Bioelectric Basis of Behavior in Protozoa. Department of Biology, University of California, Los Angeles, California 90024

Transcription

1 Bioelectric Basis of Behavior in Protozoa YUTAKA NAITOH Department of Biology, University of California, Los Angeles, California SYNOPSIS. Locomotor responses of ciliate protozoans to external stimuli primarily depend on changes in ciliary motion evoked by the stimuli. Certain regions of the protozoan cell jjiuuuce a iecepiur potential in response to stimulation. The receptor potential electrotronically spreads to the entire cell membrane and generates an overall electric response due to the electrical excitability of the general membrane. The ionic mechanisms for electrogensis are basically identical to those in nerves, muscles, and receptors of metazoan organisms. The ionic movements across the membrane associated with the electrogensis modify directly and/or indirectly the concentration of certain cations within the cell. The catonic concentration change brings about a modification of the contractile activity of cilia, which in turn results in a change in the ciliary movements. Cilia on different locations of the cell have intrinsically ionic concentrations. This fact, together with the morphological specialization of cilia in different locations on the cell, contributes to the complexity and adaptiveness of locomotor responses found in the ciliated protozoa. INTRODUCTION The locomotor behavior of many protozoans depends on the movement of their cilia or flagella. The cilia change their beating direction (direction of effective power stroke), beating frequency, and beating form spontaneously or in response to many kinds of external stimuli. These changes in ciliary motion are responsible for much of the locomotor behavior of these protozoans (Jennings, 1906). Since a protozoan is unicellular, detection of environmental changes and signaling of these changes to effector organs (cilia) to evoke an adaptive locomotor behavior of the organism must all be performed without benefit of the specialized cellular components or network of a nervous system. Our electrophysiological studies on protozoan cells (Naitoh and Eckert, 1974) have revealed that changes in ciliary motion are closely correlated with electrogenesis in the surface membrane. Certain regions of the membrane are functionally differentiated to receive external stimuli (receptor regions). At those regions stimuli are transduced into electric signals (receptor Suport for this work came from U.S. Public Health Service grant NS and National Science Foundation grant GB I thank Dr. R. Eckert for comments on the manuscript. 883 potentials). Depolarizing receptor potentials are amplified by regenerative conductance changes to evoke a large electric response over the entire surface membrane. The ionic bases for these electric responses are similar in principle to those found in nerves, muscles, and receptor cells of multicellular organisms. Activities of the cilia depend much on their cationic environment. Therefore, the movement of ions through the membrane associated with excitation influences the movements of the cilia and thus controls the behavior of the organism. Some locomotor behaviors other than those involving ciliary motion, such as contraction of the cell body in Paramecium (Kinosita et al., 1964), tentacle movement of Noctiluca (Eckert and Sibaoka, 1967), and protoplasmic streaming in Amoeba (Tasaki and Kamiya, 1964) are also found to be associated with membrane excitation. Bioluminescence of Noctiluca is controlled by an action potential which is triggered by mechanical stimulation of the cell body (Eckert and Sibaoka, 1968). The bioelectric control of behavior, therefore, seems to be a very wide-spread phenomenon in the Protozoa. This paper will deal with the mechanism by which bioelectric events in the membrane of the common ciliate Paramecium

