Contraction in Stentor coeruleus induced by electric stimulation

Camp. Biochem. Physiol., 1971, Vol. 40A, pp. 639 to 648. Pergmwn Press. Printed in Great Britain

CONTRACTION

IN STENTOR COERULEUS ELECTRIC STIMULATION LIS ENGDAHL

Zoophysiological Laboratory B, August Copenhagen, 13 Universitetsparken, (Received

INDUCED

BY

NIELSEN Krogh Institute, 2100 Copenhagen

26 March

The University 0, Denmark

of

1971)

Abstract-l.

The contraction in Stentor coeruleus was studied by superposition-photomicrography. 2. The applied electric stimulation was by square-wave pulses of O-l-2.0 msec. 3. The shortening was completed in about 9 msec and was an all-or-none phenomenon. 4. The latency between stimulation and the onset of contraction was l-3 msec. 5. Summation of two subliminal stimuli was demonstrated.

INTRODUCTION

ELECTRICcurrent has long been known to induce a rapid change of Stentor from the extended trumpet-like shape into a spherical state, but the mechanism coupling the electric stimulation and the contraction is not known. Contraction in Stentor coeruleus has been induced electrically by either “induction currents” undefined in time and intensity (Roesle, 1903) or direct currents lasting at least 100 msec (Bannister & Tatchel, 1968; Jones et al., 1970), far exceeding the shortening time in the animal. The present study applied square-wave stimuli of 0.1-2.0 msec and the reactions in S. coeruleus were recorded by superposition-photomicrography. Threshold curves for the contraction were determined, and the time course of the shortening was estimated in relation to the latent period between stimulation and the onset of contraction. Finally, the summation of two subliminal stimuli was studied. The results of the present work are compared to experimental findings in nerve and muscle suggesting that a propagated electrical membrane disturbance may be part of the excitation mechanism in S. coeruleus. MATERIALS

AND

METHODS

Stentor coendeus was cultured in flat plastic boxes (28 cm x 30 cm base area) which Grains of wheat and rice together with contained about 1 1. of sterilized pond water. cultures of Colpidium colpodum and Paramecium caudatum were added as a food supply for Stentor. The culture was maintained by transferring 1 part into 4 parts of fresh medium every 3 weeks. Animals from S- to %weeks-old cultures were used for the experiments. 639

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Figure la shows a diagram of the experimental arrangement. The animals were transferred in their culture medium into the test-chamber. The test-chamber was 0.2 cm high and constructed to allow photomicrography in immediate relation to electric stimulation. The electrode chambers and the agar bridges contained the filtered culture medium.

l-l

a)

Camera

Microscope

l4-

Cliff. ompt. To CR0

I

LElettrode

for stimulation

Agarbridge

Control

Electrode

for registration

Testchambtir

II

ll b)

I ;

l-l L Flash

Flashes Stimulus

delay

FIG. la. Block diagram of the experimental arrangement. Specimens in the testchamber were photographed by electronic light flashes in immediate relation to the electric stimulation. The timer measured the time interval from the stimulus to the second light flash. (b) The time course of events showing one light flash before and one after the electric stimulation. A homogeneous electric field was induced by a square-wave stimulator (Ministim, DISA, Copenhagen) with the current delivered through a pair of platin~~platinum black-plate electrodes placed in troughs outside the test-chamber proper. The field strength was measured by two parallel platinum/platinum black-thread electrodes (O*lS mm in dia.) situated in the test-chamber 2 cm apart and perpendicular to the field. They were connected to the differential input amplifier of an oscilloscope (Tektronix 502 A). The rise time of the pulses was less than 0.05 msec. The camera was mounted upon a Zeiss microscope with planar objective (x l/N.A. 0.04) and a photo-objective ( x 8). The animals were photographed by a single flash from an electronic stroboscope (Strobotac, General Radio, flash duration 3 Psec). They were then electrically stimulated and, after a certain time delay, exposed to another single flash, superposing the second picture on the first. This series of events is pictured in Fig. lb. It was triggered by an eIectronic circuit designed for the purpose; the time delay from the first flash to the stimulus was about 20 msec and from the stimulus to the second flash 70

FIG.

