The Journal of Experimental Biology 198, 1743–1754 (1995) 1743 Printed in Great Britain © The Company of Biologists Limited 1995 CHEMOSENSITIVITY OF THE OSPHRADIUM OF THE POND SNAIL LYMNAEA STAGNALIS HEINER WEDEMEYER AND DETLEV SCHILD* Physiologisches Institut, Universität Göttingen, Humboldtallee 23, D 37073 Göttingen, Germany Accepted 14 April 1995 Summary The osphradium of the pond snail Lymnaea stagnalis was group of 15 neurones that lay next to the issuing osphradial studied to determine the stimuli to which this organ nerve, to determine whether ganglion cells were involved responds. The following stimuli were tested: hypoxia, in olfactory signal processing. All neurones tested hypercapnia, a mixture of amino acids, a mixture of responded to at least one of the three mixtures of odorants. citralva and amyl acetate and a mixture of lyral, lilial and Both excitatory and inhibitory responses occurred. ethylvanillin. Our results indicate that the osphradium of the pond The mean nerve activity consistently increased with snail Lymnaea stagnalis is sensitive to elevated PCO∑ as well elevated PCO∑, whereas hypoxia produced variable effects. as to three different classes of odorants. In addition, at least The nerve activity became rhythmic upon application of some neurones within the osphradium are involved in the citralva and amyl acetate, but it increased in a non- processing of olfactory information. rhythmic way upon application of the other two odorant mixtures tested. Key words: Lymnaea stagnalis, osphradium, chemosensitivity, Whole-cell patch-clamp recordings were made from a olfaction, PCO∑. Introduction Since its first description by Lacaze-Duthier (1872), the involvement of the osphradium in respiratory behaviour. function of the osphradia of snails has been a topic of debate. Kamardin (1976a) and Sokolov and Kamardin (1977) reported, Sensitivity to mechanical stimuli was proposed for marine on the basis of recordings from the right internal pallial (RIP) prosobranchs by Copeland (1918), Hulbert and Yonge (1937), nerve, that the osphradium of Lymnaea stagnalis responded to Yonge (1947), Kohn (1961) and Welsch and Storch (1969), but low partial pressures of oxygen (PO∑). This could possibly the osphradia of Buccinum undatum and Paludina vivipara parallel the function of the glomus I cells of the vertebrate have been shown to respond to chemical and not to mechanical carotid body (López-López et al. 1989), which directly activate stimuli (Wölper, 1950; Bailey and Laverack, 1963, 1966; the respiratory network in the brainstem via the Welsch and Storch, 1969). Comparative and evolutionary glossopharyngeal nerve. However, morphological studies aspects of gastropod osphradia are discussed in detail by Croll (Spengel, 1881; Benjamin, 1971; Benjamin and Peat, 1971; (1983) and Haszprunar (1985a,b, 1987a,b). Studies of the Kamardin, 1976b; Zaitseva, 1982) showed the presence of transduction mechanisms of the receptor neurones have not yet sensory neurones in the osphradial epithelium, although the been undertaken. odorants that were attractive in Y-maze experiments In opisthobranchs, Stinnakre and Tauc (1969) reported (Michelson, 1960) did not change the spike rate in intracellular evidence for the presence of osmoreceptors within the recordings from osphradial ganglion cells (Bailey and osphradium and Jahan-Parwar et al. (1969) recorded central Benjamin, 1968). Nor did a functional osphradium seem to be effects after chemical, osmotic and mechanical stimulation of essential for directional movement towards the source of a food the osphradium of Aplysia californica. A role for the extract (Townsend, 1973). Kamardin (1983, 1988) studied the opisthobranch osphradium in the control of respiratory homing behaviour of different snails, including two pumping was suggested by Croll (1985), Levy et al. (1989) and pulmonates, and suggested that the osphradia might be Levy and Susswein (1993). These authors found the organ to involved in this kind of behaviour. Recently, Nezlin et al. be sensitive to hypercapnia and hypoxia. (1994) described leucine-enkephalin- and methionine- In pulmonate snails, Lankester (1883) suggested an enkephalin-immunoreactive cells within the osphradial *Author for correspondence. 1744 H. WEDEMEYER AND D. SCHILD ganglion that project their neurites into the sensory part of the (Diamond General, Ann Arbor, Michigan, USA) placed in the epithelium. The physiological roles of these cells are not yet Petri dish bath next to the osphradium. During hypoxia known. Taken together, the evidence regarding the function of experiments, PO∑ was between 7 and 11 mmHg. For patch- osphradia in aquatic pulmonate snails has remained highly clamp recordings, the patch pipette was filled with a solution controversial. containing (in mmol l21): NaCl, 2; potassium aspartate, 37; In this study we have reinvestigated this question. First, we KCl, 5; MgCl2, 2; Hepes, 10; EGTA, 1; K2ATP, 1; Li2GTP, tried to determine the type of stimulus that changes