THE ELECTROPHYSIOLOGY OF CNIDOCYTES

J. txp. Biol. 133, 215-230 (I9S7)

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Printed in Great Britain © The Company of Biologists Limited 1987

THE ELECTROPHYSIOLOGY OF CNIDOCYTES BY PETER A. V. ANDERSON AND M. CRAIG McKAY Wliitney Laboratory and Departments of Physiology and Neuroscience, University of Florida, St Augustine, FL 32086, USA Accepted 3 June 1987 SUMMARY

1. Electrical properties of cnidocytes isolated from the hydroid Cladonema and the scyphomedusa Chiysaora were examined using current- and voltage-clamp recording techniques. 2. The stenoteles of Cladonema produced action potentials when depolarized above OmV. The inward current that produced the action potential was a Na + current. These cells also possessed an A-current and a K-current. 3. Atrichous isorhizas from Chrysaora did not spike and did not have any inward currents. All cells examined had K-currents, some had A-currents also. 4. Very few cnidocytes discharged during the course of the recordings, irrespective of the degree to which they were depolarized or hyperpolarized, or the presence or selective blockade of any ionic currents. When discharge did occur it could never be correlated with any obvious electrophysiological event. 5. Recordings from cnidocytes in situ in tentacles of the siphonophore Physalia indicate that these cells do not spike. Their current/voltage relationships were linear. They too did not discharge in response to changes in membrane potential, suggesting that the failure of isolated cnidocytes to discharge cannot be attributed to the isolation procedure.

INTRODUCTION

Cnidocytes, the major diagnostic feature of members of the phylum Cnidaria, are unique cells. For this reason and because of the medical problems caused by their stings, they have attracted considerable attention from biologists from various disciplines, resulting in an extensive literature on their structure, ultrastructure, development and discharge mechanisms (for reviews see Lenhoff & Hessinger, 1987; Mariscal, 1974, 1984). Briefly, cnidocytes are cells whose interior is dominated by a large secretory product, the cnida, sometimes termed the capsule or cnidocyst. The cnida is a hardened capsule that contains an inverted tube. With appropriate stimulation, usually contact with some prey organism or potential predator, the operculum that seals the apical end of the cnida opens and the tube everts. Depending on the class of cnidocyte, the tube will either merely entangle the target or penetrate it and release a venom. Key words: cnidocytes, Cnidaria, voltage-clamp, sodium current, potassium current.

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P. A. V. ANDERSON AND M. C. MCKAY

The sequence of steps linking application of the stimulus to discharge of the cnida is little understood. It is known that the appropriate stimulus has both a chemical and a mechanical component (Glaser & Sparrow, 1909; Mariscal, 1974) and it is generally believed that these stimuli are transduced by the ciliary apparatus that adorns the apical end of many cnidocytes. There are two classes of ciliary apparatus: ciliary cones, which are found only in anthozoans, and cnidocil apparatuses which occur in all other classes (Mariscal, 1974). It is not clear, however, whether all aspects of the stimulus are transduced by these presumed sensory structures or whether other cell types in the tissue are involved. Indeed, spirocytes lack a ciliary apparatus altogether, suggesting either that other less obvious structures on the cnidocyte are responsible for stimulus transduction or that the transduction is carried out by accessory cells that communicate with the spirocyte. The reason for this uncertainty as to the site of the receptors stems from the fact that cnidarian cells are typically small and, because cnidarians are of the tissue level of organization, their tissues are complex. The tentacle of a typical cnidarian is composed of many cell types, some of which, such as neurones and epithelial cells, could interact with one another and with cnidocytes, thereby serving as a pathway for excitation of the cnidocytes. Indeed, there are several reports that the behavioural state of an animal can influence the sensitivity of the cnidocytes to discharge (Mariscal, 1973; Smith, Oshida & Bode, 1974). This implies a degree of endogenous control of the discharge, which is supported by several reports of neuro—cnidocyte synapses (Hufnagel, Kass-Simon & Lyon, 1985; Westfall, 1973a,b; Westfall, Yamataka & Enos, 1971). While the use of preparations of isolated cnidae circumvents some of the problems posed by whole tissues and has provided considerable information about several aspects of the biology of these cells (for a review of the methods, see McKay & Anderson, 1987), the isolated cnidae, by definition, are not bathed in cytoplasm and are not enclosed by a cell membrane. Since it is the cell membrane that interacts with the environment and with other cells, its presence is essential if a thorough understanding of the location of the receptors is to be achieved. Furthermore, both the cell membrane and the cell's cytoplasm must be present if the entire pathway by which a stimulus to the cnidocyte gives rise to cnida discharge is to be fully understood. Here we report on the electrophysiological properties of cnidocytes isolated from a hydroid and a scyphomedusa, and others in situ in a siphonophore. The study was undertaken primarily to examine the commonly perceived idea that cnida discharge might be triggered or otherwise controlled by electrophysiological events in the cell. This idea may have arisen from the observation that one of the most effective ways to evoke cnida discharge by isolated cnidae or intact tissues is with electrical stimulation (Holstein & Tardent, 1984). The present results indicate that although intact, isolated cnidae exhibit an array of voltage-dependent ionic currents, neither these nor any other obvious electrophysiological events evoke cnida discharge. To control against the possibility that these findings may reflect the fact that the cells weri isolated, recordings were also obtained from cnidocytes in situ.

