The Journal of Experimental Biology 203, 757Ð764 (2000) 757 Printed in Great Britain © The Company of Biologists Limited 2000 JEB2284

BISTABLE MEMBRANE POTENTIAL OF THE CILIATE COLEPS HIRTUS

PIA RUDBERG AND OLAV SAND* Department of Biology, University of Oslo, PO Box 1051 Blindern, N-0316 Oslo, Norway *Author for correspondence (e-mail: [email protected])

Accepted 24 November 1999; published on WWW 26 January 2000

Summary In normal recording solution, the swimming pattern of prolonged the shoulder, which occasionally stabilised at the the freshwater ciliate Coleps hirtus, belonging to the class shallow membrane potential (−30 mV). The membrane Prostomatea, consists of alternating periods of nearly linear potential could be shifted to the deep level by brief forward swimming and circular swimming within a small hyperpolarising current injections. Similar biphasic area. Current-clamp recordings were performed to membrane properties have not been reported previously in elucidate the mechanism for this behaviour. No members any ciliate. The bistability of the membrane potential was of this class have previously been studied using abolished in Ca2+-free solution containing Co2+ or Mg2+. In electrophysiological techniques. The ciliates were Ca2+-free solution containing 1 mmol l−1 Ba2+, brief maintained in culture and fed on the planctonic alga depolarising current injections at the deep potential level Rhodomonas minuta. The membrane potential showed evoked all-or-nothing action potentials with a prolonged spontaneous shifts between a more negative (deep) level of plateau coinciding with the shallow potential. We conclude approximately −50 mV and a less negative (shallow) level that the deep membrane potential in C. hirtus corresponds of approximately −30 mV. The input resistance and to the traditional resting potential, whereas the shallow capacitance at the more negative level were approximately level is a Ca2+-dependent plateau potential. In normal 400 MΩ and 120 pF respectively. C. hirtus displayed a solution, the direction of the ciliary beat was backwards at pronounced inward rectification, which was virtually the deep potential level and forwards at the shallow insensitive to 1 mmol l−1 Cs+ and almost completely blocked membrane potential, probably reflecting the two main by 1 mmol l−1 Ba2+. Depolarising current injections failed phases of the swimming pattern. to evoke graded, regenerative Ca2+ spikes. However, current-induced depolarisations from the more negative Key words: Coleps hirtus, ciliate, electrophysiology, membrane potential level (−50 mV) showed a pronounced shoulder potential, bistability, action potential, Ca2+, inward rectification, during the repolarising phase. Increased current injections swimming.