2. Section

of a superposition-photomicro~r~ph showing the reaction in s. One animal was extended when exposed to the stimulation. light flash (1) and contracted when the second flash was delivered (1’); 2 denotes an animal which was also extended at the second light flash.

coeruleusto electric first

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CONTRACTION IN STENTOR COERULEUS

msec unless otherwise indicated. This last time interval exceeded the contraction time, but was considerably less than the elongation time of the animals (about 2 min). The stimulation was applied with intervals of 3 min, and the animals were submitted to no more than fifteen stimuli before being replaced. In each photomicrograph the reactions of about fifteen animals were recorded. To obtain a suthcient number of observations the stimulation at a given strength and duration was repeated at least once. RESULTS

Figure 2 shows a field from a typical superposition-photomicrograph. An animal that contracted in the time interval between the two flashes appears extended during the first flash (1) and contracted (1’) at the second flash; in both positions the animal is seen as a grey image. An animal that remained extended between the two flashes resulted in two superimposed exposures seen as a black image (2). This method was used to distinguish between contracted and noncontracted animals. The number of contracted animals were calculated as a percentage of the total number of animals. Figure 3 shows the result of ten experiments (with a total of 1720 animals) using five stimulus durations at different field strengths. After experiments at one field strength, the animals in the test-chamber were replaced with new animals

Stimulus duration: 0,l mscc b 0.2 ” x 0.5 u 0 l,o

1

2

3

+'

'+

4

Field strength [ V/cm]

FIG. 3. Relation of field strength and stimulus duration to the fraction of contracted animals in a population of S. coeruleus. Each curve represents a constant stimulus duration. The total number of animals was 1720.

from the same batch. Each of the five curves represents one stimulus duration. The figure shows that a greater fraction of the animals contracted when the field strength was increased at a constant stimulus duration. No animals contracted at zero field strength; thus the light flashes did not induce contraction in Stentor. The animals oriented themselves at random in the test-chamber. To examine whether the orientation influenced the threshold value, a number of photomicrographs were taken at a stimulus strength that would induce contraction in 50 per

LIs

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ENCDAHL

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cent of the animals, The animals were separated into three groups according to their orientation. Group 1 included animals having their long axes within a sector of + 30” parallel to the field and their oral end facing the anode. Group 2 included animals with their long axes within the same sector as in group 1, but their oral ends facing the cathode. Group 3 was made up of animals with their long axes within a sector of + 30” perpendicular to the field. Thus some animals were disregarded as belonging to any of the groups. The total number of animals were counted for calculation of the percentage of randomly oriented animals which contracted at the applied stimulus. The results of a typical experiment at constant stimulus intensity and duration are given in Table 1. It is seen that the largest percentage of contracted animals is TABLE ~-EXCITABILITY

Group 1 Group 2 Group 3 Randomly

oriented

IN S. coeruleus ACCORDINGTO ORIENTATION IN THE HOMOGENEOUS ELECTRIC FIELD-A TYPICAL EXPERIMENT No. of animals

No. of contracted animals

Fraction of animals that contracted (%)

42 44 65 216

34 6 33 116

81 14 51 54

Group 1: animals parallel to the field and oral ends towards the anode. Group 2 : animals parallel to the field and oral ends towards the cathode. Group 3 : animals perpendicular to the field. Stimulation throughout the experiment was 0.1 msec, 2.3 V/cm.

in group 1. Thus the animals are most sensitive to electric stimulation when oriented with their oral ends towards the anode. Experiments in which about 50 per cent of the animals contracted were used to examine whether the contraction in Stentor can be considered all or none. The length of the contracted animal (L,) was expressed as the percentage of the length of the extended animal (L,). The number of animals were counted and grouped in classes with the range of 5 per cent. Neither in this nor the following experiments was selection made according to orientation of the animals in the field. The histogram (Fig. 4) shows that there were two groups of animals, one group showing a change to about 30 per cent of the initial length and the other essentially no change. No animals were found between the two extremes. Thus the contraction in S. coeruleus as a result of electric stimulation seems to be an all-or-none phenomenon. The procedure was slightly modified to estimate the time course of contraction. The time delay for the second flash with respect to the stimulus (see Fig. lb) was varied from 1 to 10 msec, this time interval being measured by an electronic timer (Type TC6, Advance Electronics) with a time resolution better than 10 psec. The applied stimulus lasted for 0.1 msec and was strong enough to ensure that all

