: 12 1ight:dark cycle. Boxes were lined with moist paper towels and changed once a week. Animals were fed a thin pancake of Reechnut Vegetable Chicken Dinner baby food which was spread out across the surface of a Petri dish (for details, see Maneri, 1985). Animals 17-24 mm in shell diameter were used. Each was anesthetized by temperature (3”C, 10min). The shell was removed and the animal was further dissected in a clear Sylgard dish containing chilled saline (3°C). A saline consisting of 58.3 mM Na+, 4.2 mM K+, 7.0 mM Ca2+, 4.6mM Mg2+, 80.3 mM Cl-, 0.4mM H,PO,, 5.0mM CO, and 20mM dextrose was used.
Dissection The retracted superior tentacles were exposed via a dorsal longitudinal incision in the head. Each tentacle was excised and subsequently pinned (eye dorsal) in a fresh dish of chilled saline using 0.001” tungsten pins. Optic nerve and olfactory nerve were then revealed by gentle ~~~is~tion along the midline. Close to the eye the olfactory nerve spread into the digitate ganglion. Here, the optic nerve looped away from the olfactory nerve. The olfactory nerve and digitate ganglion were crushed and the optic nerve cleaned of surrounding connective tissue. The thin layer of skin covering the eye was then removed. Dissection was done at 3°C under white light using a fiber optic. Experiments were initiated only after the completely dissected preparation had been in total darkness for at least 2 hr. Recording Extracelluiar recordings were made from the loop of the optic nerve near the eye. Broken tip glass suction electrodes were used. Optic nerve impulses were amplified by either a capacitatively coupled (low pass, 1OHz) amplifier (DAM 6A, WP Instruments) or a DC coupled amplifier (Pl6 Grass Instruments). Impulses were simultaneously displayed on an oscilloscope (Textronix 5112) and on a storage (Textronix 5113) oscilloscope. The stored display was photographed using a Polaroid oscilloscope camera gextronix). For some of the experiments, amplified optic nerve signals were led through a window discriminator (WP Instruments 121) and the resultant square wave pulse displayed as a dot (RASTER) display via-the Z-axis input of the storage oscilloscope. Thus, depending on the location of the window discriminator, many or few raster dots could be displayed during an optic nerve response to photic stimulation. Stimulation Light was presented via a fiber optic light pipe (DolanJenner model 150). A high intensity light (Sylvania DNE) was used. This was focused on the eye and surrounding tissue via a light pipette (Dolan-Jenner). The maximum intensity of the light (“G” light intensity) was 4.3 x 10’ W/cm measured 3 cm from the end of the liber optic. This value was determined, (and the calibration of neutral density hlters was carried out), using a alibrated IL500 Photometer/Radiometer (International Light). Since the light system was continuously on, a mechanical solanoid armature in the light path acted as a shutter. This shutter was triggered by a laboratory stimulator (Grass S44). Neutral density and colored tilters (Kodak) (blue, green, yellow, orange and red) were inserted in various combinations into a holder in the light path. The occurrence and intensity of the stimulus (light) was monitored at the eye via a photocell (IR 200) whose signal was led to one channel of the oscilloscope. The time between consecutive stimuli was I-2min. All experiments were carried out at room temperature (22°C).
RESULTS
Optic nerve response to light stimulation
A response occurred on the optic nerve subsequent to photic (light) stimulation (Fig. I A). This response occurred each time the eye was stimulated and appeared as a complex burst of activity. Using a short duration (100-500 msec) stimulus pulse, the burst consisted of an initial phasic (first) response of relatively large amplitude potentials followed by lower amplitude tonic potentials. First responses were similar, though not identical, each time the eye was stimulated (Fig. 1B). Sometimes a small amplitude DC shift was seen at the beginning of the response (Fig. 1A). Eflect of decreasing stimulus intensity
The first response’s delay (measured from stimulus onset to the first spike on the optic nerve trace or to the first dot on the RASTER trace), was relatively constant for almost any given stimulus intensity (Figs 1B and 2). As light intensity decreaed, response delay increased (Fig. 2) and the number of action potentials (indicated by the number of RASTER dots) decreased (Fig. 2). When optic nerve responses were compared over an intensity range of 4-6 log units, delay varied linearly with log stimulus intensity (Fig. 3). Other parameters, such as number of RASTER dots (spike frequency), RASTER burst duration ( = duration of RASTER phasic and tonic response), and the average RASTER frequency (number of RASTER dots/burst duration = average spike frequency of the initial phase and subsequent tonic response) showed no consistent relationships over this same intensity range. However, if the extremes were compared for any of these parameters, the expected trend was seen, so with decreasing light intensity, the number of dots decreased, RASTER burst duration decreased, and average RASTER frequency decreased. The delay at 0 intensity was 260-640 msec, depending on the animal. This delay increased to over 2 set at very low light intensities in one animal (Fig. 3). One eye showed saturation at high intensities (Fig. 3, dashed line). EfSect of stimulus duration
The effect of stimulus duration was studied by keeping the stimulus intensity consta.nt and increasing the stimulus duration to almost 7 sec. By using the window discriminator and photographing the storage oscilloscope at successively slower sweep speeds (5sec/cm in Fig. 6), a second feature of the optic nerve response was revealed. When the stimulus duration was varied between 100 and 6700 msec, both response delay and initial response duration were relatively constant (Fig. 4). The initial phasic (first) response was followed by lower amplitude tonic potentials, as before (in Figs 1 and 2) and was displayed as a continuous stream of RASTER dots). However, by using the slower sweep speed the first response, with its phasic and tonic component, was seen to be followed by a period of decreased activity (no RASTER dots). Then, a second response occurred (Fig. 4).
