J. Exp. Biol. (1967), 47. 433-446With 3 plates and 7 text-figuraPrinted in Great Britain

BIOELECTRIC REGULATION OF TENTACLEMOVEMENT IN A DINOFLAGELLATE

ROGER ECKERT AND TAKAO SIBAOKA*

Department of Zoology, Syracuse University, Syracuse, New York, andMarine Biological Laboratory, Woods Hole, Massachusetts

(Received 19 April 1967)

INTRODUCTION

Studies of contractile responses and their control by electroionic mechanisms havebeen performed primarily on vertebrate muscle (Huxley & Taylor, 1958; Hodgkin &Horowicz, 1960; Sandow, Taylor & Preiser, 1965); however, mechanical responses corre-lated with bioelectric potentials also occur in a number of other cells in both the animaland plant (Sibaoka, 1966) kingdoms. The Protozoa, especially, include many examples.

Mechanical phenomena in the protozoa which have been examined by electro-physiological or ionic methods include the reversal of ciliary beat in Paramecium(Kamada, 1940; Kinosita, 1954; Naitoh, 1964, 1966), metachronal coordination inOpalina (Okajima, 1953; Naitoh, 1961), streaming in Amoeba (Bingley & Thompson,1962; Bingley, 1966), and stalk contraction in Vorticella and Carchesium (Hou &Briicke, 1931; Ueda, 1954; Sugi, 1959, i960). All unicellular forms which have thusfar lent themselves to electrophysiological methods exhibit some electrical and/orionic correlates with mechanical activity. Because of the phyletic and functionaldiversity of motile systems found in these organisms their investigation should con-tribute to broadly based concepts of the control and coordination of cell movement.

The dinoflagellate Noctiluca has two functionally separate excitable systems. One,evoked by stimulation, triggers luminescence (Eckert, 1965 a, b, 1966, 1967; Eckert &Reynolds, 1967), and the other, normally spontaneous, regulates movements of thefood-gathering tentacle (Hisada, 1957; unpublished observations of Watanabe andHagiwara mentioned by Bullock & Horridge, 1965; Eckert, 1965 c). The existence inone cell of two functionally distinct excitable systems for the regulation of qualita-tively different secondary responses is in itself of interest and will be considered inmore detail elsewhere.

In this paper attention is focused on some elementary properties of the tentacle-regulating potentials (TRPs), and the movements of the tentacle which occur inresponse to the potential pattern. Finally, brief evidence is presented for the structuralbasis of tentacle contractility, and for the essential role of calcium ions in tentaclecontraction. A preliminary report has appeared elsewhere (Eckert, 1965 c). The ac-companying paper (Sibaoka & Eckert, 1967) further explores the electrical propertiesof the TRPs and their site of origin.

• On leave of absence from Biological Institute, Faculty of Science, Tohoku University, Sendai,Japan.

434 ROGER ECKERT AND TAKAO SIBAOKA

MATERIAL AND METHODS

Experiments were performed on specimens from a culture of the luminescent formof iV. mtliaris collected in 1962 from the North Sea. A non-luminescent form, collectedfrom Puget Sound (Eckert & Findlay, 1962) was used to a limited extent for compari-son. Unless specified otherwise, all references to Noctiluca are to the luminescentform. Cultures were maintained in autoclaved natural sea water at 17-190 C. withillumination of about 50 foot-candles, and were fed regularly with the alga Dtmaliella.Specimens of Noctiluca ranged in diameter from 300 to 600 fi. They were typicallysubreniform in configuration with the tentacle emerging from the sulcus (Text-fig. 2).Most of the cell interior is occupied by an extensive notation vacuole filled with a clearfluid. A thin complex layer of cytoplasm occurs between the vacuole and the pellicle.The morphology, development, and taxonomy of Noctiluca are discussed by Kofoid& Swezy (1921) and Pratje (1921).

CRO trace A

CRO trace B

s.l. _LText-fig. 1. Experimental apparatus for simultaneous recording of tentacle movement, stimu-lating current and intravacuolar potential. Specimen was held under sea water to the end of asuction pipette. Phase contrast was employed to focus the image of the tentacle on continuouslymoving film in a kymograph camera. A second lens was used to project the image of a cathode-ray tube through the rear surface of clear-base recording film. Tentacle movement was photo-graphically stopped about three times per second by stroboscopic illumination. The movementof the film alone provided the time base for the CRT traces. A bridge circuit was used in con-junction with a unit-gain amplifier and a differential amplifier for simultaneous recording ofpotential and passage of current with the intravacuolar capillary micro-electrode. Recordingsobtained by this method are shown in Plate 3. b.b., Bridge balance; d.a., differential amplifier;s.l., strobe lamp;/./., front lens; r.L, rear lens.