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animals would contract. The initial length of an animal was designated L, and the shorter length La. At each flash delay the mean percentage of La/L, was calculated. Figure 5 shows the mean length in Stentor, expressed as the percentage of the original length of the animals, plotted vs. the Aash delay. By examination of the material some animals showed signs of shortening at 1*5 msec after stimulation,

5b

LL L, 1 percent

1

FIG. 4. Distribution of relative lengths in S. coe~uEeusafter electric stimulation. The length of the animal after stimulation (L2) was calculated as the percentage of the initial length (L,). The total number of animals was 152. Stimulus O-1 msec, 2.3 V/cm.

and the total number of animals were in the process of shortening at 3.5 msec after stimulation. The whole process of shortening was completed within 9 msec. This represents the contraction time of the total population. The contraction in some

Flash delay fmsccl

FIG. 5. The time course of contraction in S. coen.&us. The ordinate represents the mean lengths of the animals at the second light Rash (La) as a percentage of their initial length (Li). Bars indicate* one standard deviation. The abscissa indicates the time delay from the electric stimulus to the second light flash. The total number of animals was 289. Stimulus 0.1 msec, 5 V/cm.

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animals may have been faster or slower as seen from the considerable spread around the individual points. The reactions in Stentor to two identical stimuli delivered with time intervals close to the latency time were determined. The stimuli were O-1 msec in duration and the interval between their starting points varied from 0.2 to 4-O msec. Each batch of animals was subjected to a full sequence of varying intervals and was then discarded. The field strength was kept constant throughout the experiments. Application of one light flash before, and one 70 msec after stimulation, resulted in the usual superposition-photomicrographs and the fraction of contracted animals was evaluated as formerly described.

+

.: 0

1;

1

1 Time

2 interval

3 I msec

1

I

FIG. 6. Effect of two stimuli on the fraction of contracted animals. Two stimuli (0.1 msec) were delivered with the time intervals shown on the abscissa (+ ). n , Indicates one stimulus of 0.2 msec and 0, one stimulus of O-1 msec. The field strength of the stimuli was 1.6 V/cm. The total number of animals was 892.

Figure 6 shows that the shorter the time interval between stimuli the larger was the fraction of the animals which contracted. Thus in a number of animals one stimulus “facilitated” for the next to induce contraction, provided that the two stimuli were delivered within a short time. The figure also shows that the fraction of animals which contracted after two stimuli of 0.1 msec delivered with a O-2msec interval was approximately the same as the fraction obtained, when the stimulation was given as a single pulse of O-2 msec. DISCUSSION

Earlier work on the excitability in protozoa has been based upon observations of single animals. The application of superposition-photomicrography allows the reactions of a relatively large number of animals to be studied simultaneously. The application of a homogeneous field across the test-chamber ensures that all animals will be submitted to comparable conditions. The all-or-none reaction in Stentor found by the present technique (Fig. 4) agrees with the description given by Roesle (1903) of a complete contraction