Optic nerve responses in Dyakia sfriata
Fig. 1. Response of Dyakia optic nerve to photic stimulation. In B, three responses separated by 1 min are shown. Stimulus duration in A and B, light = 450 msec. Intensity = 0.8 O.D. units. The second response consisted exclusively of low amplitude tonic potentials. (The optic nerve showed activity during the period of decreased activity, but this activity was below the window of the window discriminator.) The second response increased in duration as the duration of the flashes increased (Figs 5 and 6). The period of decreased activity showed no consistent relationship to the stimulus duration.
Response to light of dyerent wavelengths Dyakia optic nerve responded to all wavelengths of
light presented (Fig. 7). As colored light intensity decreased, delay increased (data not presented here). Although the delay to blue light was sometimes relatively less than the delay to green and yellow light (Fig. 7), this was not always the case. However, the delay to blue, green or yellow light was always much less than the delay to orange or red (Fig. 7). There were aiso fewer RASTER dots (i.e. less nerve activity) with orange and red light than there were with blue, green or yellow light (Fig. 7). Because of the complexity of the optic nerve
JONAI-HAN C~PELAND
Fig. 2. Response of optic nerve to three different intensities of light: A, full intensity (O.D. = 0); B, reduced by 90% from full intensity (O.D. = - 1) and C, reduced by 99% from full intensity (O.D. = -2). Calibration: upper voltage values indicate light trace calibration for A, B and C respectively; lower voltage value indicates voltage value of optic nerve trace. The Brst three traces in A, B and C were recorded simultaneously. Thereafter,with subsequent stimulations, the first two traces were eliminated and only the RASTER display shown.
rise, it was not possible to measure spectral sensitiivity of single units at high stimulus intensities. (Tests at low intensities, where single units were seen, gave inconsistent results.) However, threshold sensi-
tivity for the entire optic nerve response could be estimated by defining threshold as the value of the light intensity that was just capable of producin ga weak (2-4 dots) first response whose delay varied by
Optic nerve responses in D$cia striata
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less than 100 msec. Using these criteria for threshold, the eyes responded to blue, green and yellow light at very weak intensities (between - 2.5 and - 4.4 O.D. units) and to orange and red light between 0 (full intensity) and - 1.OO.D. units (Fig. 8).
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DISCUSSION
Optic nerve response
,200 B I
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Fig. 3. Effect of decrease in light intensity on delay. Five different preparations were used. Mean and 1 SD shown at each light intensity for each eye. Dashed line indicates eye which showed saturation at high intensities of light.
When stimulated with light, Dyakiu’s optic nerve shows a complex burst of activity. A phasic first response is followed by tonic activity (Figs 1, 2 and 4-6). Additionally, when stimulus durations are increased to behaviorally relevant ranges (Copeland and Maneri, 1984), beyond the usual physiological range normally employed in gastropod vision studies, and furthermore, when a window discriminator circuit is used to simplify the recorded response, the optic nerve response is seen to be even more complex than a phasic on-response followed by tonic activity. The response consists of first response, period of decreased activity, and second response (Figs 4-6). Either because a different type of analysis was attempted, or because longer (behaviorally relevant) stimulus durations were used, or because Dyakiu’s optic nerve response is, indeed, more complex than that of other stylommatophorans, Dyukia’s optic nerve response to light seems to be different from other pulmonates.
Fig. 4. Effect of stimulus duration on optic nerve response. As in Fig. 2, first three traces were recorded simultaneously. Thereafter, the first and second traces were eliminated and only the RASTER display shown. Optic nerve response consists of first response, period of decreased activity (no RASTER dots), and second response. Note time calibration equals 1 sec.
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Fig. !i. Effect of stimulus duration on optic nerve response. As in Fig. 4. Note time calibration 5 sec.