2 884 YUTAKA NAITOH govern the locomotor responses of the specimen to mechanical stimulation. BEHAVIORAL RESPONSES OF Paramecium TO STIMULI Avoiding response In pond water or in culture medium, the beating direction of cilia of Paramecium caudatum is largely toward the posterior, so that the specimen swims forward. When the forward-swimming specimen bumps against a solid object (Fig. 1/4,1) it temporarily reverses the beating direction of cilia on the cell surface, so that the specimen swims backward for a short distance (Fig. 1/4,2). The cilia gradually resume their original normal (posteriorly pointing) direction, so that the backward swimming halts, and the specimen begins to swim forward again. Before resuming forward swimming, the specimen rotates around its posterior end due to strong beating of oral cilia (Fig. 1/4,3). Consequently, the direction of resumed forward swimming is different from the original direction (Fig. 1/4,4). Thus, the specimen, by one or more trials, can avoid the mechanical obstacle with which it FIG. 1. Behavorial responses of Paramecium. A, Avoiding response following collision with an obstacle. The specimen temporarily reverses the beating direction of its cilia to swim backward for a short distance, then resumes forward locomotion in a different direction, thus avoiding the obstacle. B, Escape response following entrapment in a narrow chink of debris in the culture. The cilia beat vigorously in forward swimming direction, allowing the specimen to escape from the chink, a, Anterior end of the specimen. e s :=: es -= FIG. 2. Responses of cilia to intracellularly applied electric current (AJ3) and to mechanical stimulation (C,D). An outward current through the membrane induces reversed beating of cilia on the whole cell surface (A), which is identical with the ciliary response evoked by mechanical stimulation of the anterior (a) membrane (C). An inward current through the membrane induces an increase in beating frequency of the cilia in the normal direction (B), which is identical with the ciliary response to mechanical stimulation of the posterior membrane (D). Small arrows across the membrane shown in A and B indicate the general direction of electric current, r, Glass rod for mechanical tap of the membrane. originally collided. Jennings (1906) called this response the "avoiding reaction." The tactic behaviors of ciliates depend primarily on this locomotor response. An avoiding response can be induced experimentally by touching the anterior region of the specimen with a fine glass needle (Fig. 2C). A strong touch induces strong and long-lasting backward swimming, while a weak touch induces only a brief halting of forward swimming. Close observation of ciliary motion upon the application of a mechanical stimulus revealed that the direction of effective power stroke

3 BIOELECTRIC BASIS OF BEHAVIOR IN PROTOZOA 885 of cilia changed clockwise to fully reversed direction with increasing intensity of the stimulus (Dryl and Grebecki, 1966; Machemer, 1972). Therefore, a weak stimulus induces only slight clockwise shift of the beating direction, which causes a slight reduction of forward momentum of the specimen. Escape response When a mechanical touch by a glass needle is applied to the posterior region of the specimen, all the cilia on the cell surface temporarily beat at a higher frequency than before the stimulus, but the beating direction does not change (Fig. 2D). This results in rapid forward swimming of the ciliate, facilitating its escape from the source of stimulus ("escape response"). The increase in beating frequency becomes more prominent with stronger stimuli. In cultures, the escape response is most readily observable when the specimen is trapped in a narrow channel within rather soft debris (Fig. 15). Mechanical stimulation of the posterior region of the specimen by the debris results in an increase in the beating frequency, resulting in rapid headlong progress through the channel until the specimen swims out of it. Under these conditions the mechanical stimulus provided to the anterior region does not induce an avoiding response, because the threshold for the avoiding response is higher than that for the escape response (Naitoh and Eckert, 1969). For the same reason, a light tap of the culture vessel induces a sudden increase in the forward-swimming velocity of all the specimens in the culture. 1 Responses to electric current It has long been known that when an electric current is applied to a medium in which paramecia are suspended, the specimens change their orientation so as to i Some ciliate protozoa, such as Urostyla sp., show an avoiding response with their culture vessel is lightly tapped (Naitoh, unpublished). In these species the mechanical sensitivity of the head region may be higher than that of the posterior region. FIG. 3. Responses of cilia to an electric current applied externally to Paramecium. 1, Forward-swimming specimen before electric stimulation. 