Electrical recordings and photographic studies were carried out at temperatures of20-25° C. in natural or artificial sea water. Specimens were held to the end of a suctionpipette over the objective of an inverted-type phase contrast microscope (Eckert1965 a).

Vacuolar potentials were recorded with conventional 3 M-KCl-filled glass capillaryelectrodes of 15-50 MD resistance, and a neutralized capacity high-impedance d.c.

Bioelectric regulation of tentacle movement in a dinoflagellate 435

electrometer. A second internal electrode was used to pass polarizing current pulseswhen required, although in some experiments (PI. 3), a single polarizing-recordingelectrode was used in one arm of a Wheatstone bridge (Frank & Fuortes, 1956).Electrode tips were always positioned by visual control within the flotation vacuolewhich comprises the major cellular volume. It should be noted that the potentialswere recorded and currents passed between vacuole and sea water across two mem-branes, the plasmalemma and the vacuole-limiting membrane (Eckert, 1965 a, b).

Several minutes after insertion the electrode tip became engulfed by streamingcytoplasm, causing a drop in the recorded potential to about zero, and a great reduc-tion in the size of the action potentials. The experiment was then terminated, for thetip had become electrically isolated from the cell interior, presumably by the forma-tion of a continuum of the plasma membrane along the shank of the electrode and overthe interior of a miniature vacuole which formed around the tip. To delay these events,electrodes were inserted as far as 100 fi into the vacuole.

The relationships between potential changes and movements of the tentacle wereexamined by photographically superimposing the display of a cathode-ray tube upona time sequence of images of the tentacle (Text-fig. 1).

Ion substitution experiments were conducted with the specimen in a perfusionchamber of 0-5 ml. capacity. A valve permitted selection of either steadily flowingnormal sea water or experimental solutions.

A limited number of electron micrographs of the tentacle were obtained incidentallyin the course of another investigation on Noctiluca. Specimens were fixed with gluter-aldehyde, stained with lead acetate, and embedded in Maraglas.

RESULTS

Anatomical

The tentacle of Noctiluca is about 200 fi long, and has a cross-section of about6 x 10 /i. It has no structural or developmental relationships to flagella, but is, instead,a specialized evagination of the soma. A true flagellum of unknown function occursin the oral pouch.

Electron micrographs of the tentacle in transverse section show it to be slightlyconcave on the ventral surface and convex on the dorsal surface (Text-fig. 2). Likethe pellicle of the soma, that of the tentacle consists of a mosaic of convexities over-lying a layer of nearly homogeneous fine structure (PI. 1). Along the inner surface ofthe homogeneous layer (on the dorsal surface of the tentacle in our sections) are pro-files having the appearance of longitudinal microtubules with external diameters ofapproximately 200 A. The matrix surrounding the microtubules is continuous withthe cytoplasm of anastomosing strands which penetrate the lumen-like vacuole of thetentacle. A membrane limits the cytoplasm which is exposed to the vacuolar fluid.

Behaviour of the tentacle

As the tentacle waves about, small food organisms are caught in a layer of mucuscovering the end of the organelle. They are brushed off by a movement of the tentacle(in the flexed position, Text-fig. 2) through the sulcus and are ingested, via the cyto-pharynx, into food vacuoles. The coordinated movements of the tentacle serve no

436 ROGER ECKERT AND TAKAO SIBAOKA

effective locomotor function, translocation of the organism in the horizontal plane beingentirely passive while vertical position is influenced by changes in the density of thefluid in the flotation vacuole (Krogh, 1959).

The movements of the tentacle fall into two principal categories. The most preva-lent is an almost uniformly rhythmic series of spontaneous extensions and flexions inan approximately sagittal plane (PI. 2). Flexion is defined as the bending of the tentaclealong the proximal third of its length so as to bring the distal end into or close to theventral sulcua (Text-fig. 2, PI. zc, d), whereas extension involves an arching of the

Dorsal sideApical trough

Anterior

Sulcus

Sulcus

Dorsal(convex)

Ventral(concave)

Posterior

Cytoplasmic mass

Sulcus.

Extension

Flexion

Ventral side

Text-fig, a. Some gross features of Noctiiuca. Bending directions of tentacle areindicated at the right.

tentacle in the opposite direction, toward the apical trough (Text-fig. 2, PI. 2e-h).The duration of a spontaneous flexion-extension cycle (beginning of flexion to end ofextension) ranges from 4 to 12 sec.

The second type of movement is a tight coiling of the proximal half or more of thetentacle about its base, followed after several seconds by relaxation, This response,which only occura when the cell is mechanically or electrically stimulated so as toelicit the flash-triggering action potential (Eckert, 1965 a, b), will not be examinedhere.

