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Modulation of Hydra attenuata rhythmic activity
J. Photochem. Photobiol. B: Biol., 15 (1992) 307-315
Modulation of Hydra attenuata rhythmic activity VI. Combined effects of background and pulse light wavelength Cloe Taddei-Ferretti+,
V. Di Maio, C. Musio and A. Cotugno
Istituto di Cibernetica de1 CNR, via Toiano 6, I-80072 Arco Felice, Napoli (Italy) (Received
January
17, 1992, accepted
April 10, 1992)
Abstract Hydra is a photosensitive organism, although lacking identified photoreceptive organs. The behaviour of Hydra in undisturbed conditions is characterized by a repetitive body con-
traction-elongation activity. A transient alteration of the period length of such natural rhythmic activity is evoked by a light pulse stimulus and results in an immediate phase shift of the activity cycle. The sign and amplitude of such a photoresponse is a function of the light pulse intensity, application phase and wavelength. A persistent alteration in the period length of the rhythmic activity can be produced by a change in the background illumination wavelength. Experiments were performed to study the effects of modifications of the light wavelength of both the pulse and the background illumination. The results show that the persistent modification of the period length mentioned above does not occur isotropically along all phases of the cycle, giving an insight into the complexity of the mechanism responsible for the photoresponse.
Hydra, photoresponsiveness, periodic behaviour.
Keyworok
wavelength,
background-stimulus
interaction,
1. Introduction
The Coelenterate Hydra at rest is characterized by a behaviour consisting of an uninterrupted sequence of alternate body contractions and elongations, carried out by two antagonistic effector layers: this periodic behaviour results in a repetitive motor pattern sequence [l]. Three types of repetitive bioelectric event are correlated to the periodic body behaviour, in phase with it: (i) a big slow wave (a few millivolts, period length a few minutes, i.e. equal to that of the behavioural contraction-elongation sequence) [2]; (ii) a series of contraction pulses (a few tens of millivolts) [l, 31, organized as a contraction pulse train (about 30 s duration), arising as soon as the body contraction begins, lasting for the duration of such contractions and linked to a definite phase (with a definite trend) of the big slow wave; (iii) a series (a few minutes duration, i.e. the big slow wave duration minus the contraction pulse train duration) of rhythmic +Author to whom correspondence
loll-1344/92/$5.00
should be addressed.
0 1992 - Elsevier
Sequoia.
All rights reserved
308
Fig. 1. Example of phase advance of a contraction pulse train after a light stin~lus. A 10 s light pulse (indicated in the bottom trace) on a dark background, producing 3000 lx on a Hydra attenuafa, immediately interrupts a train in progress and anticipates the next one. Note the existence of a second pulse of the first train, which has not been recorded perfectly by the pen (AC recording). pulses (a few tenths of a millivolt) [4,5], arising at the beginning of the body elongation, lasting for the duration, and linked to the phase of the big slow wave, but exhibiting the opposite trend with respect to (ii). Although Hydra does not bear organized identified photoreceptive structures, it is photosensitive. The photosensitivity is revealed by a persistent variation in the length of the total contraction-elongation period evoked by a change in the background conditions, such as darkness, white light or wavelength variation of monochromatic light 16, 7j and also by a transient period length variation evoked by a light pulse stimulus, which results in an immediate positive or negative phase shift of the activity cycle to a new (delayed or advanced) phase (after which the amount of the phase alteration fades within a few - usually two - cycles and the previous behavioural period length is restored) (Fig. 1) [l, 3-6, 8, 91. Photosensitivity varies during the behavioural period in such a way that different phase response curves can be constructed for pulses of white light or darkness (i.e. pulses of opposite polarity) [6, lo]. For a fixed phase of stimulus application, the amount and polarity of the evoked phase shift depend on the stimulus duration, intensity, polarity [6], and wavelength [9]. Furthermore, all other conditions remaining unchanged, phase shifts of opposite directions (i.e. response times of different amounts) are obtained with short (450 run) and long (550-600 nm) wavelengths of the stimulus [9]. Combined effects of pulse light stimuli of different wavelengths, applied either simultaneously or in immediate sequence, have been reported [ll, 121. Carotenoids, taken in with food, have been found in epitheliomuscular cells of Hydra and their concentration depends on the illumination level [13]. Nevertheless, the molecular mechanism of Hydra photosensitivity remains unknown [14]. This paper reports an investigation into the possible interaction between the influence of the background light stimulation and the light pulse stimulation. 2. Materials
and methods
A Swiss strain of Hydra attenrrata was used. Tests were also performed using an African strain of H. attenuata and a strain of aposymbiotic H. magnipapillata, in order to compare the results with those obtained from the Swiss strain. The animals were
309
cultured in a solution described by Ham et al. [15], at 18 “C, under a circadian 600 Ix light-dark cycle. They were fed twice a week with Artemia salina nauplii, washed 2 h after the meal and used for experiments from the day after feeding. Before the start of an experiment, each animal was kept in total darkness for 1 h and the period length of the behavioural cycle was recorded. If the temporal distribution of the contraction pulse trains was so even that evaluation of the intervals between the trains was meaningless (Fig. 2(a)), the animal was not tested further. However, if the evaluation was meaningful (Fig. 2(b)), the animal was used in the experiment. All three types of bioelectric event correlated to periodic behaviour can be a useful monitor of the body behaviour itself. The first contraction pulse of a contraction pulse train was arbitrarily chosen by us as a recognizable point of the behavioural pattern appearing repetitively. A phase value of “0” was assigned at this reference point. The potentials were picked up using a polyethylene suction Ag/AgCl electrode, an Ag/AgCl indifferent electrode and a PAR 113 pre-amplifier and displayed on a Tektronix 5031 oscilloscope and a YEW 3047 pen recorder. The parameter measured was either the period length of the behavioural activity cycle in undisturbed conditions, or the elapsed response time between the end of an applied light pulse and the first contraction pulse of the next train. In the second case, two undisturbed contraction pulse trains were allowed to pass before the application of a further pulse. The light source was a Philips 6824, 12 volt, 100 watt lamp. The wavelength was varied by means of Balzers interference filters (5 cm diameter, 5 nm band, maximum transmittance at 400, 450, 500, 550, 600, 650 and 700 nm). An output of 7 X lo-’ J s-i cmw2, measured with a digital photometer Tektronix 516 with radiometric head 56502, was provided in all cases by using suitable neutral Balzers filters.