2, Upon an application of electric current, the cilia nearest the cathode (-) reverse their beating direction, while the cilia nearest the anode ( + ) beat in the normal direction. The beating of cilia in opposite direction acts to rotate the specimen until its anterior end (a) points toward the cathode (3). swim toward the cathode. It was soon discovered that cilia on the cell surface near the cathode show reversed beating, whereas the cilia on the surface near the anode beat in the normal direction but with a higher frequency (Kamada, 1931). The opposite beating direction of cilia on the two sides of a single specimen apparently yields a torque which rotates the specimen until its anterior end points toward the cathode (Verworn, 1889; Jennings, 1906) (Fig. 3). An electric current enters the cell through the membrane near the anode and leaves the cell through the membrane near the cathode. Thus, it was concluded that an outward current through the membrane induces reversed beating, while an inward current evokes an increase in beating frequency of the cilia in normal direction. In the case of an external application of electric current, the distribution of the current density and direction across the mem-

4 886 YUTAKA NAITOH brane are complicated due to a non-linear change in the membrane resistance in response to the current (Naitoh and Eckert, 1968) and complexity of cell shape. An application of electric current to the cell membrane through a microelectrode inserted into the cell makes an analysis far simpler. The applied current produces an almost uniform change in the electric potential across the entire membrane (see later section) and causes a ciliary response of a given type depending on the polarity of the current. As a matter of fact, an application of sufficient outward current produces reversed beating of cilia on the entire membrane (Naitoh, 1958) (Fig. 2A). The direction of effective power stroke changes clockwise to fully reversed direction with increasing current intensity. The ciliary response to an outward current is quite similar to that evoked by mechanical stimulation of the anterior membrane (Fig. 2C). On the other hand, application of inward current through the membrane through the microelectrode induces an increase in the beating frequency of cilia on the entire cell membrane (Naitoh, 1958) (Fig. 25). The degree of the increase is larger when the current intensity is higher. The ciliary response to an inward current is similar to that evoked by mechanical stimulation of the posterior membrane (Fig. 2D). ANALYSIS OF THE BIOELECTRIC MECHANISMS Resti?ig membrane potential Paramecium has an internally negative resting potential as do nerve and muscle cells. This was first found by Kamada (1934) and confirmed later by others (Yamaguchi, 1960; Naitoh and Eckert, 1968). The magnitude of the negative potential decreases with increasing external concentration of various cations (K+, Rb+, Na+, Ca 2+, Mg 2+, etc.), although the degree of decrease is different for different cation species (Fig. 4). This indicates that the membrane shows relatively little specificity for any of the various cations tested. K+ and Rb+ are the most permeable among the cations tested, and Na+, Ca 2+, and Mg 2+ are less perme- (mv) IONIC CONC (mm) FIG. 4. Concentration effects of various cations on the resting membrane potential of Paramecium. The elfects of cations other than Ca 2t were all determined in I he presence of 1 ITIM Ca 2f. able. Therefore, according to Hodgkin and Horowicz (1959), the level of the membrane potential of Paramecium in a mixture of KC1 (4 mm) and CaCl 2 (IITIM) is presumably between the equilibrium potential for K + (E K ) and that for Ca 2+ (E Cn ), but closer to E K due to higher permeability of the resting membrane to K+. The internal K+ concentration of Paramecium is approximately 20 mm KC1 (Naitoh and Eckert, 1973), which corresponds to an E K value of about 40 mv. The Ca 2+ concentration of the cytoplasm of Paramecium appears to be less than 10-7 M (Naitoh and Kaneko, 1973). Accordingly, E Ca approximates +120 mv. The actual membrane potential measured in this mixture is about 20 mv. Based on these data together with the value of the resting conductance (4 x 10~ 8 mho; Eckert and Naitoh, 1970) the ratio of permeability of the membrane for K + to that for Ca 2+ can be calculated to be about 10. Response of the membrane to electric current When a small outward (depolarizing) electric current pulse is applied to Paramecium through an inserted microelec-