Cnidocyte electrophysiology

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MATERIALS AND METHODS

Recordings were obtained from cnidocytes of Physalia physalis (Hydrozoa: Siphonophora), Cladonema sp. (Hydrozoa) and Chrysaora quinquecirrha (Scyphozoa). All animals were maintained in the laboratory under appropriate conditions; Chrysaora and Phvsalia usually only survived for a few days and were used immediately after collection. Cnidocytes were isolated from the various tissues enzymatically (McKay & Anderson, 1985, 1986). Chrysaora tentacles were treated with L-cysteine-activated papain, until microscopic examination revealed that sufficient cnidocytes had been released. This usually took 40—60min. The released cnidocytes, which were heavier than the other cell types, were then separated from any debris and other cell types by density centrifugation in a Percoll-containing medium. The supernatants were discarded and the pelleted cnidocytes transferred to either sea water or saline for recordings. The technique used for isolating cnidocytes from Cladonema was a simpler version of that given above. The excised hydranths were soaked in a Ca 2+ - and Mg2"*"free saline (Table 1) containing O-Smgrnl"' of L-cysteine-activated papain. After lOmin they were transferred to the final recording solution (Cyanea saline or sea water) and triturated with a fine pipette until individual cnidocytes were released from the capitate tentacles. Once again, the cnidocytes were denser than other cell types and settled to the bottom of the dish. Recording techniques Electrophysiological recordings were carried out using conventional microelectrode recording techniques and the whole-cell configuration of the patch-clamp technique (Hamill et al. 1981). Microelectrode recordings were obtained from cnidocytes in the tentacles of Physalia using microelectrodes filled with 3 mol 1~ KC1 with impedances in the range 15-25 MQ. To obtain the recordings, a short piece of tentacle was excised from the animal, anaesthetized in a 1:1 mixture of isotonic (0-37 mol I"1) MgCl2 in sea water, then stretched out and pinned firmly to a layer of Sylgard (Dow Corning, Midland, MI) on the bottom of a Petri dish, using cactus spines (Opuntia sp.). The bathing medium was then replaced with normal sea water for recordings. Recorded signals were amplified with a conventional d.c. amplifier equipped with a Wheatstone bridge circuit for current injection, and displayed on a Nicolet 2090 digital oscilloscope. Lucifer Yellow injection was carried out in the manner described previously (Anderson & Schwab, 1981). Whole-cell patch pipette recordings were obtained using Dagan 8900 patch-clamp amplifiers equipped with 0 - l GQ headstages. Pipettes were prepared from borosilicate glass (Boralux, Rochester Scientific, Rochester, NY) using a two-stage puller (Narashige PP83). They were coated with a layer of cured Sylgard but not firepolished (Corey & Stevens, 1983), since we have found that, for unknown reasons, omitting the polishing results in better seals onto coelenterate cells. The coated

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electrodes were filled with one of the solutions given in Table 1. With these pipette solutions, the electrodes had impedances of 5-8 MQ. Once any offset currents had been neutralized, the pipette tip was pushed gently against the cell surface, usually near the basal end, and light suction applied. Seal formation was usually aided by the application of a negative potential to the interior of the pipette and, under these conditions, seal resistances in the range 1 —10 GQ could be obtained fairly routinely. Once an adequate seal had been obtained, capacitive transients introduced by the electrode were neutralized. Breakthrough into the intracellular configuration was achieved with additional suction. For voltage-clamp recordings, compensation was made for 40-60 % of the series resistance error by circuitry in the amplifier. For current-clamp recordings, the amplifier was switched to the current-clamp mode and sufficient current was injected into the cell to establish a membrane potential of —70 mV. Square current steps were then injected into the cell and any d.c. offsets attributable to pipette resistance neutralized with a Wheatstone bridge circuit in the amplifier. Membrane potential was then readjusted if necessary. Current-clamp recordings obtained with patch pipettes were displayed in the same way as the microelectrode recordings. Voltage-clamp experiments were performed and the data digitized (80kHz), stored and manipulated by an IBM AT computer equipped with pClamp software (Axon Instruments, Burlingame, CA). With some cells and under some conditions, leakage currents were not linear at hyperpolarized membrane potentials, making their subtraction difficult. Such instances are noted. With the other cells, leakage and capacitive currents were removed from the records either by digital addition of currents generated by hyperpolarizing voltage steps onehalf the amplitude of those used to generate ionic currents, or by scaling and subtracting the currents generated when cells that had been hyperpolarized to —120 m V were depolarized with voltage steps one-half the amplitude of those used to generate ionic currents. Solutions The compositions of the various solutions used in this study are given in Table 1. Artificial salines were based on a saline developed for the scyphomedusa Cyanea (Anderson & Schwab, 1984). The pH of all external solutions was adjusted to 7-4 with NaOH or HC1, that of all internal solutions was adjusted to 7-0 with KOH or CsOH. All experiments were conducted at room temperature (21-24°C).

