Hydrobiologia 530/531: 97–105, 2004. D.G. Fautin, J.A. Westfall, P. Cartwright, M. Daly & C.R. Wyttenbach (eds), Coelenterate Biology 2003: Trends in Research on Cnidaria and Ctenophora. 2004 Kluwer Academic Publishers. Printed in the Netherlands.
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Mechanoreception and synaptic transmission of hydrozoan nematocytesw Ulrich Thurm*, Martin Brinkmann, Rainer Golz, Matthias Holtmann, Dominik Oliver & Thiemo Sieger Institute for Neuro- and Behavioral Biology, University of Mu¨nster, Badestr. 9, D-48149 Mu¨nster, Germany (*Author for correspondence: Tel.: +49-251/83-22386, Fax: +49-251/83-23896, E-mail:
[email protected])
Key words: Corynidae, Hydra, mechanoelectric transduction, hair cell, chemoreception, synaptic exocytosis
Abstract Mechanoelectric transduction and its ultrastuctural basis were studied in the cnidocil apparatus of stenotele nematocytes of marine and freshwater Hydrozoa (Capitata and Hydra) as a paradigm for invertebrate hair cells with concentric hair bundles. The nematocytes respond to selective deflection of their cnidocil with phasic-tonic receptor currents and potentials, similar to vertebrate hair cells but without directional dependence of sensitivity. Ultrastructural studies and the use of monoclonal antibodies allowed correlating the mechanoelectric transduction with structural components of the hair bundle. Two other types of depolarising current and voltage changes in nematocytes are postsynaptic, as concluded from their ionic and pharmacological characteristics. One of these types is induced by mechanical stimulation of distant nematocytes and sensory hair cells. It is graded in amplitude and duration, but different from the presynaptic receptor potential. Adequate chemical stimulation of the stenoteles strongly increases the probability of discharge of their cnidocyst, if the chemical stimulus precedes the mechanical one. Simultaneously, the probability of synaptic signalling induced by mechanical stimulation is increased, reaching nearly 100%. The chemoreception of the phospholipids used could be localized in the shaft of the cnidocil, because of the water-insolubility of the stimulant. This chemical stimulation itself does not cause a receptor potential; its action is classified as a modulatory process. Electron microscopy of serial sections of the tentacular spheres of Coryne revealed synapses that are efferent to nematocytes and hair cells besides neurite–neurite synapses, each containing 3– 10 clear and/or dense-core vesicles of 70–150 nm diameter. The only candidates to explain the graded afferent signal transmission of nematocytes and hair cells are regularly occurring cell contacts associated with 1(–4) clear vesicles of 160–1100 nm diameter. Transient fusion and partial depletion of stationary vesicles are discussed as mechanisms to reconcile functional and structural data of many cnidarian synapses.
Introduction Hydrozoan nematocytes exhibit the configuration of mechanosensitive hair bundles that is characw
Review contributed to the Symposium on Neuro-Anatomy and -Physiology of Coelenterates; 7th International Conference on Coelenterate Biology, Lawrence, Kansas, USA; July 6–11, 2003.
teristic for epithelial mechanosensory cells of most invertebrate phyla. This is the concentric hair bundle (Fig. 1b). In contrast to the multicellular ciliary cone of anthozoan nematocytes (Watson & Mire, 2004), the hair bundle of hydrozoan nematocytes, the cnidocil apparatus, is a product only of the nematocyte (Slautterback, 1967). The cnidocil apparatus closely resembles
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Figure 1. Schemes of (a) a hydrozoan nematocyte (in textbook resolution), (b) a concentric hair bundle of invertebrate hair cells (collar receptors), and (c) an eccentric hair bundle of vertebrates. In b and c bottom: longitudinal sections; top: cross sections at the level of membrane connections (composite section in c). Arrows indicate directions of stimulatory deflections for de- or hyperpolarizing receptor potentials (adapted from Thurm et al., 1998a, b).