5 BIOELECTRIC BASIS OF BEHAVIOR IN PROTOZOA (mm) FIG. 5. A, Electric responses of the membrane (Vm) trie response. B, Concentration effects of Ca"* on the to outward current pulses of different intensities peak value of the response (Vp). C, Concentration (I). dvm/dt is the first-time derivative of the elec- elfects of K + on Vp. trode, the membrane behaves as an ohmic resistance in parallel with a capacitance. That is, the potential change due to the pulse exhibits a simple exponential time course (Fig. 5A,a). If the depolarization exceeds a certain level, the passive electric change is accompanied by a further depolarization which may continue to develop even after cessation of the pulse (Fig. 5A,b). This indicates the onset of an active membrane response by the depolarization. The peak value (Fig. 5A,Vp), as well as the maximum rate of rise (Fig. 5.<4,dVm/dt) of the active response increases to saturated levels with increasing intensities of the stimulus pulse (Fig. 5A,c). The ionic mechanism of the active response was investigated by examining the effects of cation concentrations on the saturated peak value (Naitoh et al., 1972). This value increases with a logarithmic increase in the external Ca 2+ concentration with a slope of about + 25 mv per tenfold increase in concentration (Fig. 5B). This approaches the predicted Nernst slope (29 mv) for a divalent cation specific electrode. These findings strongly suggest that a depolarization of the membrane produces an increase in the permeability of the membrane to Ca- + ions, which leads to an inward current carried by Ca 2+ down its electrochemical gradient in accordance with the ionic hypothesis of Hodgkin, Huxley, and Katz (Hodgkin, 1957). The inward Ca 2+ current, which is responsible for the upstroke of the active response can be demonstrated more directly with the voltage-clamp technique (Fig. 6). This also reveals that depolariza- Vrrv 40 mv 5X IO" 9 A 10 ms FIG. 6. Membrane current (Im) in the voltageclamped Paramecium. The intensity of the initial inward (Ca 2 *) current increases with increasing depolarization of the membrane (Vm). Delayed outward (K+) current markedly increases with the membrane depolarization over a certain level.

6 888 YUTAKA NAITOH tion leads to a delayed outward K + current across the membrane as in muscle and nerve cells. The K + current is responsible for the downstroke of the active response (Fig. 5A,c). The electric response of the membrane to an inward (hyperpolarizing) electric current is relatively passive. However, recent evidence indicates that in response to a hyperpolarization, Ca 2+ permeability of the membrane decreases, while K+ permeability gradually increases (Naitoh, unpublished). This gradual increase in K+ permeability might be a cause for a delayed anomalous rectification found in Paramecium upon an application of electric current (Naitoh and Eckert, 1968). Response of the membrane to mechanical stimulation A mechanical stimulus applied to the (mv) B anterior region of Paramecium evokes a transient depolarization of the membrane (Fig. 1A, upper part), while the same mechanical stimulus evokes a transient hyperpolarization of the membrane when it is applied to the posterior region (Naitoh and Eckert, 1969) (Fig. 1A, lower part). Both potential responses increase in amplitude to saturated values with increasing intensities of stimulation. In order to examine the electric conductance change during these potential responses, a train of small electric pulses was injected into the cell during the potential response (Fig. 8). The fact that superimposed deflections produced by the current pulses during the early phase of each potential response are smaller than those on the resting membrane potential indicates that the membrane conductance is higher during the early portion of the electric response to mechanical stimulation (Eckert Vpa?.. Vpp [K]=2mM - c - o Vpo tca] = I mm -60 FIG. 7. A, Electric responses of the membrane (Vm) to mechanicial stimulation of the anterior (a) (upper figures) and the posterior (lower figures) ends of Paramecium. Sm, Electric pulses applied to a piezoelectric phonocartridge (T) which drives a [Co] CK3 glass rod against the cell surface. B, Concentration effects of Ca 2+ on the peak values of the responses (Vpa and Vpp). C, Concentration effects of K + on Vpa and Vpp.