RESULTS Morphology of isolated

cnidocytes

The isolation procedures produced a mixture of cnidocyte types, representing the variety found in the intact tissues. The morphology of the different types o^ cnidocyte was varied, as would be expected from the well-recognized variations in

Cnidocyte electrophysiology

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Table 1. Composition of media (in mmol l~ ) Salt NaCl KC1 CaCl2 MgCl2 MgSO 4 Hepes NaHCO 3 Na 2 SO 4 CsCl TEAC1 Glucose EGTA Na z EDTA 4-aminopyridine

Externa 1 solutions Saline* Ca 2+ /Mg 2+ -free 390 13-4 9-5 24 5 10 — — —

— —

430 9

Internal solutions Cs+/TEA+ Normal

— — 10 5 20 —

140 1 — — 10 — — —

— 20 —

696 11 — —

30 — 1 — — 10 — — 70 70 636 11 — 2

• From Anderson & Schwab (1984).

capsule morphology (Mariscal, 1974). Only the stenoteles of Cladonema and the atrichous isorhizas of Chrysaora (Fig. 1) were examined in any detail. In all cases, the cnidocytes were easily distinguished from the surrounding tissue, and any debris, by the presence of the highly refractile cnida. The tube was usually visible within the cnida (Fig. 1) and in some cases, especially in the large stenoteles from Cladonema, the stereociliary complex at the apical end of the cell was easily discerned. Cnidocytes freshly isolated from Cladonema (Fig. 1A) usually had a long cytoplasmic extension at their basal end but these retracted within 15 min of isolation. Electrophvsiology of cnidocytes in situ Microelectrode recordings were obtained from the large (28 /zm in diameter) cnidocytes in the tentacles of Physalia. These recordings were obtained easily and were stable so long as the tentacle had been adequately pinned out. With sea water as the bathing medium, the mean resting potential of 15 cells was — 58-8 ± l-6mV (±S.E.M.). Current/voltage (i/V) plots, obtained by injecting depolarizing and hyperpolarizing current into the cell through the bridge-balanced microelectrode were completely linear and showed no evidence of delayed rectification. The average input impedance was 36-5 ± 13-3 MQ (±S.E.M.; N = 3). The time constant of these cells, determined from the slope of a semi-logarithmic plot of the charging curve created by the injection of hyperpolarizing current, was 1-3-1 -4 ms. Physalia cnidocytes did not discharge during these recordings, irrespective of the degree to which the cell was depolarized or hyperpolarized. Because cnidocytes are quite small and the cell is chiefly occupied by the impenetrable capsule, there is always some question as to whether recordings are

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obtained from the cnidocytes or from some other cell type. To confirm the recording site, the impaled cells were filled with Lucifer Yellow (Fig. ID). In every case, Lucifer Yellow was found in a cnidocyte, as defined by the presence of a cnida, and was restricted to that cnidocyte. There was no evidence of dye coupling to adjacent cells.

Fig. 1. Light micrographs of isolated cnidocytes of the type used in this study. (A,B) Nomarski micrographs of stenoteles from Cladonema. Freshly isolated cells (A) usually possessed a very obvious cytoplasmic protrusion at the basal end (arrowhead) but this was later retracted and became only barely visible (B). Notice the very obvious cnidocil apparatus (c) and the spines (s) on the inverted tubule. (C) Nomarski micrograph of an atrichous isorhiza from Chrysaora. In these cells the tubule is irregularly coiled and gives the cnida a granular appearance. (D) A phase/fluorescence micrograph of part of a tentacle from Physalia. A Lucifer Yellow-filled cnidocyte is indicated with an arrowhead. Note that dye has not passed to any of the surrounding cells. Scale bars in all cases = 10/