The spontaneous recurring excursion cycles of the tentacle were examined by cine-matography in specimens oriented so that the tentacle moved principally in the planeof focus of the camera lens. Plate 2 shows a sequence of 8 frames taken at 1 sec.intervals from one complete flexion/extension cycle. The major bendings are seen tooccur in a region about one-quarter the total length of the tentacle distal from the base;however, the entire length of the tentacle exhibits some capacity for endogenousbending.

Recovery of the cell from electrode penetration

The electrical time constant and d.c. resistance of the cell (recorded across pellicle,plasmalemma, cytoplasm, and vacuolar membrane), and the vacuolar potential wereinitially all low after insertion of electrodes. This was presumably due to damage

Bioelectric regulation of tentacle movement in a dmoflagellate 437

caused by the insertion, which was invariably accompanied by dimpling of the pellicle,due to its mechanical resistance. There followed a period of 150-500 sec., duringwhich the d.c. resistance and the time constant gradually increased (Text-figs. 3, 6).The plateau value for specific resistance (assuming the cell to be a smooth sphere,and treating the two membranes and pellicle as a single membrane) was character-istically 1 x 10s ohm-cm.2. The effective resistance between vacuole and sea waterwas of the order of 12 x io6 ii. The membrane capacitance, calculated from the timeconstant and the 'specific' membrane resistance was approximately o ^ x i o ^ F . /cm.8.* The effective capacitance between vacuole and sea water was of the order of2-3 x io-8 F.

- 1 0 1-

•a

Io

1 - 2 0 -1

- 3 0

1

\

1 1

i I

I

i

- 16

- 12

- 8

100 200 300 400Time after electrode insertion (sec.)

500

Text-fig. 3. Recovery with time of vacuolar potential (upper plots) and membrane resistance(lower plots) of three representative specimens, a, b, and c, after injury produced by insertionof recording and polarizing electrodes into the vacuole. The end of each curve indicates the timeat which the first spontaneous TR spike occurred.

The gradual increase in cell resistance was accompanied by a parallel increase inthe negativity of the vacuole. A cell was considered to have' recovered' when no furtherincrease in resistance or time constant took place. The first negative-going spikealways appeared spontaneously toward the end of the recovery period when the vacu-olar potential had attained a level of —20 to - 3 0 mV. (Text-fig. 5 A, Cj).

Spontaneous tentacle-regulating potentials

Spontaneously recurring potential changes were routinely recorded from thenotation vacuole of the luminescent form of Noctiluca during movements of the tentacle.The evidence presented below indicates that these potential changes are involved

• Chang (i960) determined average specific resistance and capacitance in the nonluminescent formof Noctiluca of 1-4 x io1 ohm-cm.1 and 1-3 x io"1 F./cm.1, respectively. Assuming the measurementswere made on cells which had recovered from impalement, this indicates a difference of about 70-foldbetween the resistances of the luminescent and non-luminescent forms. This discrepancy is an orderof magnitude greater than the resistance increase which occur in the luminescent form during recoveryfollowing impalement.

ROGER ECKERT AND TAKAO SIBAOKA

in the control of tentacle movements, hence they are termed tentacle-regulatingpotentials (TRPs).

The spontaneous potentials exhibited several shapes. However, they all appearedto be variations of a basic form which was seen in about half the specimens examined.The basic TRP, shown diagrammatically in Text-fig. 4, recurred 5-15 times perminute, and exhibited a maximum d.c. level of —45 to — 60 mV., which persistedfor 1 or more seconds following the spike. This post-spike d.c. level usually underwentoscillatory potential changes of several millivolts. It was followed by a slow depolari-

Pre-spike1 positive wave

Post-spike —negative level

- IOC-Spike

-1 sec-J-Spike

Text-fig. 4. Diagram of the basic TRP wave form. The vacuolar potential alternates betweentwo levels, the pre- and post-spike d.c. levels. When the pre-spike level has drifted to — 20 to— 30 mV., the spike-like transient ensues, followed by the post-spike d.c. level which prevailsfor a time. The sequence has a form reminiscent of an inverted cardiac potential cycle. The firstspontaneous spike occurs as recovery from insertion of the electrode nears completion, andarises from a d.c. level similar to that of the pre-spike wave, as seen in Text-fig. 5 A and Ct.

zation to a level of —10 mV. or more, sometimes overshooting zero. The potentialthen showed a slow negative shift lasting one-half to several seconds, terminated at— 20 to —30 mV. by the spike. The cycle was then repeated.