7 BIOELECTRIC BASIS OF BEHAVIOR IN PROTOZOA 889 Vm 100 mt FIG. 8. Conductance changes during the electric responses of the membrane to mechanical stimulation (Sm). A train of current pulses (Si) was injected into the cell to superinpose resulting IR drops on the responses. Membrane potential change (Vm) in response to anterior stimulation (A) and posterior stimulation (B). et al., 1972; Naitoh and Eckert, 1973). This means that mechanical stimulation makes the membrane more permeable to certain ions. The identity of the ionic species responsible for the membrane responses was determined by examining concentration effects of various cations on the saturated peak values of the responses. The peak value of posterior hyperpolarizing response (Fig. 7/4/Vpp). increases linearly with logarithmic increase in K+ concentration with a slope of about 50 mv (Fig. 7C), which approximates the value of 58 mv predicted from the Nernst K+-diffusion potential. When the membrane is kept hyperpolarized by inward current injection, the posterior response diminishes its amplitude and reverses its polarity (to depolarizing direction) if the level of hyperpolarization is beyond a certain potential. This reversal potential is consistent with the equilibrium potential for potassium, E K. This evidence indicates that mechanical stimulation of the posterior membrane produces a local increase in permeability to K + ions. Since E K is more negative than the resting potential level, K + current flows outward across the membrane, producing a hyperpolarization. The peak value of the anterior depolarizing response (Fig. 7/4,Vpa) increases linearly with a logarithmic increase in Ca 2+ concentration with a slope of 25 mv (Fig. IB), which approximates the 29 mv slope predicted for a Nernst Ca 2+ - diffusion potential. Examination of the depolarizing response to mechanical stimulation of the anterior end revealed that it consists of two components (Eckert et al., 1972). The first is a relatively slow depolarization, which is followed by the second, a faster spike-like depolarization (Fig. 1A). The rate of rise and amplitude of the early slow component also increases with increasing stimulus intensity. General features of the fast component as well as its Ca 2+ dependency are very similar to those of the Ca 2+ response evoked by a depolarizing current. The fast component, therefore, seems to be a regenerative Ca 2+ response evoked by the initial slow depolarization induced directly by mechanical stimulation. Although direct evidence is still lacking for the identity of the ionic species carrying the initial inward mechanoreceptor current, Ca 2+ is the most probable candidate. Cable properties The inside of Paramecium is virtually isopotential. This was demonstrated by the injection of an electric current into one end of the specimen while the potential response of the membrane was recorded separately from both ends of the specimen (Eckert and Naitoh, 1970). The two recorded potentials were compared with each other to determine the decay and the time delay of the response along the longitudinal axis of the specimen. The membrane responses recorded from both ends of the specimen are virtually identical in their magnitude and time course. This result is consistent with the relatively large value of space constant of Paramecium (1400 ;uxn; 5 times longer

8 890 YUTAKA NAITOH than the long axis), which can be calculated according to the standard cable equation of Hodgkin and Rushton (1946) by introducing the measured values of membrane resistance and cytoplasmic resistance. Because of the isopotential condition of the cell interior the mechanoreceptor potential evoked at one end of a paramecium spreads electronically to the rest of the cell membrane to evoke a distributed electric response by the entire membrane. CATIONIC CONTROL OF CILIARY MOTION IN DETERGENT-EXTRACTED CILIA Electron microscopic studies on cilia revealed that ciliary apparatus is tightly covered by a membrane which is continuous with the surface membrane of the cell body (Fawcett, 1961). It is, therefore, not unreasonable to suspect that ciliary activity may be influenced by the ionic fluxes which occur across the membrane during its electric activity. In this context it is important to know the effects of the ciliary apparatus of the ions involved in the membrane electric