the hair bundle of oldest mechanosensory hair cells (Tardent & Schmid, 1972; Golz & Thurm, 1994; Holtmann & Thurm, 2001b) and its basic organization is retained up to the lower chordates (Hu¨esker & Thurm, 1994). Hydrozoan nematocytes may be the best accessible paradigm to study concentric hair cells. In several hydrozoa we found that nematocytes transmit afferent synaptic signals to other cells. This means, their mechanosensory transduction controls exocytotic processes not only at the apical side, i.e. the discharge of the cnidocyst, but also at the basolateral side, i.e. the release of transmitter, as in sensory cells. This raised the question of the structural basis of this output; afferent synapses had not been reported so far for hydrozoan nematocytes. The result challenges the concept of synapses of coelenterates. Different from mechanosensory cells, exocytotic activity of nematocytes is known to be under additional control of a second, the chemical, modality (for Hydra: Lentz & Barrnett, 1962). The mode of interaction of mechanical and chemical modalities proved to be a revealing issue.
Material and methods Our objects for structural as well as for physiological studies were freshwater Hydra (mostly H. vulgaris) and marine Corynidae (Stauridiosarsia producta, Coryne tubulosa, and Dipurena reesi). Transmission electron microscopy was optimized to stabilize and to stain cytoplasmatic and extracellular connecting structures and cytomembranes (Golz & Thurm, 1991). The noncontractile tentacular spheres of the capitate Corynidae allowed stable electrical recordings from nematocytes in their natural connectivity while the tentacle was isolated and held by a suction capillary (Brinkmann et al., 1996; Fig. 2). Tentacles of Hydra, isolated for electrical recordings, were immobilized by 1 lM HA-1077, a blocker of myosin phosphorylation (Lawonn, 1999). A glass probe that was moved in two dimensions by piezo-electric drivers and was opto-electronically controlled in feedback loops achieved mechanical stimulation of the individual cnidocil. Intracellular electrical measurements were done by one-electrode currentor voltage-clamp.
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Figure 2. Scheme of the arrangement for stimulation and intracellular recording from nematocytes in isolated tentacular spheres of Corynidae (as seen in focal plane). CA, cnidocil apparatus; Mc, microcapillary electrode; Nc, nematocyst; Cy, cytoplasm; Pe, pellicle; SP, stimulating probe (from Brinkmann et al., 1996).
Results and discussion Ultrastructure of the cnidocil apparatus The regular concentric arrangement of the stereovilli surrounding a cilium differs from the eccentric configuration of vertebrate hair bundles (Fig. 1c). In vertebrates, tip links connect stereovilli of different length and are assumed to control the mechanosensitive ion channels (Hudspeth, 1989). They are not present in hydrozoan concentric hair bundles as our comparative studies with various methods showed. This is associated with the fact that the stereovilli are arranged in one ring only and are mostly of the same length. At their tips, they are tightly interconnected with their neighbors, thus forming a stiff cone (Fig. 1b). When force is applied to the cone from the side, it is not deflected with internal shearing but is tilted as a whole (Brinkmann et al., 1994). In its center, the cilium or cnidocil is radially linked to the stereovilli by chains of extraand intracellular elements that connect the ciliary microtubular doublets to the microvillar actin core. These chains traverse the ciliary and stereovillar membranes (Golz & Thurm, 1991; Brinkmann et al., 1994; Golz, 1994; Thurm et al., 1998a). The mechanosensory input of nematocytes A purely mechanical stimulation of a nematocyte normally does not elicit the discharge of its cni-
docyst. Therefore, the electrical and sensory properties of nematocytes could be studied with repetitive stimulation. Since only stenotele cells were studied intracellularly (they are the only type in Corynidae), the data shown may not be valid for all types of nematocytes. Forces directed against the cnidocil apparatus cause phasic-tonic voltage responses, of depolarizing sign only (Brinkmann et al., 1996; Lawonn & Thurm, 1998; Fig. 3). The adaptation that occurs during about 200 ms of response decays within 200–500 ms after the end of the stimulus (shown by test stimuli). The peak response amplitude can reach 50 mV in Stauridiosarsia. The responses are due to an increase in nonspecific cation conductance with a reversal potential around 0 mV. They can be blocked by the ion-channel blocker streptomycin. These properties as well as the kinetics (latency