Because the response delay is constant from stimulus onset (Figs 1, 2 and 4) when stimulus duration is increased over a very large range (Figs 5 and 6), this response may be considered as an on-response. On-responses have also been found on the optic nerve of the terrestrial pulmonates Helix aspersa
(Gillary, 1970) Otala fatea (Gillary and Wolbarsht, 1967; Goldman and Hermann, 1967) and Limax jlaous (Suzuki et al., 1979) and the aquatic pulmonates Helisoma trivolvis (Patton and Kater, 1972) and Lymnea stagnalis (St011and Bijlsma, 1973). However, these on-responses appear as a compound
Optic nerve responses in Dyakia striata
397
LimaxJlaous (Kataoka, 1975) and Ariolimax californicus (Eaken and Brandenburger, 1975) and, using a
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RESPONSE
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DURATION
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DURET
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OIlRATION AC7lVlTI
Fig. 6. Effect of stimulus duration on optic nerve response. Left vertical axis for second response duration; right vertical axis for first response duration and for period of decreased activity. Same data as Fig. 5. Mean and 1SD shown. Similar results were found in two additional preparations.
action potential in Helix (Gillary, 1970) or as a slow wave form with a spike discharge riding on it in Helisoma (Patton and Kater, 1972). In the stylommathophorans Limax (Suzuki et al., 1979), Lymanea @toll and Bijlsma, 1973) and Otulu (Gillary and Wolbarsht, 1967; Goldman and Hermann, 1967), the on-response appears as a complex spike discharge. In these animals, however, only short duration flashes of light were used for stimuli. The physiological or anatomical causes of this response complexity in Dyakia are unknown, as is also the case for the somewhat simpler responses recorded in other pulmonates. Part of the response complexity could be due to the presence of both compound action potentials and single unit discharges in the recordings. Such potentials could be produced by the presence of different types of receptors or secondary cells. These receptors or secondary cells could adapt at different rates to continual or repeated stimulation. Although neither anatomical cross section of the optic nerve or structure of the retinal receptors and second order neurons are known for Dyakia, clues may be obtained from other pulmonates. With respect to optic nerve, both Helix aspersa (Eaken and Brandenburger, 1967) and Limax maximus (Gelperin, unpublished observations) possess several hundred small axons in the optic nerve, making the recording of compound action potentials a possibility. In the eye itself, two different photoreceptor types have been found in Helix aspersa (Brandenburger, 1975),
slightly larger phylogenetic perspective, five different photoreceptor types have been found in the eye of the opisthobranch gastropod Aplysia californica (Herman and Strumwasser, 1984). Additionally, second order cells, called ganglion cells, have been reported in the periphery of the retina in Helix aspersa (Brandenburger, 1975). Also, the eye of Agriolimax reticulatus contains an “accessory retina”, possibly infra-red sensitive (Newell and Newell, 1968) and the Aplysia californica eye, in addition to having five different receptor types, also possesses many afferent neurosecretory neurons (Herman and Strumwasser, 1984). Thus, the response complexity may be due to a heterogeneity of receptor and second order cell types, their different response properties, and different rates of adaptation. Using white light, the intensity-delay function for the first response of the on-response in Dyakia (Fig. 3) shows an inverse relationship between intensity and delay over 6 log units. A similar relationship has also been found for all wavelengths tested. Similar intensity-delay functions for white light have been shown over 6 log units in Lhux (Suzuki et al., 1979) and 4 log units in Otula (Goldman and Hermann, 1967). The threshold response, -6.0 log units in one individual (Fig. 3), to white light in Dyukiu, may actually be l-2 orders of magnitude lower than that of Limaxflavus (Suzuki et al., 1979). This is difficult to compare, however, because in Limax flavus a brighter stimulus was used (different 0 log intensity) and threshold was defined differently (Suzuki et al., 1979). phe very low number of preparations showing any response at all in Otala (Goldman and Hermann, 1967) can be explained by Goldman and Hermann’s methods, i.e. dissection in white light and immediate testing versus 3 hr dark adaptation time in Limax (Suzuki et al., 1979) and 2 hr here.] This 6 log unit response range encompasses much of the range of light intensities that Dyakia might experience in the field. Direct sunlight at sea level at noon at mid-latitudes has a radiant flux density of5 x 104~Wcm-2,moonlight is3 x lO-‘~W~rn-~, and the energy of starlight is 3 x 10-4pW cm-2 (reviewed in Nichol, 1978). The dynamic range of stimulus intensities that could evoke responses on the optic nerve in Dyakiu was from 4.3 x 1ticrnm2 (0 intensity) to 1.2 x 10m2PW cmw2 ( - 6 intensity) measured at 3 cm from the source. The in vitro preparation, then, is sensitive enough to be stimulated by moonlight. This means that the in viuo preparation, probably more sensitive, would be likely to be stimulated by the weak flash of a conspecific snail. What these sensitivities and background luminances translate into in terms of range and form vision in Dyakia cannot be estimated until the radiant energy emitted by the luminescent organ is measured, the optical characteristics of the eye are assessed, and appropriate psychophysical experiments are done. Second response
Using a stimulus of several seconds, only the second response period’s duration was directly pro-
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COPELAND
399
Optic nerve responses in Dyakiu striutu
the results in fireflies see, for example, Biggley et al., 1967; La11 et al., 1980). Visually guided behavior in snails
*
i
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0 o
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I
Fig. 8. Threshold sensitivity of four eyes to colored light of
different wavelength. Threshold defined in text.