events. For this reason we examined the effects of various cations on the ATPreactivated cilia of detergent (Triton X- 100)-extracted models of Paramecium. Since the detergent destroys the diffusion-limiting properties of the membrane, the externally applied cations have direct access to the ciliary apparatus without membrane intervention (Naitoh and Kaneko, 1973). In a mixture of ATP and Mg 2+, models of Paramecium swim forward by their reactivated ciliary beating in normal direction as live specimens do in the absence of depolarizing stimuli (Fig. 9B). Ca 2+ ions are unnecessary for the reactivation of ciliary beating. The beating frequency and, therefore, the forward swimming velocity of the model depend on the concentrations of both ATP and Mg 2 +. The direction of the effective stroke gradually shifts clockwise to the fully reversed direction with increasing Ca 2+ concentration in the ATP-Mg 2+ mixture up to a calcium concentration of 5 x 10~ 5 M. A slight change in the beating direction of V ATP, Mg ATP.Mg.Co ATP, Co FIG. 9. Schematic illustrations of ciliary motion in detergent-extracted models of Paramecium. A, An extracted model in a reference (50 DIM KC\) solution. Cilia in this solution remain immobile. The position of the non-beating cilia approximates the end of the effective power stroke of beating cilia in normal direction. B, In a mixture of ATP and Mg 2 * all the cilia on the model are reactivated to beat metachronously in the normal direction. The model swims forward. C, The ATP-Mg 2+ reactivated cilia beat in reversed direction when Ca 2+ ions are added to the ATP-Mg 2 * mixture. The model swims backward. D, On application of Ca 2+ together with ATP (without Mg 2 *) the non-beating cilia swing once to point anteriorly, a, Anterior end of the model. cilia evoked by low Ca 2+ concentration results in only a slowing down of forward swimming velocity. Reversed beating of cilia evoked by higher Ca 2+ concentration makes the models swim backward (Fig. 9C). The effect of Ca 2+ ions on the motion of the reactivated cilia is similar to the effect of membrane depolarization on the motion of live cilia. If Ca 2+ ions are applied to the models together with ATP, but without Mg 2+ ions, ciliary beating does not occur, but the nonbeating cilia swing once clockwise so as to point anteriorly, the direction corresponding to the end of the effective power stroke of fully reversed beating cilia (Fig. 9D). These findings lead to the conclusion that the ciliary apparatus of Paramecium has two kinds of motile components: one is responsible for cyclic ciliary bending and requires Mg 2+ as a cofactor for its activation. The other, which is calcium dependent, governs the orientation of the cilium in its cyclic movements. Each of these components is believed to include an ATPase, activated by the corresponding divalent

9 BIOELECTRIC BASIS OF BEHAVIOR IN PROTOZOA 891 cation, responsible for the transduction of chemical into mechanical energy. COUPLING OF THE ELECTRIC RESPONSES IN THE MEMBRANE TO CILIARY ACTIVITY The ionic requirements for reversed beating of cilia in response to electric stimulation has lung been studied (Bancroft, 1906; Kinosita and Murakami, 1967). It was concluded that only Ca 2+ ions in the external solution are essential for the response. Other cations antagonize the effect of Ca 2+. Simultaneous recording of both electric and ciliary responses clearly demonstrated that reversed beating of cilia is initiated by depolarization only if the regenerative calcium response is evoked in the membrane (Machemer and Eckert, 1973). The degree of ciliary response is larger when the calcium response is greater. Based on these findings together with the fact that ATP-Mg reactivated model cilia beat in reversed direction in the presence of Ca 2+ ions, it is concluded that Ca 2+ ions carried across the membrane during the regenerative depolarization of the membrane might bring about an increase in the cytoplasmic Ca 2+ concentration, which activates the Ca-sensitive motile component in the ciliary apparatus. The activation of this component, in turn, results in a reversed beating of cilia (Eckert, 1972; Eckert and Naitoh, 1972). The mechanism by which hyperpolarization of the membrane activates the Mg 2+ - sensitive beating mechanism in ciliary apparatus to increase the beating frequency remains unknown. A