Helgol Mar Res (2007) 61:135–142 DOI 10.1007/s10152-007-0061-z ORIGINAL ARTICLE Functional neuroanatomy of the rhinophore of Archidoris pseudoargus Adrian Wertz · Wolfgang Rössler · Malu Obermayer · Ulf Bickmeyer Received: 4 December 2006 / Revised: 18 January 2007 / Accepted: 18 January 2007 / Published online: 15 March 2007 © Springer-Verlag and AWI 2007 Abstract For sea slugs, chemosensory information repre- Keywords Serotonin · Chemosensory · Calcium sents an important sensory modality, because optical and imaging · Sea slug · Mollusc acoustical information are limited. In the present study, we focussed on the neuroanatomy of the rhinophores and pro- cessing of olfactory stimuli in the rhinophore ganglion of Introduction Archidoris pseudoargus, belonging to the order of Nudi- branchia in the subclass of Opisthobranchia. Histological Chemical signals play a prominent role in most organ- techniques, Xuorescent markers, and immunohistochemis- isms. Sea slugs, living in shallow waters at the North sea try were used to analyse neuroanatomical features of the should primarily rely on chemical and mechanical rhinophore. A large ganglion and a prominent central lym- senses, as optical information is limited and the sensitiv- phatic channel are surrounded by longitudinal muscles. ity of long-range acoustic stimuli without a swim bladder Many serotonin-immunoreactive (IR) processes were found or a sophisticated ear is probably low. Archidoris around the centre and between the ganglion and the highly belongs to the order of nudibranchia, and its rhinophores folded lobes of the rhinophore, but serotonin-IR cell bodies were shown to be sensitive to mechanical stimulation were absent inside the rhinophore. In contrast to the condi- (Agersborg 1922). Anatomical studies by Storch and tions recently found in Aplysia punctata, we found no evi- Welsch (1969), for the Wrst time, suggested similarities to dence for the presence of olfactory glomeruli within the the osphradium (Welsch and Storch 1969; Wedemeyer rhinophore. Using calcium-imaging techniques with Fura II and Schild 1995) and that the posterior tentacles, the rhi- as a calcium indicator, we found diVerential calcium nophores, may serve as chemoreceptive organs. Using responses in various regions within the ganglion to stimula- electrophysiological recordings and neuroanatomical tion of the rhinophore with diVerent amino acids. The lack backWll techniques, Bicker et al. (1982) investigated of glomeruli in the rhinophores induces functional ques- mechano- and chemoreception in Pleurobranchaea cali- tions about processing of chemical information in the fornica. In Aplysia, the rhinophore epithelium was sug- rhinophore. gested to be chemoreceptive (Audesirk 1975; Emery and Audesirk 1978), and the function of the rhinophore as an Communicated by H.-D. Franke. olfactory organ was described by Audesirk and Audesirk (1977). Ablation of the rhinophores of Aplysia was A. Wertz · U. Bickmeyer (&) shown to cause a decrease in the time spent for mating Biologische Anstalt Helgoland, Alfred Wegener Institut für and the egg laying behaviour, suggesting the sensitivity Polar- und Meeresforschung, Kurpromenade 201, 27483 Helgoland, Germany of the rhinophores for pheromones (Levy et al. 1997; e-mail: [email protected] Susswein and Nagle 2004; Cummins et al. 2004). In a recent study using calcium-imaging techniques, Wertz A. Wertz · W. Rössler · M. Obermayer et al. (2006) showed that amino acids are detected and Department of Behavioral Physiology and Sociobiology, Biozentrum, University of Würzburg, Am Hubland, processed by the rhinophores of Aplysia punctata sup- 97074 Würzburg, Germany porting their olfactory function. 123 136 Helgol Mar Res (2007) 61:135–142 Compared to vertebrates and insects, the olfactory path- phosphate buVered saline (PBS, pH 7.2). For labelling with way of molluscs has not been investigated deeply. In terres- Xuophore-conjugated phalloidin, immunohistochemistry, trial gastropods, the importance of the procerebrum for tracing with biocytin markers and nuclear staining, the rhi- olfactory information processing was shown (Delaney et al. nophores were embedded in 5% low-melting point agarose 1994; Gelperin 1999; Gelperin and Tank, 1990), and the (Agarose II, Amresco, Solon, OH, USA, No. 210-815) and neuroanatomy and function of the cerebrum with respect to sectioned in a frontal or sagittal plane at 150 m with a olfactory information processing was reviewed by Chase vibrating microtome (Leica VT 1000S, Wetzlar, Germany). (2000). However, only little is known about the olfactory Free-Xoating agarose sections were preincubated in PBS pathway from the sensory cells to higher centres. In the with 0.2% Triton X-100 and 2% normal goat serum (NGS: stylommatophoran