portional to stimulus duration (Fig. 6). Thus, at behaviorally significant durations, the flash duration of a Dyakia could be encoded by the time between the beginning of the first response and end of the second response. Behavioral tlashes produced by Dyakia at 28°C are 0.5-6.0 set duration at 28°C (Copeland and Maneri, 1984). Depending upon the Q10 of the physiological processes associated with the optic response, the behavioral data here suggests an even broader range of stimulus durations might have been tested physiologically. (However, some visual responses measures, such as average ERG amplitude, increase from 10 to 20°C and decrease from 20 to 30°C (Gillary and Wolbarsht, 1967; Gillary, 1970) whereas others, such as ERG delay, decrease between 10 and 20°C (Gillary and Wolbarsht, 1967). Effect of colored light
The optic nerve response is most sensitive to blue, green and yellow light (Figs 7 and 8). These results are similar to those of Gillary and Wolbarsht (1967). where the dark adapted ERG’s constant response action spectrum was most sensitive in the blue-green (490 nm), and those of Suzuki et al. (1979), where the dark adapted optic nerve response showed a peak sensitivity to blue (480nm). (In this latter study, the light adapted response showed a shift in peak sensitivity to 460 nm if the amount of light adaptation exceeded 3 log units. Suzuki et al. (1979) interpreted this as providing neurophysiological evidence for two kinds of photosensory cells in the retina of Climax flavus.)
The color of Dyukiu’s in oioo bioluminescence is yellow-green (Copeland and Maneri, 1984). This lies well within the very broadly defined range of greatest spectral sensitivity found here (Figs 7 and 8). This observation, that the spectral sensitivity maximum of the bioluminescence and the sensitivity maximum of the eye are similar, has been found in other bioluminescent systems (reviewed in Nichol, 1978) (for
Slug and snail behavior has usually been thought to be largely guided by mechano- and chemoreception (Runham and Hunter, 1970). Only simple behaviors (tropisms, taxes, locomatory rhythms) were considered to be photically guided (Kerkut and Walker, 1975) and true visual behavior was not known. However, Otala lactea shows a body orientation guidance system to a slot of darkness (22.5”C x 45” at 25 cm) in a band of uniform brightness (Hermann, 1968). Additionally, Helix uspersu shows some preference for vertical bars subtending a visual field of 14.5-24.6” (versus horizontal or diagonal bars) (Hamilton and Winter, 1982). Also, Euparina pisuriu, closely related to Helix, may return to vegetation from which it has been displaced via visual orientation (Zanforlin, 1976). Additionally, prosobranch snails like the intertidal Littorina irrorata and Techtariurn muricatus and the subtidal Turbo castanea show l’esponse thresholds to 0.3-6.5” dark vertical bars (Hamilton and Winter, 1982, 1984). Thus, the visual world of snails may be considerably more complex than originally thought and simple form vision may be possible. Dyakia, being a bioluminescent terrestrial snail, is probably a reasonable candidate for possessing an unusual visual system. It might be appropriate that, if Dyukia’s bioluminescence is involved in a social role (Copeland and Maneri, 1984; Counsilman et al., 1987), the visual system should be specialized when compared to other terrestrial pulmonates. At the level of recording used here, the visual system of Dyukiu seems similar to that of other terrestrial pulmonates. However, the level of analysis used here suggests a complexity of response to photic stimulation (first response, period of decreased activity, second response) not found in other pulmonates. Furthermore, Dyukiu’s eye seems more sensitive than other pulmonate eyes. Thus, this visual system may, indeed, be unusually adapted to receive bioluminescently produced light. Acknowledgements-This work was supported in part by a grant from National Geographic Society (2716-83). The author thanks Dr George Daston and MS Maryellen Daston for their criticisms of the manuscript, Dr. James Counsilman for collection and shipment of the snails, and Mr David Lichtman for the loan of the Photometer/ Radiometer. REFERENCES
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