tremendous increase in the beating frequency occurs in association with reversed beating of cilia evoked by calcium response in live specimens (Kinosita et al., 1965). Therefore, it might be conjectured that the beating frequency is also controlled by the Ca 2+ concentration. However, reversed beating of ATP-reactivated model cilia evoked by Ca 2+ ions is not associated with an increase in the beating frequency (Naitoh and Kaneko, 1972). This suggests that the increase in the beating frequency associated with the calcium response is not due to the direct action of Ca 2+ on the motile apparatus, but through an indirect mechanism which might be located in one of the detergent-extractable components of the cell. SUMMARY Although our experimental evidence does not yet provide a complete explanation of the control mechanisms underlying the behavioral response of Paramecium to mechanical stimulation, we can tentatively summarize (Fig. 10) the mechanisms based on the foregoing findings as follows: 1) Avoiding response: A mechanical stimulus to the anterior membrane evokes an increase in the permeability of that membrane to ion j+ (probably Ca 2+ ). j + ions move into the cell to make the membrane depolarized (a depolarizing mechanoreceptor potential). The depolarization spreads to the whole membrane due to the cable properties of the cell. The calcium conductance (GQ, 2 *) of the entire membrane increases in response to the depolarizing receptor potential. External calcium ions move into the cell. This makes the membrane more depolarized and more permeable to calcium ions. This results in a large regenerative depolarizing response. Ca 2+ inflow associated with the response increases the cytoplasmic Ca 2+ concentration ([Ca 2+ ]j). The calcium-sensitive mechanism for reversed beating of the cilia is activated by the increased [Ca 2+ ]i. The activation makes the cilia beat in the reversed direction and produces backward swimming of the specimen. The increase in cytoplasmic Ca 2+ concentration due to Ca 2+ inflow indirectly (through an unknown Mg- + -related mechanism) stimulates the beating mechanism of cilia. This results in a large increase in the frequency during reversed beating. The large depolarization of the membrane due to the regenerative increase in G 2+ Ca induces a delayed increase in the K+ conductance (G K +) of the membrane. K + ions move out of the cell according to their electrochemical gradient. The outflow of K+ ions acts to repolarize the membrane, turning off the regenerative calcium response. [Ca 2+ ]j is decreased by

10 892 YUTAKA NAITOH i Anterior membrane Gj* increased (possibly GQQ*) j + moved into the cell Mechanical stimulation I Depolarizing receptor potential Electrotonlcally spread General membrane depolarized Regenerative r-> 1 Co response = I Ca + Viewed into the cell I Further depolarization- CCa ++ l increased Reversal mechanism activated Beating direction reversed i Gcn'decreased Ca response terminated = Beating mechanism activated Beating frequency increased I Quick backward swimming II Avoiding response! Reversal mechanism deactivated I J Normal beating resumed I G K *increosed K + moved out of the cell - Membrane repolarized Beating mechanism deactivated I Forward swimming restored FIG. 10, Summary of mechanisms controlling loco- I Posterior membrane GR* increased K* moved out of the cell Hyperpolarizing receptor potential Electrotonically spread General membrane hyperpolarized Beating mechanism activated Beating frequency increased Forward swimming accelerated Escape response i G«+ decreased Resting potential resumed Beating mechanism deactivated Normal beating resumed Normal forward swimming restored motor behavior of Parmecium. pumping out and/or sequestering of Ca 2+, and reversed beating ceases. The cilia resume their normal beating direction, and forward swimming is restored. 2) Escape response: A mechanical stimulus applied to the posterior membrane evokes an increase in the permeability of that membrane to K+ ions. K + ions move out of the cell, thereby producing a hyperpolarization of the membrane (hyperpolarizing receptor potential) which electrotonically hyperpolarizes the entire cell membrane. The hyperpolarization activates the beating mechanism of cilia through an unknown Mg 2+ -related coupling mechanism. The activation of a beating mechanism results in an increase in the beating frequency of cilia. Thus, forward swimming is accelerated. The G K + increase of the membrane by a mechanical stimulus is