Introduction Within the eight different classes of ciliates, approximately found in Bursaridium, from the class Colpodea, which 8000 species have so far been described (Corliss, 1994). generates spontaneous all-or-nothing action potentials with a However, electrophysiological properties have only been distinct threshold in normal solution (Berg and Sand, 1994). studied in a surprisingly small number of species, mainly Spontaneous action potentials with a plateau phase lasting up within the genera Paramecium (Eckert and Naitoh, 1972), to 3Ð4 s have also been reported in Stylonychia in solutions Stylonychia (Machemer and Deitmer, 1987), Didinium (Pape containing artificially high concentrations of Ca2+ (Machemer, and Machemer, 1986; Pernberg and Machemer, 1989) and 1970). Euplotes (Lueken et al., 1996). The presence of voltage- The ciliate genus Coleps belongs to the class Prostomatea, sensitive Ca2+ channels is a general feature in these ciliates, and no species within this class has previously been studied and the direction of the ciliary beat is controlled by the using electrophysiological methods. The swimming pattern of cytosolic Ca2+ concentration (Machemer and Sugino, 1989; C. hirtus in normal solution consists of forward swimming Pernberg and Machemer, 1995a,b). The detailed electrical covering relatively large distances interrupted by periods membrane properties, however, show great variation among of circular swimming within a small area. We have made different ciliate classes. In most cases, depolarising current current-clamp recordings from C. hirtus using standard injections evoke regenerative Ca2+ spikes lacking all-or- microelectrodes, and report exceptional electrophysiological nothing characteristics in normal solution, for instance in properties of this ciliate. The membrane potential showed Paramecium (see Machemer, 1988), belonging to the class spontaneous shifts between two semistable levels. The more Oligohymenophorea. A striking exception to this pattern is negative level corresponds to the traditional resting potential, 758 P. RUDBERG AND O. SAND whereas the less negative level is a Ca2+-dependent plateau animals were kept in position using a microsuction pipette with potential. The transition between these membrane potential a tip diameter of 10Ð15 µm. The suction pipette was connected levels was correlated with the reversal of the direction of the to ambient, subatmospheric or superatmospheric pressure via ciliary beat and may explain the characteristic swimming a solenoid valve (Jonsson and Sand, 1987). A selected animal behaviour of this ciliate. was sucked onto the tip of the pipette by activating the solenoid valve, using a manual trigger. The subatmospheric catching pressure was between −1.0 and −1.5 kPa, whereas the holding Materials and methods pressure during the recordings was reduced to −0.5 kPa. The Animals animals are coated with a gel-like material, which tended to Coleps hirtus Nitzsch is a barrel-shaped freshwater ciliate clog the pipette. Between each catch, the pipette was therefore feeding on dead or living algae, flagellates, rotifers or other cleaned using a short flush of medium through the tip. This was protozoa. The genus is characterised by an elaborate achieved by connecting the pipette to a pressure of endoskeleton consisting of superficial rows of calcium approximately 5 kPa. carbonate plates (Fig. 1). The animals were obtained from a The movements of both freely swimming animals and lake near Oslo and kept in culture. They were fed on the ciliates penetrated by microelectrodes were studied using a − planktonic alga Rhodomonas minuta, as described by video camera recording at 25 frames s 1. The general direction Klaveness (1984). The algae were grown in standard growth of the ciliary beat of penetrated cells was determined in a medium (Guillard and Lorenzen, 1972) at 17 ¡C with a suspension of carmine particles. A rough estimate of the beat 14 h:10 h light:dark photoperiod. The mean length of the direction was obtained from the movements of the insoluble animals in our cultures was 42 µm and the mean width was dye grains (diameter 0.5Ð1 µm) surrounding the cell. 29 µm (N=60). The cultures of Coleps hirtus and Rhodomonas Simultaneous recordings of the membrane potential and animal minuta were generously provided by Dr Dag Klaveness. and particle movements were stored on separate channels of the same recorder. Recordings Results are reported as means ± S.D. Prior to the recordings, the animals were transferred from the growth medium to a recording solution containing (in −1 Results mmol l ): CaCl2, 1; NaCl, 1; KCl, 1. The solution was adjusted to pH 7.2 with 1 mmol l−1 Tris/HCl. In some Swimming behaviour experiments, the CaCl2 in this normal recording solution was In normal recording solution, the swimming behaviour of C. replaced with BaCl2, CoCl2 or MgCl2. In one series of hirtus consisted largely of two alternating main patterns: experiments, 1 mmol l−1 CsCl was added to the normal forward swimming and circular movements. The forward solution. During the experiments, the animals were viewed swimming usually displayed a component of lateral through an inverted microscope. oscillations of small amplitude. Swimming speed was The electrical properties of the surface membrane were 0.4Ð1.0 mm s−1. At irregular intervals, the periods of forward studied using conventional microelectrodes and standard swimming were interrupted by circular movements within a recording equipment. The electrodes were filled with 4 mol l−1 restricted area (Fig. 2). The duration of these two swimming potassium acetate adjusted to pH 7.2 with acetic acid, and the patterns varied from a few seconds to more than a minute. electrode resistance was 40Ð80 MΩ. During the recordings, the The main purpose of the present investigation was to elucidate the cellular mechanisms behind this characteristic swimming pattern. In other ciliate species, the direction of the ciliary beat is controlled by the cytosolic Ca2+ concentration and, hence, the membrane potential. The natural approach was therefore to study the electrophysiological properties of C. hirtus.