the activity 0.5; cyclic AMP, 0.01. The combination of cyclic AMP and of the RIP nerve, by recording the activity of this nerve under ATP largely prevented the so-called ‘calcium wash-out’ hypoxia and hypercapnia and upon application of olfactory (Kostyuk et al. 1986). stimuli. The results show that the osphradium of L. stagnalis Three different solutions (A, B and C) of odorant stimuli is unique, in that it is sensitive to odorants as well as to changes were prepared by adding odorants to solution LR1. Solution A in PCO∑. Second, we used the whole-cell configuration of the contained the following L-amino acids (final concentrations in patch-clamp technique to determine whether osphradial mmol l21): arginine, 0.6; cysteine, 0.2; isoleucine, 0.4; leucine, ganglion neurones were involved in the signal processing of 0.4; lysine, 0.4; methionine, 0.1; phenylalanine, 0.2; threonine, the responses to odorants. As this turned out to be the case, the 0.4; tryptophan, 0.1; tyrosine, 0.1; and valine, 0.4. Solution B osphradium of L. stagnalis could be unique also because both contained the odorants citralva and amyl acetate (100mmol l21 primary and secondary olfactory signal processing may take each). Solution C contained the odorants lyral, lilial and place in the same organ. ethylvanillin (100 mmol l21 each). Stock solutions of citralva, amyl acetate, lyral, lilial and ethylvanillin were made by dissolving the odorants in methanol. The final concentration of Materials and methods methanol in solutions B and C was 0.01 %. All stimulus Preparation solutions were applied to the Petri dish bath by superfusion Specimens of Lymnaea stagnalis L., collected originally in (exchange time was about 1 s). The application of methanol a local pond and maintained for many generations under alone had no effect upon membrane current, membrane voltage laboratory conditions, were kept in aquaria at 15–17 ˚C and fed or RIP nerve activity. on lettuce supplemented with tropical fish food. Electrophysiological experiments were performed on Nerve activity recordings isolated osphradia from snails weighing approximately 2 g. The activity of the RIP nerve was recorded using suction After anaesthesia in iced water, the tissue above the central electrodes with a tip diameter of about 75 mm, amplified with nervous system was removed and the central ganglion ring was an EPC7 amplifier (List, Darmstadt, Germany) in current- extirpated by cutting all nerves close to the ganglia except the clamp mode. Data were recorded on video tape after pulse code RIP nerve. The mantle was then folded back and the modulation (Sony PCM501) and on a chart recorder. The osphradium, with a small section of the RIP nerve attached to activities were off-line low-pass-filtered at 1 kHz (eight-pole it, was extirpated together with a small piece of adjacent tissue, Bessel filter, Frequency Devices) and sampled at 8 kHz using large enough to fix the preparation with minute needles on a an A/D converter (Burr & Brown, MPV952) in a VME silicon layer within a Petri dish. The connective tissue sheet computer (Eltek, Darmstadt, Germany). The digitized data above the neurones was removed by microdissection. were then analysed using a SUN workstation. For the detection of spikes, a custom-built program written in ‘C’ language was Solutions used. The algorithm assigned a spike event to the first of four The isolated osphradia were continuously superfused successive bins of an activity histogram (bin width, 1 ms), (4 ml min21, gravity feed) with standard Lymnaea Ringer whenever 32 successive data points (corresponding to a period 21 (LR1) containing (in mmol l ): NaCl, 42; KCl, 1.5; CaCl2, 4; of 4 ms) were above a threshold value. For the purposes of this MgCl2, 2; glucose, 5; Hepes, 10. Two other solutions were paper, we call these histograms ‘raw data histograms’. The used where indicated: LR2 (NaCl, 29; KCl, 1.5; MgCl2, 15; threshold was chosen to lie just above the peak noise level of glucose, 5; Hepes, 10; in mmol l21) and LR3 (NaCl, 36; KCl, the nerve activity signal. The validity of the algorithm, i.e. its 1.5; CaCl2, 4; MgCl2, 2; NaHCO3, 7.5; glucose 5; Hepes, 10; ability to detect all spikes above the threshold value, was in mmol l21). Solutions were adjusted to pH 7.8 with NaOH. determined by comparing the original nerve activity traces and Solution LR2 contained an elevated concentration of Mg2+ and the raw data histograms. Finally, the the raw data histograms no added Ca2+, corresponding to an actual calcium were transformed into histograms with a broader bin width concentration of about 20 mmol l21, measured with a Ca2+- (5 s) by adding the events of 5000 adjacent bins from the raw sensitive electrode (Ingold, Steinbach, Germany). data histograms. The resulting histograms thus gave the nerve To increase PCO∑ without changing PO∑ or pH, solution LR3 activity averaged within bins of 5 s. In some cases, we chose was bubbled with a mixture of 98 % O2 and 2 % CO2 and a bin width of 10 s (e.g. Fig. 6).