In the basic TRP the vacuolar potential alternated between two levels, the quasi-stable post-spike level, and the less well defined and relatively more positive pre-spikelevel. Prior to the first spontaneous spike after penetration of the electrode there wasa long period during recovery in which the cell was electrically in the 'pre-spikestate'. Following the initial spike the vacuolar potential remained at the post-spikelevel except for a pre-spike wave of variable duration (Text-figs. 4, 6).

In a common variation of the basic TRP (observed in about half the specimens) thepost-spike level was either very short or absent, i.e. the pre-spike wave occurred im-mediately following the spike. A series of such TRPs is shown in Text-fig. 5 C1 to Ce.When the vacuolar negativity was especially high the frequency of the spikes some-times increased to 3 per sec. while the pre-spike waves became shorter in duration andlower in amplitude. When the vacuolar negativity grew still higher, the rapidly recurr-ing spikes damped out (Text-fig. 5 C6). Sometimes the wave form characteristic of themore slowly recurring basic TRP would reappear (Text-fig. 5 C6).

There were intermediates between the slowly and rapidly recurring wave forms.The post-spike level was sometimes maintained for a relatively short time, and termi-nated with a small negative wave (Text-fig. 5-D6) which occasionally was also ob-served on the slowly recurring wave form (Text-fig. 5 B, C6).

There was a positive correlation between the maximum positivity attained by thepre-spike wave and the maximum negativity attained by the subsequent spike (Text-

Bioelectric regulation of tentacle movement in a dinoflagellate 439

fig. 5 C2 to CB). The maximum rate of potential change during development of thenegative transient was 0-4-4-0 V./sec. (average, i-8 V./sec. from twenty measurementsof six specimens), with the peak of the spike reaching a level of — 75 mV. or more.

- 4 0

- 8 0 -

- 8 0 -

- 4 0

- 8 0 - :

- 4 0 -

-80 H

Text-fig. 5. Spontaneous TRPs. A, Basic wave form, showing the first spike, which appeared165 sec. after electrode impalement. B, Basic wave form with long-duration post-spike d.c.level. Pre-spike positive wave is of short duration and overshoots zero. Ci-C,, Rapidly recurringTRPs obtained from a specimen at 105, 115, 123, 125, 171 and 352 sec., respectively, afterelectrode impalement. Clt the first and second spikes after impalement, Ct, basic TRP after along post-spike negative level. Dlt Dt, Intermediate forms obtained from one specimen at 113and 427 sec. after impalement. The small irregularities toward the end of the trace in C, arethe first signs of cytoplasmic encapsulation of the electrode tip.

Exp. Biol. 47, 3

44° ROGER ECKERT AND TAKAO SIBAOKA

Tentacle regulating potentials evoked by current pulses

Before recovery from the electrode insertions had reached completion, the passageof constant current pulses in either the inward or outward direction resulted inexponential potential drops (Text-fig. 6A, B). When the vacuolar potential hadrecovered to about — 20 mV., the passage of outward current still produced onlypassive drops (Text-fig. 6 C, E), whereas inward current evoked the TR spike (Text-fig. 6D).

Text-fig. 6. TRP responses to outward and inward current pulses in one cell at several timesduring and after recovery of the cell from electrode insertion. Upper trace: polarizing currentmonitor; upward deflexion indicates outward current. Lower trace: potential changes recordedwith a second electrode in the vacuole. The d.c. levels in millivolt* negative immediately priorto application of the current pulses were 7 in A and B, 18 in C, 19 in D, 26 in E, 46 in F, and52 in G and H. In F and H, the spike which terminated the TRP occurred outside the frame.Polarizing current pulse was 100 msec, ata-o x io~° A., except C and D which were 1-5 x io~*A.

After the post-spike d.c. level had been established by a spontaneous spike, anadequate outward current pulse evoked a potential wave form similar to that of thebasic spontaneous TRP (Text-fig. 6E,G) except that the early shape of the pre-spike

Bioelectric regulation of tentacle movement in a dinqflagellate 441

wave was modified by the direct response to the current pulse.* The shape of thesubsequent pre-spike wave differed according to the degree of current-induceddisplacement (Text-fig. 7). At the lowest effective outward current levels the pre-spikewave continued to shift somewhat positively after the end of the current pulse, givingthe evoked potential a shape indistinguishable from the basic spontaneous TRP(Text-fig. 7 A). The TRP also developed upon the termination of an inward currentof nanoampere strength or greater in the post-spike state (Text-fig. 6H), but nospike was evoked directly as it was in the pre-spike state (Text fig 6D).