Passive electrical membrane properties and membrane rectification The initial recordings revealed a bimodal distribution of the membrane potential in normal solution. In approximately 70 % of the cells, the membrane potential was between −50 and −60 mV (deep membrane potential), whereas the remaining − 10 µM cells displayed membrane potentials between 20 mV and −30 mV (shallow membrane potential). To study the resistance, Fig. 1. A drawing of the endoskeleton of Coleps hirtus based on capacitance and rectifying properties of the membrane, hyper- scanning electron micrographs from Klaveness (1984) and Foissner and depolarising current pulses of varying magnitude and et al. (1994). 250Ð500 ms duration were injected, and the data were plotted Bistable membrane potential of Coleps hirtus 759

membrane time constant measured from potential deflections within this range was 48±19 ms (N=42), giving a mean membrane capacitance of 114±35 pF (N=42). The I/V relationship shows marked outward rectification for potentials above −25 mV and pronounced inward rectification for potentials below −60 mV. The potential deflections with large hyperpolarising current injections reached a peak value before settling at a stable level towards the end of the pulse (inset) because of the time-dependence of the activation of the ion channels responsible for the inward rectification. These 1 mm peak values are also included in the graph. The inward rectification was only moderately depressed by 1 mmol l−1 Cs+ Fig. 2. Typical swimming behaviour of Coleps hirtus in normal (data not shown), but was completely blocked by 1 mmol l−1 recording solution. Circular movements within a restricted area are Ba2+ (see Fig. 9). interrupted by periods of forward swimming with small lateral movements. The swimming speed during forward locomotion was Absence of graded Ca2+ spikes 0.4Ð1.0 mm s−1. The total duration of this tracing was approximately 2.5 min. On the basis of previous studies of the electrophysiological properties of ciliates, current-induced, graded Ca2+ spikes or all-or-nothing Ca2+-dependent action potentials might have as current/voltage (I/V) diagrams. Fig. 3 presents the I/V been expected in response to depolarising current injections. relationship for a cell displaying a resting potential of −48 mV However, in C. hirtus, depolarising current injections evoked during the recording period. The potentials recorded 200 ms membrane responses more like passive potential deflections after the start of current injections are plotted in the graph, and than active, regenerative responses. Fig. 4 presents recordings the inset shows a sample of superimposed original recordings. from two different cells, with stable membrane potentials of The slope of the I/V relationship was nearly linear between −60 −50 mV (Fig. 4A) and −20 mV (Fig. 4B). It is evident from and −30 mV, corresponding to a constant membrane resistance. Fig. 4A that the response to depolarising current injections The mean input resistance of the cells in this potential range cannot be completely explained by the passive electrical was 386±141 MΩ (mean ± S.D., N=61). The mean value of the properties of the membrane. The cell displayed an after- depolarisation outlasting the current injection by several Current (pA) hundred milliseconds; a similar active component of the membrane response was evident in approximately 90 % of the -600 -400 -200 200 400 600 cells with a deep membrane potential. After-depolarisations were never observed in cells with a shallow membrane -20 potential. Depolarising current injection into these cells were 0 instead usually followed by a moderate after-hyperpolarisation -20 -40 -40 (Fig. 4B). -60 (mV) The shape and duration of the after-depolarisation varied m

-80 E -60 greatly among cells. Fig. 5 presents a recording from a cell -100 with a stable membrane potential of approximately −60 mV. A 200 pA small, depolarising current injection caused a passive -80 100 ms membrane response, whereas larger current injections evoked after-depolarisations of increasing duration. At a level of -100 current injection exceeding 70 pA, the post-stimulus membrane potential stayed at a stable, depolarised level for approximately -120 19 s. Depolarising current injection may therefore induce a prolonged, depolarised plateau phase of the membrane potential. It is therefore reasonable to explain the bimodal -140 Membrane potential (mV) distribution of the membrane potential in C. hirtus in the following manner. The proper resting potential is relatively Fig. 3. Current/voltage (I/V) relationship for a cell in normal deep and is within the range of resting potentials observed in recording solution. The recordings were obtained from the deep other ciliates. Under certain conditions, C. hirtus may generate (−50 mV) membrane potential level. Data points show the membrane potential 200 ms after the start of current injection (filled symbols) active membrane depolarisations of extremely long duration. and at the peak of the hyperpolarisation (open symbols). Note the These plateau potentials are dependent on voltage-sensitive ion existence of both inward and outward rectification. The inset shows a channels and correspond to the action potentials observed in sample of superimposed, original recordings. Em, membrane some species of ciliate. However, in C. hirtus, the plateau potential. phase of the action potential may be prolonged to such an 760 P. RUDBERG AND O. SAND