CONTRACTION IN STENTOR

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resulting from “induction currents”. In addition, Roesle reported a larger sensitivity of specimens having their oral ends close to the needle-shaped anode. Table 1 shows that also in a homogeneous electric field Stentor showed the lowest threshold value with the oral end facing the anode. In other contractile protozoa the sensitivity to electric current is also influenced by the position of the stimulating electrode. For example, Paramecium showed the lowest threshold value with their anterior ends towards the anode (Hisada, 1952) and Sp irostomum with their long axes parallel to the field (Jones et al., 1966). Differences in electrical excitability according to orientation may be due to local differences of the electrical properties of the cell membrane. Local variations in membrane resistancehave been found in Opalina by Naitoh (1958). Also, the peculiar shape of Stentor may influence the electric field in the cytoplasm during stimulation, even with uniform membrane characteristics. The contraction in Spirostomum was completed within 4 msec (Jones et al., 1966) and in I’mticeZZawithin 5 msec (Jones et al., 1967). The contraction time in Stentor was reported by the same authors slightly to exceed these values (Jones et al., 1970). All these figures were based upon individual events recorded by high-speed cinematography. In the present study the contraction time in the population of Stentor was found to be 9 msec and thus seems to agree with the order of magnitude, as determined by the former method. The latency between stimulationand onset of contraction in Sterttm was found to be between 1 and 3 msec in the present report. The latency time in Spirostomum was reported by Jones et al. (1966) to be up to 30 msec. Ettienne (1970) found values from 13 to 25 msec also in Spirostomum. The variance in latency time determined in the present work to those of other authors may be true differences in latency time in different protozoa or may tentativelybe caused by differences in the time course of the electric stimulation. Jones et aZ. (1966), in another work, mentioned a rise time of 24 msec of the applied current. This is a long rise time, as compared to the rise time of less than 0.05 msec in the present work, but insu.fIicientdata are given to extend the discussion of this point. The mechanism which couples the stimulation and the contraction in Stentor is not known. The present work has applied striation similar to that used in nerve and muscle physiology and thus allows a direct comparison between the excitation in Stentm and in nerve and muscle from higher animals. The following will show some similaritiesbetween Stentor and the well-known excitable systems. Figure 7 shows the relation between the field strength and duration of stimuli resulting in contraction of 50 per cent of the Stentors. The values of field strength are taken from Fig. 3, and can be considered threshold values for a certain fraction of the animals. The strength-duration curve for threshold stimuli in the nerve has a similar shape, being hyperbolic. This relationshipis understood when a certain amount of electric charge is needed to depolarize the membrane for initiatingan action potential (Katz, 1939).

La

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An interpretation of Fig. 7 along these lines would be that a certain amount of electric charge must be supplied to elicit the contraction in Stentor. In the nerve two subthreshold stimuli applied within a few msec after each other may excite the cell, provided that their sum equals or exceeds the threshold

I 2

1 Stimulus

duration

lmsecl

FIG. 7. Relation of strength to duration for stimuli giving contraction in 50 per cent of the specimens in a population of S. coeruleus. The data were obtained from the curves in Fig. 3.

value, and the sum of charge may be smaller the less the time interval is between the two stimuli. This phenomenon is known as subliminal summation (Katz, 1939). In Stetatur certain stimuli resulted in contraction of a fraction of the population but were of subthreshold value to the rest. When two such identical stimuli were applied in close time succession, the fraction of contracted animals increased; the more, the shorter the time interval (Fig. 6). This experiment showed that in Stentor a subliminal summation could also be found. The contraction of striated fast vertebrate muscle is an all-or-none phenomenon. This is due to the all-or-none type of action potential which elicits the contraction (Huxley & Taylor, 1958). The action potential precedes by a certain interval the onset of shortening in striated muscle (Abbott & Ritchie, 1951), giving a latency time between the stimulation and the onset of contraction. In Stentor the contraction was an all-or-none reaction. A short electric pulse of about one one-hundredth of the contraction time, and completed before the onset of contraction, could elicit an “~1-contraction” (Fig. 5). This suggests that the current flowing during stimulation cannot directly be responsible for the course of contraction. Stentor thus shows similarities to nerve and muscle in having subliminal summation, a hyperbolic shape of the strength-duration curve and an all-or-none contraction with a latency time after stimulation. On this basis a propagated

CONTRACTION IN STENTOR

electrical membrane

disturbance

COERULEUS

647

is suggested as part of the excitation process in

Ste?ztor.