Text-fig. 7. The influence of stimulus current strength on the wave form of the TRP. Uppertrace: polarizing current monitor. Current duration ioo msec.: strength; A, 0-5; B, 1-5; C,i-8, and D, 2'O x io~* A. Lower trace: vacuolar potential. Potential levels immediately prior toeach current pulse were 48 in A, 44 in B, 45 in C, and 47 mV negative in D. With low-currentstimuli the evoked TRP is nearly indistinguishable from the spontaneous wave form. Withincreased current-induced displacements the repolarization slope of the pre-spike wavebecomes steeper.

Following recovery of the cell, inward or outward currents applied during the pre-spike wave had the same effect as during the maintained pre-spike d.c. level prior tothe first spontaneous spike (Text-fig. 6D). That is, outward current elicited onlypassive drops while an inward current could evoke a TR spike directly.

Correlation of tentacle movements with potential changes

In the absence of electrical activity (e.g. during recovery from electrode insertion)tentacle movements were normally absent. With the beginning of spontaneous TRPs,movements of the tentacle also appeared. Plate 3 demonstrates the temporal correla-tion between these movements and the basic TRP.

• Hisada (1957, fig. 6) shows an action potential following the termination of an outward currentpulse. Although potential and time scales are not indicated, both the appearance of the exponentialrise and the shape of the action potential indicate that the latter was the flash-triggering action potentialevoked in an unrecovered specimen by the break of a strong outward current. Comparisons may bemade with Text-figs. 6 and 7 of the present paper, and fig. 1 of the accompanying paper (Sibaoka &Eckert, 1967).

28-2

442 ROGER ECKERT AND TAKAO SIBAOKA

The positive going pre-spike wave was always followed by flexion, with a latencygenerally below i sec. from the beginning of the positive wave to beginning of flexion.Flexion was completed in another 2 or 3 sec, although this was often after the pre-spikewave had terminated in the spike. The flexed position was maintained for a variabletime, loosely related in duration to the duration of the positive wave. (Note especiallythe third and fourth strips of the lower panel, PI. 3.) Extension normally began 1 to 2sec. after the spike, and once completed, the extended or semi-extended position wasmaintained until the next pre-spike deflection. The rapid cyclic potential patterns(Text-fig. 5 C) were accompanied by minor, ill-defined motions of the tentacle. Thecontractile process is apparently too slow to follow these potential cycles.

Movements elicited by spontaneous potentials or by TRPs evoked by stimulatingcurrent of either polarity were indistinguishable from one another (PI. 3, upper paneland unpublished records). Stimulating currents too small to evoke the TRP were alsoineffective in eliciting tentacle movement (PI. 3, strips 3 and 4 in upper panel).

Specimens of the nonluminescent form of Noctiluca did not exhibit vacuolar poten-tial changes concomitant with tentacle movements, moreover, they exhibited neithervacuolar d.c. potentials of more than several millivolts nor current-evoked TRPs.Similar observations were made by Chang (i960). Infrequently, a specimen of theluminescent form also failed to exhibit TRPs with movements of the tentacle. Thereason for this is not known; but, whenever the basic TRP wave form was seen inrecordings such as those of PI. 3, there was a consistent temporal correlation betweenTRPs and movements of the tentacle.

Calcium requirement for tentacle movement

During the course of an investigation of the ionic requirements of the luminescentresponse of Noctiluca it was noted that tentacle movement ceased when the organismwas exposed to low-calcium (trace amounts only) artificial sea water. Upon exposureto the essentially Ca-free sea water the movements weakened, and within severalseconds they ceased with the tentacle in a position straight out from the soma. Duringthe period of Ca-free perfusion normal spontaneous TRPs continued. When thecalcium concentration was returned to normal tentacle movements were resumed.

DISCUSSION

There are a number of well-documented examples of excitable cells which havefunctionally distinct areas of membrane; it is nevertheless unusual for a cell to haveevolved two distinctly different regenerative potential wave forms. Except for theunorthodox polarity of the spike component, the tentacle-regulating potential haslittle in common with the action potential which triggers the flash (Eckert, 1965 a, b).The TRP wave form is slower, more complex, and more variable. The flash-triggeringpotential is elicited by either an inward current or at the break of an outward currentpulse at any stage during or after cellular recovery from impalement. The TRPcomplex, on the other hand, is initiated in the post-spike condition by outward currentor at the break of an inward current pulse. In the pre-spike condition only a portionof the TRP complex (spike) can be elicited. The flash-triggering potential occursonly in response to stimulation, while the TRP normally occurs spontaneously. The

Bioelectric regulation of tentacle movement in a dinoflagellate 443

evolution of the two forms of regenerative activity was undoubtedly influenced by re-quirements for controlling the two secondary responses, Light emission and contraction.