Fig. 4. Membrane potential 0 0 (Em) responses to A B

depolarising current -20 -20 (mV) (mV) injections in normal m E -40 m -40 recording solution. The E superimposed recordings at -60 100 ms varying current intensities 200 pA are from different cells, with stable membrane potentials of −50 mV (A) and −20 mV (B). Note the lack of regenerative responses in both cells and the existence of after- depolarisations following the current injections in A. extent that the membrane potential of the cell is best described injections in approximately 50 % of the cells. The mean value as bistable or biphasic. of the deep potential level, corresponding to the resting membrane potential, was −52±6 mV (N=69). The mean value Shifts between shallow and deep membrane potential of the shallow membrane potential level, or the plateau During long-lasting recordings of several minutes, potential, was −29±6 mV (N=65). The mean value of the spontaneous shifts between shallow and deep membrane observed shifts between these two semi-stable levels of the potential levels were frequently observed, supporting the membrane potential was 23±6 mV (N=47). In recordings in theory outlined above. An example of a shift from the shallow which current injections were not performed, the membrane to the deep level is shown in Fig. 6, which also presents potential was at the deep level for 78 % of the total recording potential responses to depolarising current injections at both time. the shallow and the deep membrane potentials. Initially, the membrane potential was approximately −30 mV, and Mechanism of the regenerative plateau potential depolarising current injection evoked a nearly passive response Fig. 8 shows potential responses to hyperpolarising current lacking an after-depolarisation. Shortly after the injection, the injections. The first injection was performed at the deep membrane potential spontaneously hyperpolarised and membrane potential level, and the potential response was stabilised at a level of approximately −50 mV. From this level, passive. However, after termination of the hyperpolarising depolarising current injection evoked a potential response with response, the membrane potential spontaneously shifted to a the characteristic after-depolarisation. The time course of the stable, shallow level. The hyperpolarisation evoked by current spontaneous shift from the shallow to the deep membrane injection at this plateau potential clearly deviated from a potential was similar to the shape of the after-depolarisation, passive response. Closure of voltage-sensitive ion channels supporting the idea that the shallow membrane potential level during the hyperpolarizing phase reduced the membrane and the after-depolarisation are dependent on a similar, active conductance and accelerated the hyperpolarization (Fig. 8, component. Depolarisations from the shallow level will then arrow a). Opening of voltage-sensitive ion channels during the lack an after-depolarisation, since the active component is repolarising phase (Fig. 8, arrow b) induced the regenerative already activated. response responsible for the plateau potential. In the cell depicted in Fig. 7, hyperpolarising current On the basis of the known electrophysiological properties of injections from a shallow membrane potential induced a shift other ciliates, it is reasonable to suggest that the depolarisation of the membrane potential to a semi-stable deep level. In from the deep membrane potential to the plateau potential is normal medium, recordings were obtained from 94 cells, and due to Ca2+ influx through voltage-sensitive Ca2+ channels. To shifts between the shallow and deep membrane potential levels test this hypothesis, the Ca2+ in the recording solution was could be induced by depolarising or hyperpolarising current replaced with either Co2+, which blocks Ca2+ channels, or

0 -20

-40 (mV)

-60 m E Fig. 5. Current-induced, semi-stable -80 after-depolarisation. This cell had a resting membrane (Em) potential of 100 pA −60 mV, and successive responses to depolarising current injections of 200 ms increasing magnitude are displayed. 0 At a current intensity exceeding -20 70 pA, the after-depolarisation was -40 (mV) -60 m transformed to a prolonged plateau. E -80 The broken lines indicate a 15 s break in the recording. Bistable membrane potential of Coleps hirtus 761

Fig. 6. Recording showing a 0 spontaneous shift from the shallow -20 to the deep membrane potential

-40 (mV) (Em). The response to depolarising m current injection is shown at both -60 E membrane potential levels. An 500 pA -80 100 ms after-depolarisation is only evoked at the deep membrane potential. Note the similar time course of the spontaneous potential shift and the termination of the evoked after- depolarisation.