Action potentials as seen in nerve and muscle have not been described in protozoa, as pointed out by Jahn (1966). Electrical activity in a broader sense has been demonstrated in association with mechanical activity in some protozoa. For example, in Paramecium a transient hyperpolarization was recorded by intracellular microelectrodes and observed in connexion with the spontaneous contraction of the animal (Kinosita et al., 1964). In Noctihca a spike of an all-or-none type was recorded in relation to the stroke of the tentacle (Hisida, 1957). This spike was also a hyperpolarization but the interpretation was complicated by the existence of the central vacuole in the organism (Jahn, 1966). Further suggestions about the membrane phenomenon in Stentor seem premature; only the higher sensitivity to anodal stimulation (Roesle, 1903, and the present work) and the reports of transient hyperpolarizations in other protozoa point to a possibility of a propagated hyperpolarization in Stentor. When considering an electrical membrane phenomenon as part of an excitationcontraction coupling in Stentor, the relatively low concentration of ions in the pond water should be taken into consideration. The isotonic extracellular medium bathing nerve and muscle allows for the propagation of an impulse in higher animals. The low ionic strength in the external medium of Stentor represents a special physiological problem so that an electrical membrane phenomenon in this organism may be different from that known from nerve and muscle. SUMMARY

The contraction in S. coeruleus as a result of electric stimulation has been studied. A new application of superposition-photomicrography allowed the reaction of numerous animals to be studied at the same time. The contraction was an all-or-none phenomenon completed in about 9 msec. The latency between stimulation and the start of contraction was l-3 msec. The summation of two subliminal stimuli was demonstrated. The excitation in Stentor is compared to that in nerve and muscle from higher animals. Due to the similar properties of the two systems a propagated electrical membrane disturbance is suggested as a part of the excitation mechanism in S. coeruleus. Acknowledgements-The original culture of S. coeruleus and the feeding cultures were kindly donated by Dr. Knud Max Moller, of the Carlsberg Laboratories, Copenhagen, Denmark. REFERENCES ABBOTTB. C. & RITCHIEJ. M. (19.51) The onset of shortening in striated muscle. r. Physiol., Lond. 113,336-345. BANNISTERL. H. & TATCHELLE. C. (1968) Contractility and the fibre systems of Stentor coeruleus. J. Cell Sci. 3, 295-308. ETTIENNEE. M. (1970) Control of contractility in Spirostomum by dissociated calcium ions. J. gen. Physiol. 56, 168-179.

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HISADAM. (19.52) Induction of contraction in Paramecium by electric current. Annotnes ~001. jap. 25,415-419. HISADAM. (1957) Membrane resting and action potentials from a protozoan, Noctiluca scintillans. J. cell. camp. Physiol. 50, 57-71. HUXLEY A. F. & TAYLOR R. E. (1958) Local activation of striated muscle fibres. J. Physiol., Lond. 144,426-441. JAHN T. L. (1966) Contraction of protoplasm-II. Theory: Anodal vs. cathodal in relation to calcium. J. cell. Physiol. 68, 135-148. JONESA. R., JAHN T. L. & FONSECAJ. R. (1966) Contraction of protoplasm-I. Cinematographic analysis of the anodally stimulated contraction of Spirostomum ambiguum. J_ cell. Physiol. 68, 127-134. JONESA. R., JAHNT. L. & FONSECAJ. R. (1967) Contraction of some peritrichs. J. Protozool. 14, suppl. 41. JONESA. R., JAHN T. L. & FONSECAJ. R. (1970) Contraction of protoplasm-III. Cinematographic analysis of the contraction of some heterotrichs. J. cell. Physiol. 75, l-8. KATZ B. (1939) EZectric Excitation of Nerve. Oxford University Press, Oxford. KINOSITAH., DRYL S. & NAITOH Y. (1964) Changes in the membrane potential and the responses to stimuli in Paramecium. J. Fat. Sci. Tokyo Univ. sec. IV. 10,291-301. NAITOHY. (1958) Direct current stimulation of Opalina with intracellular microelectrode. Annotnes 2001. jap. 31, 59-73. ROESLEE. (1903) Die Reaktion einiger Infusorien auf einzelne Induktionsschllge. 2. allg. Physiol. 2, 139-168. Key Word Index-Stentor

coeruleus ; electric stimulation ; contraction.