The vacuolar potential appears to have two quasi-stable states, the pre- and post-spike levels (Text-fig. 4), both of which are maintained for variable durations. As thepre-spike potential approaches a value of — 20 to — 30 mV. from the positive side thespike ensues and the potential temporarily remains in the more negative state (— 45 to— 60 mV.) until the potential, for an unknown reason, drifts upward again to the pre-spike level. Thus, the current-evoked response depends on the prevailing d.c. level(Text-fig. 6); when the vacuolar potential is at or near one of the two quasi-stablelevels only active transients toward the other level can be evoked.

The variety of recorded TRP wave forms can tentatively be ascribed to quantitativevariations in stability and interaction of pre- and post-spike d.c. levels. Since un-disturbed specimens floating in culture normally exhibit the well-defined tentaclemovements associated with the basic TRP, some of the variant potential patterns areassumed to include components arising in response to the experimental conditions.

There are differences between our interpretations and those of Hisada (1957). Heconcluded that the vacuolar fluid had a negligible potential with respect to the sea-water reference, and that the growth of the potential subsequent to impalement(compare his fig. 2 and our Text-fig. 3) was due to progressive covering of the elec-trode tip by cytoplasm, with the final level representing the potential difference be-tween cytoplasm and sea water. We consistently observed the growth of the negatived.c. potential while the electrode tip was exposed to the vacuolar fluid, and we ascribethe gradual rise in vacuolar potential to recovery of membrane resistances and/ordecrease in electrical shunting as the damage of electrode penetration was repaired.The transient negativity he recorded on penetrating the perivacuolar cytoplasm(Hisada, 1957, p. 66) was noted in our experiments as the flash-triggering action poten-tial evoked by the mechanical stimulus associated with electrode penetration.

It is curious that low cellular resistance, low vacuolar d.c. potential, and the absenceof recorded TRPs exist normally as a characteristic set of features in the non-lumi-nescent population of NoctUuca miUaris. If the TRPs recorded in the vacuole of theluminescent form are electrotonic potentials resulting from the spread of current fromactive membrane in or near the tentacle, a lower effective resistance in the somabetween vacuole and sea water (as found in the non-luminescent form) should resultin correspondingly lower TRP amplitudes recorded from the vacuole; however, theyshould still be of a detectable magnitude. Electrotonic potentials might be reduced toinsignificant levels if there were a large impedance to current flow into the vacuole.The following paper (Sibaoka & Eckert, 1967) examines the origin and some electricalproperties of the TRP in more detail.

The microtubules seen in electron micrographs of the tentacle have not been foundin the cytoplasm of the soma, and are therefore suspected of being associated withmovements of the tentacle. Their diameters, ranging from 175 to 210 A, are somewhatsmaller than those of ciliary fibrils (240 A., Gibbons & Grimstone, i960). As judgedfrom its posture in low-calcium sea water, the tentacle is unbent in its relaxed state.Bending should occur if microtubules along one side of the organ contracted. Acombination of differential contraction, turgor, and structural elasticity of the organcould probably produce the movements observed.

444 ROGER ECKERT AND TAKAO SEBAOKA

While the temporal relationship between TRPs and movements of the tentacle areobvious, details of the causal relationship remain obscure. It is reasonable to supposethat the potentials are closely related to the mechanism of tentacle movement, perhapsin a manner analogous to the control of contraction by the action potential in striatedmuscle fibres (Podolsky & Costantin, 1964).

SUMMARY

1. Recurring extensions and flexions of the food-gathering tentacle of Noctilucamxiiaris occur spontaneously. Identical movements can be evoked by appropriateelectrical stimulation.

2. Spontaneous recurring potential wave forms (TRPs) were recorded from thevacuole of the luminescent form of Noctiluca during movements of the tentacle.The basic TRP wave form consists of a characteristic negative-going spike whicharises at — 20 to — 30 mV. from the slowly redeveloping negativity of a pre-spikedepolarization, and is followed by a quasi-stable post-spike d.c. level of relativevacuolar negativity (—45 to — 60 mV.).

3. The TRP complex, similar in shape to that which occurs spontaneously, followsan intracellularly applied current pulse of either polarity if the vacuolar potential isat the post-spike level. The duration of the evoked pre-spike wave is related to thecurrent intensity and duration. During the pre-spike state outward current is ineffec-tive, although a TR spike occurs in response to inward current

4. The TRP is distinct in its behaviour and wave form from the flash-triggeringpotential, which can be evoked in the same cell, even though both exhibit all-or-nonespikes.

5. Simultaneous recordings of intracellular potentials and movements of the tentacleshowed a consistent temporal relationship between potential changes and subsequentmovement. Extension of the tentacle begins 1-2 sec. after the spike and flexion beginswithin 1 sec. after beginning of the pre-spike wave.