Mg2+, which cannot permeate the channels. In solutions mean value of the shifts between this deep level and the plateau containing Co2+, the resting membrane potential was potential was 52±12 mV. −57±5 mV (N=4), and in solutions containing Mg2+, the resting In other ciliates that exhibit graded Ca2+ spikes, such membrane potential was −50±4 mV (N=7). Potential responses regenerative responses may be transformed to all-or-nothing to depolarising current injections were passive, with no sign of action potentials in Ba2+-containing solution. We therefore after-depolarisations. Neither electrically induced plateau investigated whether Ba2+-dependent action potentials can also potentials nor spontaneous shifts to a shallow membrane be evoked in C. hirtus. Since the membrane potential in Ba2+- potential were observed. The complete elimination of plateau containing solution was stable at the shallow level, at which potentials and membrane potential shifts in these solutions the Ca2+ channels are activated, these experiments were supports the hypothesis that voltage-sensitive Ca2+ channels performed during continuous injection of hyperpolarising are responsible for the biphasic membrane properties of C. current. This treatment forced the membrane potential to stay hirtus. at a hyperpolarized level, at which the Ca2+ channels are The voltage-sensitive Ca2+ channels are permeable to Ba2+, closed. Brief depolarising current pulses superimposed on the which is also a general inhibitor of K+ channels. Furthermore, direct holding current then evoked all-or-nothing action while Ca2+-dependent inactivation of Ca2+ channels is an potentials with a prolonged plateau coinciding with the shallow important element in the termination of Ca2+-dependent potential (Fig. 11). depolarisations, Ba2+ does not inactivate Ca2+ channels. If Ca2+ channels were involved in the activation of the plateau potential, Direction of the ciliary beat and swimming behaviour this shallow membrane potential would be expected to dominate To determine the direction of the ciliary beat, intracellular in a recording solution in which Ca2+ is replaced with Ba2+. In recordings were performed after the addition of the hydrophobic such a solution, the membrane potential was −14±9 mV (N=27) dye Carmine Red to the solution. In the initial experiments, the and spontaneous shifts to a deeper potential level never occurred. membrane potential was monitored without injecting current, Fig. 9 presents the I/V relationship for a cell in Ba2+-containing and movements of the cilia were correlated with potential level. solution. Compared with the I/V relationship in Fig. 3, the At the deep membrane potential, the ciliary power stroke had a general blocking of K+ channels is evident from the elimination backward direction, corresponding to forward swimming of of both inward and outward rectification. unrestricted cells. When the membrane potential spontaneously The hypothesis described above predicts that the shallow shifted to the shallow level, the power stroke reversed to a membrane potential is due to a continuous inward Ba2+ current forward direction. Similar changes in the ciliary beat were through the Ca2+ channels and it should, therefore, be possible observed when the membrane potential was shifted between the to terminate the plateau potential with a brief injection of two semi-stable levels by current injections. It is reasonable to hyperpolarising current. Fig. 10 shows an example in which assume that the periods of forward swimming, during which the such a treatment shifted the membrane potential to a deep level animals may cover fairly large distances, are correlated with the for a few seconds, after which the cell spontaneously deep membrane potential level, whereas the circular movements depolarised to the stable plateau potential. Similar experiments are associated with the plateau potential. To clarify this question, were performed on 14 additional cells, and the mean value of swimming behaviour was studied in a solution in which Ca2+ the short-lasting, deep membrane potential following the was replaced with Mg2+. The plateau potentials are absent in this hyperpolarising current injection was −65±14 mV (N=15). The solution, and the membrane potential is stable at the deep level.

0

-20 -40 (mV)

Fig. 7. Recording showing a shift -60 m E from the shallow to the deep 200 pA -80 membrane potential (Em) induced by 100 ms hyperpolarising current injection. -100 762 P. RUDBERG AND O. SAND

A 0 -20 -40

b (mV)

-60 m E -80 -100

200 pA Fig. 8. Recordings showing the effects of 100 ms hyperpolarising current injection at the 0 deep (A) and shallow (B) membrane B -20 potential (Em) in the same cell. In both cases, a semi-stable plateau potential was -40 a induced at the termination of current b (mV)

-60 m

injection. Arrows indicate when voltage- E dependent ion channels were closed (a) and -80 opened (b). The broken lines indicate a 3 s -100 break in the recording.

In this solution, the animals only displayed forward swimming, and Didinium nasutum (2.8×104 Ω cm2; Pape and Machemer, without the characteristic, brief periods of circular swimming 1986), but considerably smaller than the specific membrane observed in normal medium. resistance of Stentor coeruleus (5.3×104 Ω cm2; Wood, 1982).