11 BIOELECTRIC BASIS OF BEHAVIOR IN PROTOZOA 893 temporary. The membrane potential soon resumes its resting level, and the accelerated forward movement of the specimen slows down to normal velocity. REFERENCES Bancroft, F. W On the influence of the relative concentration of calcium ions on the reversal of the polar effects of the galvanic current in Paramecium. J. Physiol. (London) 34: Dryl, S., and A. Grebecki Progress in the study of excitation and response in ciliates. Protoplasma 62: Eckert, R Bioelectric control of ciliary activity. Science 176: Eckert, R., and Y. Naitoh Passive electrical properties of Paramecium and problems of ciliary coordination. J. Gen. Physiol. 55: Eckert, R., and Y. Naitoh Bioelectric control of locomotion in the ciliates. J. Protozool. 19: Eckert, R., Y. Naitoh, and K. Friedman Sensory mechanisms in Paramecium. I. Two components of the electric response to mechanical stimulation of the anterior surface. J. Exp. Biol. 56: Eckert, R., and T. Sibaoka Bioelectric regulation of tentacle movement in a dinoflagellate. J. Exp. Biol. 47: Eckert, R., and T. Sibaoka The flash-triggering action potential of the luminescent dinoflagellate Noctiluca. J. Gen. Physiol. 52: Fawcett, D. W Cilia and flagella, p In J. Brachet and A. E. Mirsky [ed.], The cell. Vol. II. Academic Press, New York. Hodgkin, A. L Ionic movements and electrical activity in giant nerve fibres. Proc. Roy. Soc. London B 148:1-37. Hodgkin, A. L., and P. Horowicz The influence of potassium and chloride ions on the membrane potential of single muscle fibers. J. Physiol. (London) 148: Hodgkin, A. L., and W. A. H. Rushton The electrical constants of a crustacean nerve fibre. Proc. Roy. Soc. London B 133: Jennings, H. S Behavior of the lower organisms. Columbia Univ. Press, New York. Kamada, T Polar effect of electric current on the ciliary movements of Paramecium. J. Fac. Sci. Tokyo Univ. Sect. IV 2: Kamada, T Some observations on potential differences across the ectoplasma of Paramecium. J. Exp. Biol. 11: Kinosita, H., and A. Murakami Control of ciliary motion Physiol. Rev. 47: Kinosita, H., S. Dryl, and Y. Naitoh Change in the membrane potential and the responses to stimuli in Paramecium. J. Fac. Sci. Tokyo Univ. Sec. IV 10: Kinosita, H., A. Murakami and M. Yasuda Interval between membrane potential change and ciliary reversal in Parmecium immersed in Ba-Ca mixture. J. Fac. Sci. Tokyo Univ. Sect. IV 10: Machemer, H Ciliary activity and the origin of metachrony in Paramecium: effects of increased viscosity. J. Exp. Biol. 57: Machemer, H., and R. Eckert Electrophysiological control of reversed ciliary beating in Paramecium. J. Gen. Physiol. 61: Naitoh, Y Direct current stimulation of Opalina with intracellular microelectrode. Annot. Zool. Jap. 31: Naitoh, Y., and R. Eckert Electrical properties of Paramecium caudatum: modification by bound and free cations. Z. Vergal. Physiol. 61: Naitoh, Y., and R. Eckert Ionic mechanisms controlling behavioral responses of Paramecium to mechanical stimulation. Science 164: Naitoh, Y., and R. Eckert Sensory mechanism in Paramecium. II. Ionic basis of the hyperpolarizing mechano-receptor potential. J. Exp. Biol. 59: Naitoh, Y., and R. Eckert Control of ciliary activity in protozoa, p In M. Sleigh [ed.], Cilia and flagella. Academic Press, New York. Naitoh, Y., R. Eckert, and K. Friedman. 1972, A regenerative calcium response in Paramecium. J. Exp. Biol. 56: Naitoh, Y., and H. Kaneko Reactivated Triton-extracted models of Paramecium: Modification of ciliary movement by Ca ions. Science 176: Naitoh, Y., and H. Kaneko Control of ciliary activities by adenosinetriphosphate and divalent cations in Triton-extracted models of Paramecium caudatum. J. Exp. Biol. 58: Tasaki, I., and N. Kamiya A study of electrophysiological properties of carnivorous Amoebae. J. Cell. Comp. Physiol. 63: Verworn, M Die polare Erregung der Protisten durch den galvanischen Strom. Arch. Gesamte. Physiol. 46: Yamaguchi, T Studies on the mode of ionic behavior across the ectoplasmic membrane of Paramecium. I. Electric potential difference measured by the intracellular micro-electrode. J. Fac. Sci. Tokyo Univ. Sect. IV 8:

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