6. Tentacle movement ceased in Ca-free sea water even though the cyclic potentialchanges continued normally.

7. Electron micrographs of the tentacle showed longitudinal aggregations of micro-tubules near the outer surface of the peripheral cytoplasm. It is proposed thatcontraction of these microtubules is the immediate cause of tentacle movements.

We are grateful to the staff of the Biologische Anstalt, Helgoland, Germany, forproviding the initial specimens of our Noctiluca culture, and to Dr Luigi Provosoli ofHaskins Laboratories, New York City, for providing a culture of DunalieUa. The workwas facilitated by the excellent care given the cultures by Mrs Ikuko Sibaoka and MissFrancine Brady. Miss Constance Quinn performed the experiments showing the Ca-dependence of tentacle movement. We are also indebted to Dr DeF. Mellon for acritical reading of the manuscript. Support came from the National Science Founda-ton and the National Institutes of Health, U.S.D.H.E.W., and in part from a grantby the Office of Naval Research to the Marine Biological Laboratory.

Bioelectric regulation of tentacle movement in a dinoflagellate 445

REFERENCES

BINGLKT, M. S. (1966). Membrane potentials in Amoeba proteus. J. exp. Biol. 45, 251-68.BINGLBY, M. S. & THOMPSON, C. M. (196a). Bioelectric potentials in relation to movement in Amoebae.

J. Theorct. Biol. a, 16-32.BULLOCK, T. H. & HORRUKJE, G. A. (1965). Structure and Function in the Nervous System of Inverte-

brates, vol. 1, p. 436. San Francisco and London: Freeman.CHANO, J. J. (i960). Electrophysiological studies of a non-luminescent form of the dinoflagellate

Noctiluca mUiaris. J. cell. comp. Physiol. 56, 33-43.ECKKRT, R. (1065 a). Bioelectric control of bioluminescence in the dinoflagellate Noctiluca. I. Specific

nature of triggering events. Science 147, 1140-2.ECKKRT, R. (19656). Bioelectric control of bioluminescence in the dinoflagellate Noctiluca. II. Asyn-

chronous flash initiation by a propagated triggering potential. Science 147, 1143-5.ECKHRT, R. (1965c). Spontaneous potentials and tentacle movements in the dinoflagellate Noctihica.

Abstracts of Papers XXIII, Intern. Congr. Physiol. Sci.ECKERT, R. (1966). Excitation and luminescence in Noctihica. In Bioluminescence in Progress, pp. 269—

300. (Ed. Johnson and Haneda.) Princeton University Press.ECKERT, R. (1967). The wave form of luminescence emitted by Noctiluca. J. gen. Pkysiol. 50, 2211-37.ECKBRT, R. & FINDLAY, M. (1962). Two physiological varieties oiNoctituca miliaris. Biol. Bull. 133, 494-

5-ECKKRT, R. & REYNOLDS, G. T. (1967). The subcellular origin of luminescence in Noctiluca miliaris.

J. gen. Physiol. 50, 1429-58.FRANK, K. & FUORTES, M, G. F. (1956). Stimulation of spinal motoneurons with intracellular electrodes.

J. Physiol 134,451-70.GIBBONS, I. R. & GRTMSTONE, .A V. (i960). On flagellar structure in certain flagellates. J. biophysic.

biochem. Cytol. 7, 697.HISADA, M. (1957). Membrane resting and action potentials from a protozoan, Noctiluca sdntillans. J.

cell. comp. Physiol. 50, 57-71.HODGKTN, A. L. & HOROWICZ, P. (i960). The effect of sudden changes in ionic concentrations on the

membrane potential of single muscle fibres. J. Pkysiol. 153, 370-85.Hou, C. L. & BROCKE, E. T. (1931). Reizversuche an Vorticellen. PflUgers Arch. ges. Physiol. aa6,411-17.HUXLEY, A. F. & TAYLOR, R. E. (1958). Local activation of striated muscle fibres. J. Pkysiol. 144, 426-

4i-KAMADA, T. (1940). Ciliary reversal of Paramecium. Proc. Imp. Acad. (Japan), 16, 241-7.KrNOSlTA, H. (1954). Electric potentials and ciliary response in Opalina. J. Fac. Sci. Univ. Tokyo, Sect.