60 Discussion Passive electrical membrane properties and membrane rectification 40 The mean resting membrane potential (deep potential level) − of C. hirtus in normal recording solution is 52 mV. Although 20 the reported resting membrane potentials in most ciliates are between −30 mV and −40 mV, the value in C. hirtus is not exceptional. Other ciliates with rather negative resting membrane potentials are Bursaridium difficile (−45 mV; Berg -400 -200 200 400 600 and Sand, 1994), Stentor coeruleus (−49 mV; Wood, 1992), Current (pA) Stylonychia mytilus (−51 mV; Deitmer, 1981) and Didinium -20 − nasutum ( 53 mV; Pape and Machemer, 1986). 20 Ω The mean membrane resistance of C. hirtus is 386 M . At -40 0 the resting potential, the conductance of the ciliary membrane is -20 negligible compared with that of the soma membrane (Pape and -40 (mV) -60 m Machemer, 1986). The surface area of a barrel-shaped body with -60 E the dimensions of C. hirtus, 42 µm×29 µm, is approximately -80 -100 4.7×10−5 cm2. The specific resistance of the soma membrane is -80 therefore estimated to approximately 1.8×104 Ωcm2. This value 200 pA is comparable with the membrane resistance of Paramecium 100 ms × 4 Ω 2 caudatum (1.9 10 cm ; Machemer and Ogura, 1979), -100 Euplotes vannus (2.3×104 Ωcm2; Krüppel and Lueken, 1988)

Fig. 9. Current/voltage (I/V) relationship for a cell in a Ca2+-free -120 recording solution containing 1 mmol l−1 Ba2+. In this solution, the membrane potential (Em) was less than −30 mV, and spontaneous shifts to a deeper potential level never occurred. Data points show -140 the membrane potential 200 ms after the start of current injection. Note the absence of both inward and outward rectification. The inset shows a sample of superimposed, original recordings. -160 Membrane potential (mV) Bistable membrane potential of Coleps hirtus 763

0 -20 -40 (mV)

-60 m -80 E -100 Fig. 10. Recordings showing the current-induced shift from the shallow to the deep membrane 200 pA potential (E ) in a Ca2+-free m 200 ms recording solution containing 0 − 1 mmol l 1 Ba2+. In this solution, -20 spontaneous shifts to the shallow -40 membrane potential did not occur, (mV)

-60 m

and the current-induced shifts were E always short-lasting. The broken -80 lines indicate a 6 s break in the -100 recording.

The mean membrane time constant of C. hirtus is 48 ms, normal recording solution, spontaneous shifts between these corresponding to a membrane capacitance of 114 pF. The levels occur regularly, with the animals spending membrane time constant shows little variation among different approximately 80 % of the time at the deep level. Similar ciliate species, and previously reported values are in the range biphasic membrane properties in ciliates have not been 40Ð60 ms (Naitoh et al., 1972; Pape and Machemer, 1986; reported previously. The direction of the ciliary beat is Machemer and Deitmer, 1987; Berg and Sand, 1994). backwards at the deep potential, corresponding to forward C. hirtus showed both outward and inward membrane swimming, and the direction is reversed at the shallow rectification, in common with other ciliates. In Paramecium, potential. These findings may explain the swimming behaviour the outward rectification is mainly due to Ca2+-activated of C. hirtus, which displays irregular periods of linear forward conductances and is therefore secondary to Ca2+ influx through swimming interrupted by periods of circular movements within voltage-gated channels. The inward rectification in a small area. It is reasonable to suggest that favourable Paramecium is due to at least five distinct ion conductances environmental conditions, such as an abundance of food, may (Oertel et al., 1978; Richard et al., 1986; Saimi, 1986; promote the latter swimming pattern, but this hypothesis has Hennessey, 1987; Preston et al., 1990, 1992), some of which not been tested in behavioural experiments. are Ca2+-dependent and secondary to Ca2+ influx through The shallow membrane potential level was absent in Ca2+- channels activated by hyperpolarisation. The ion conductances free solution containing Mg2+ or Co2+, which do not permeate responsible for the rectification in C. hirtus were not the Ca2+ channels, and the animals spent 100 % of the time at investigated further in the present study. However, it is the shallow potential level in a solution in which Ca2+ had been interesting to note that Cs+, which inhibits most types of replaced with Ba2+. This ion may act as a charge carrier through inward rectification in metazoans (Castle et al., 1989), did not Ca2+ channels and is also a general inhibitor of K+ channels. block the inward rectification in C. hirtus. The graded, regenerative Ca2+-dependent depolarisations reported in other ciliates may thus be transformed to all-or- Bistable membrane potential and swimming behaviour nothing action potentials in the presence of Ba2+ (Paramecium We have shown that the membrane potential in C. hirtus has caudatum, Naitoh and Eckert, 1968; Stylonychia mytilus, de a bimodal distribution, with a deep level of approximately Peyer and Deitmer, 1980; Didinium nasutum, Pape and −50 mV and a shallow level of approximately −30 mV. In Machemer, 1986; Euplotes vannus, Krüppel and Leuken,