terrestric pulmonate Achatina fulica, ICN, Biomedicals, Orsay, France, Cat. No. 191356) for 1 h Chase and Tolloczko (1993) described the anatomy of the at room temperature. DiVerent combinations of double lab- posterior tentacle and found olfactory glomeruli with simi- ellings were performed. To label serotonergic neurons, sec- larities to glomeruli found in vertebrates and arthropods tions were incubated with a primary antibody against (Hildebrand and Shepherd 1997) and Croll et al. (2003) serotonin derived from rabbit (1:4000, DiaSorin, Stillwater, described glomeruli-like structures in the nudibranch Phe- MN, USA, Cat. No. 20080, Lot No. 051007) in PBS with stilla. Likewise, the rhinophore of A. punctata possesses a 0.2% Triton X-100 and 2% NGS overnight at room temper- rhinophore ganglion and glomeruli, which are arranged ature. This antibody was used successfully in previous around the rhinophore groove (Wertz et al. 2006). For the studies in gastropod molluscs (Croll et al. 2003; Moroz genus Archidoris, there is no information available about et al. 1997; Wertz et al. 2006). After Wve rinses in PBS, sec- processing of chemosensory information or the underlying tions were incubated in Alexa Fluor 488-conjugated goat neuroanatomy within the rhinophore. In A. punctata, the anti-rabbit secondary antibody (1:250, Molecular Probes, rhinophore ganglion and the glomeruli are innervated by Eugene, OR, USA, Cat. No. A -11008). To label Wlamen- centrifugal serotonergic processes (Wertz et al. 2006). Sim- tous (f)-actin in muscles and nervous tissue, sections were ilar to results in the opisthobranchia Pleurobranchaea and incubated in 0.2 units of Alexa Fluor 568 phalloidin Tritonia (Moroz et al. 1997) and sensory organs in Phe- (Molecular probes, A-12380) in PBS overnight at 4°C. To stilla, serotonergic cell bodies were shown to be absent in stain cell nuclei, sections were incubated for 15 min in the periphery (Croll et al. 2003). 25 g/ml propidium iodide (Molecular probes, P-1304) in In the present study, we investigated the rhinophore of PBS with 0.2% Triton X-100 at room temperature. Sections Archidoris pseudoargus using histological techniques, were Wnally washed at least Wve times with PBS, trans- serotonin immunohistochemistry, and Xuorescent tracers to ferred into 60% glycerol/PBS for 30 min, and mounted on reveal general neuroanatomical features. Responses to microscopic slides in 80% glycerol in PBS. amino acids as potential odorants were measured in the rhi- nophore ganglion using Xuorimetric calcium imaging. The Staining with biocytin results of this study give a Wrst account of the chemosen- sory structure and function of the rhinophores of A. pseud- For live staining with biocytin (Molecular Probes, B1592), oargus and open up new avenues for further studies of rhinophores were transferred to a dish containing ASW, chemoreception in sea slugs. DiVerences with recent Wnd- which was then removed by a dry paper. Small amounts of ings in A. punctata are discussed. biocytin crystals were applied into the tentacle nerve using a minuten pin. After 2–4 h incubation at room temperature, the rhinophores were Wxed in 4% formaldehyde in ASW for Materials and methods 1 day at 4°C. After rinsing three times in PBS, rhinophores were embedded in agarose and sliced at 400 m thickness Tissue preparation, immunohistochemistry, with a vibratome. To visualize biocytin, slices were incu- and Xuorescent tracers bated with streptavidin conjugated with Alexa 488 Fluoro- phore (1:125, Molecular Probes, S11223) overnight. Specimens of A. pseudoargus were collected from shallow Following dehydration in an ascending series of ethanol, waters around Helgoland. Animals were of diVerent ages slices were cleared and mounted in methyl salicylate. and body sizes. For preparation, the animals were cooled on ice and Wxed in 4% formaldehyde in artiWcial sea water Histology and confocal microscopy (ASW; pH 7.5; in mM: 460 NaCl, 104 KCl, 55 MgCl, 11 W W CaCl2, and 15 Na–HEPES (N-2-hydroxyethylpiperazine- Rhinophores were xed in Bouin’s xative solution for N’-2-ethanesulfonic acid Na-salt). Before further treat- 2 days, washed with ethanol, embedded in Spurr’s resin, ments, the rhinophores were washed three times in 0.1 M and sectioned in the sagittal and frontal planes (6 m). 123 Helgol Mar Res (2007) 61:135–142 137 After standard histological procedures, plastic sections uli (Alanine, Arginine, Glutamine, Methionine, and Isoleu- were stained on a hotplate after Mallory (1900). Stained cine; all purchased at Sigma-Aldrich, Munich, Germany). sections were washed with distilled water, dried on the Amino acids were applied for 1 min at various concentra- hotplate, and mounted in Entellan (Merck, Darmstadt, tions with the peristaltic pump