4, 7. J-I4-KOFOID, C. A. & SWEZY, O. (1921). The free-living unarmored Dinoflagellata. Mem. Univ. Calif. 5,

1-562.KROGH, A. (1959). Osmotic Regulation in Aquatic Animals. Cambridge University Press.NAITOH, Y. (1961). Local chemical stimulation of Opalina. I. The mode of action of K ion to induce

ciliary reversal. Zool. Mag. Tokyo, 70, 435-46.NAITOH, Y. (1964). Ciliary responses of Paramecium to the external application of various chemical*

under different ionic conditions. Zool. Mag. Tokyo, 73, 207-12.NAITOH, Y. (1966). Reversal response elicited in nonbeating cilia of Paramecium by membrane de-

polarization. Science 154, 660-2.OKAJIMA, A. (1953). Studies on the metachronal wave in Opalina. I. Electrical stimulation with the

micro-electrode. Jap. J. Zool. 11, 87-100.PODOLSKY, R. J. & COSTANTIN, L. L. (1964). Regulation by calcium of the contraction and relaxation

of muscle fibers. Fed. Proc. 23, 933.PRATJE, A. (1921). Noctiluca miliaris Suriray. Beitrfge zur Morphologie, Physiologie und Cytologie. I.

Morphologie und Physiologie. Arch. Protistenk. 42, 1—95.SANDOW, A., TAYLOR, S. R. & PREISER, H. (1965). Role of the action potential in excitation-contraction

coupling. Fed. Proc. 24, 1116-23.SIBAOKA, T. (1966). Action potentials in plant organs. Symp. Soc. exp. Biol. ax>, 49-73.SIBAOKA, T. & ECKERT, R. (1967). An electrophysiological study of the tentacle regulating potentials in

Noctiluca. J. exp. Biol. 47, 447-59.SUGI, H. (1959). Contraction and relaxation in the stalk muscle of Carchesium. Armot. Zool. Japon. 3a,

163-9.SUGI, H. (i960). Propagation of contraction in the stalk muscle of Carchesium. J. Fac. Sci. Univ. Tokyo,

Sect. 4, 8, 603-15.UBDA, K. (1954). Electric stimulation of the stalk muscle of Carchesium. II. Zool. Mag. Tokyo, 63, 9-14.

446 ROGER ECKERT AND TAKAO SIBAOKA

EXPLANATION OF PLATES

PLATE I

Structural features of the tentacle. A, Cross-section through dorsal surface of tentacle showing pellicle(p.), homogeneous layer (h.L), microtubular layer (nut.l.), mitochondria (nj.), and vacuole (u.). Thecytoplasmic strands which contain the mitochondria and other organelles anastomose in web-likefashion through the vacuole. A', An enlarged section of A to show details of microtubular layer.Diameters of tubules range from 175 to 210 A. The homogeneous layer is substantially (4 to 10 x)thicker in the tentacle than it is in the soma. Unless the fuzzy dense line at the lower edge of the homo-geneous layer is a poorly fixed membrane the membrane at the outer surface of the layer must be theplasmalemma. B, Another section showing similar features.

PLATE 2

A complete tentacle flexion/extension cycle. Frames were selected at 1 sec. intervals from a 16 mm.cinematographic strip. Flexion began between frames a and b, and was completed in d. Extensionoccupied the period from e to h. The initial 15-20% segment of the tentacle is securely anchored to thesoma, while the remaining portion of the proximal half is most active in bending. The dark irregularmass is the contents of a food vacuole. Fine cytoplasmic strands can be seen traversing the flotationvacuole in the soma. Phase contrast.

PLATE 3

Concurrent records of cellular potentials and tentacle position, obtained with the method of Text-fig. 1.Frames were exposed stroboscopically at approximately 3/sec. on continuously moving film. Traceswere photographed from the face of a cathode-ray tube, with the movement of the film providing thetime base. Upper trace in each strip records the potential of the vacuolar fluid, while the lower trace(along bottom edge of each strip) records stimulating current passed from the intravacuolar electrode.Upward deflexion of the current trace indicates positive current (= outward current) supplied by theintravacuolar electrode. Upward motion of the tentacle is flexion.

The potential changes and corresponding tentacle movements in the first strip in the upper panelare spontaneous. A second spontaneous cycle begins toward the end of the third strip. All other poten-tials and tentacle movements in upper and lower panels were evoked by outward current. Two sub-threshold current pulses are shown in the upper panel; these showed no effect on the position of thetentacle. Potential trace was adjusted slightly upward just past the middle of the fifth strip in the lowerpanel.

Journal of Experimental Biology, Vol. 47, No. 3 Plate 1CO

ROGER ECKERT AND TAKAO SIBAOKA (Facing p. 446)

Journal of Experimental Biology, Vol. 47, No. 3 Plate 2

ROGER ECKERT AND TAKAO SIBAOKA

Journal of Experimental Biology, Vol. 47, Vol. 3 Plate 3

ROGER ECKERT AND TAKAO SIBAOKA