0 Fig. 11. All-or-nothing action potential in a Ca2+-free recording solution -20 containing 1 mmol l−1 Ba2+. The -40 membrane potential was kept at the deep (mV) level by continuous direct -60 m hyperpolarising current, and the action E potential was evoked by depolarising -80 current injection. Two recordings are 100 pA -100 superimposed to show the all-or-nothing 200 ms nature of the prolonged action potential. 764 P. RUDBERG AND O. SAND

1988), and a similar phenomenon was observed in C. hirtus. If Klaveness, D. (1984). Studies on the morphology, food selection and the membrane potential was kept at a hyperpolarised level in growth of two planktonic freshwater strains of Coleps sp. Protistol. Ba2+-containing solution by direct holding current, depolarising 20, 335Ð349. current injections evoked prolonged action potentials with Krüppel, T. and Lueken, W. (1988). Membrane excitability and distinct thresholds. membrane currents in the marine ciliate Euplotes vannus. Eur. J. On the basis of the electrophysiological data, we conclude Protistol. 24, 11Ð21. Lueken, W., Ricci, N. and Krüppel, T. (1966). Rhythmic that the deep membrane potential in C. hirtus corresponds to spontaneous depolarizations determine a slow-and-fast rhythm in the traditional resting potential, while the shallow potential walking of the marine hypotrich Euplotes vannus. Eur. J. Protistol. level is a plateau potential maintained by non-inactivating 32, 47Ð54. 2+ 2+ inward Ca current through voltage-sensitive Ca channels. Machemer, H. (1970). Korrelation zwischen Membrapotential und It is interesting to note that, although similar Fortbewegung bei Stylonychia (Hypotricha). Naturwissenschaften electrophysiological properties have not been observed in other 57, 398Ð399. ciliates, bistable membrane potentials have previously been Machemer, H. (1988). Elektrophysiology. In Paramecium (ed. H. D. reported in vertebrate motoneurones (Hounsgaard and Kiehn, Görtz), pp. 185–215. Berlin: Springer Verlag. 1989). In these neurones, serotonin induces a sustained, Ca2+- Machemer, H. and Deitmer, J. W. (1987). From structure to dependent plateau potential associated with a state of enhanced behavior: Stylonychia as a model system for cellular physiology. In excitability. The plateau potential is initiated by a combination Progress in Protistology (ed. J. O. Corliss and D. J. Patterson), pp. 213Ð230. Bristol: Biopress Ltd. of reduced K+ permeability and a non-inactivating Ca2+ Machemer, H. and Ogura, A. (1979). Ionic conductances of current. The primary effect of serotonin is probably inhibition membranes in ciliated and deciliated Paramecium. J. Physiol., 2+ + of Ca -dependent K channels. Lond. 296, 49Ð60. Machemer, H. and Sugino, K. (1989). Electrophysiological control We thank Dr Dag Klaveness for providing the cultures of of ciliary beating: a basis of motile behaviour in ciliated protozoa. Coleps hirtus and for valuable discussions. Comp. Biochem. Physiol. 94A, 365Ð374. Naitoh, Y. and Eckert, R. (1968). Electrical properties of Paramecium caudatum: all or none electrogenesis. Z. Vergl. References Physiol. 61, 453Ð472. Berg, T. O. and Sand, O. (1994). Spontaneous all-or-nothing action Naitoh, Y., Eckert, R. and Friedman, K. (1972). A regenerative potentials in the ciliate Bursaridium difficile. J. Euk. Microbiol. 41, calcium response in Paramecium. J. 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