5
6
7
8
91011
8
910
._ IT :
f *
60-
40-
20-
0 @I
.liLIsj 1
2 TIME
3
4
5
INTERVALS
6
7
(min)
Fig. 2. Distribution of the time intervals between the beginning of successive contraction pulse trains. Two Hydra otienuatu were tested in continuous darkness. Duration of the experiment: top, 340 min; bottom, 432 min.
310
The different background conditions were D, W, 400, 450, 500, 550, 600, 650 or 700 and the pulse conditions were either D, W, 450 or 550 where: D= darkness; W =white light; 400=400 nm light; 450=450 nm light; 500=500 nm light; 550=550 nm light; 600 = 600 nm light; 650 = 650 nm light; 700 = 700 nm light. Each light pulse was always 7.5 s in duration, and administered immediately after the second contraction pulse of a train, i.e. 7.5 s ( f 2.5 s, depending on the reaction time of the operator) after the start of the first contraction pulse (always roughly at the same phase, notwithstanding the period length variability). The second contraction pulse was used instead of the first in order to avoid rare isolated single-contraction pulses, but it was necessary to apply the stimulus at an early phase, where the elicited effect of a D or W pulse is greatest [6].
3. Results A preliminary set of experiments was carried out in order to test, on a greater number of animals and also using new Hydra strains, the preliminary results of TaddeiFerretti et al. [7], which described the influence of background illumination wavelength on the period length of the behavioural cycle, using the background conditions described above. It was ascertained that the period length of the behavioural sequence in undisturbed conditions varies with the wavelength of the background illumination (Table 1). Longer periods are found with 450 background illumination and shorter ones with 600. The data obtained in uninterrupted darkness and in continuous white light illumination are reported for comparison: they confirm the earlier results of TaddeiFerretti and Cordella [6], showing that in darkness the period is shorter. A second set of experiments was then performed for each animal by testing the influence of the background illumination on the response evoked by a pulse stimulus, which always maintained the same characteristics (of intensity, duration, application phase and - for each single test series - wavelength) and briefly replaced the background conditions in a step-wise manner. The background conditions were chosen a D, W, 450, 550 and in two cases 600. The pulse conditions were D, W, 450 and 550. The 450 and 550 (or 600) conditions, rather than any of the other possible monochromatic wavelength conditions were chosen, (i) for the background during pulse stimulation, and (ii) for the pulse stimuli, for different reasons. In (i), these two conditions produce two peaks in the variation of the behavioural cycle period length (high persistent period increase/low persistent period increase respectively) with respect to the length produced with D, linked to the background wavelength change from 450 to 550 (or 600) [7]. In (ii), these conditions produce two peaks in response in opposite directions (phase delay, i.e. transient behavioural period increase/phase advance, i.e. transient behavioural period decrease respectively), linked to the same pulse wavelength variation, where stimulus intensity, duration, early application phase and D background remain unchanged [9]. Comparable results have also been obtained by Ellis [16], Singer et al. [17] and Tardent et al. [Ml, with different protocols. In each experiment with a single animal, the period lengths of the contraction-elongation sequence in undisturbed background conditions were checked for 30 min and the mean period length was calculated. The response time (i.e. between the end of each pulse stimulus and the first contraction pulse of the subsequent contraction pulse train) was then checked for all
311 TABLE 1 Influence of background illumination on the period length of the behaviour cycle. Mean length (s) of the behavioural period in different undisturbed conditions (indicated by the column headings); each row represents a single animal Strain
Background illumination D
W
315 242 135 121 165 120 111 122 115 150 77 115
277 283 195 288
Mean
149
246
H. attenuata (African strain)
276
739
Aposymbiotic H. magnipapillata
220 135 177
H. attenwta (Swiss strain)
Mean
400
109
450
500
550
600
6.50
228 184
150
71
165
157
700
337 187 160 232
604 540 315 451 442 465
454
319
270
199 230 82
187
362
204
161
157
315 330 346 283 337
334 357
286 295 295 130 159
158 194
234
182
193
391
345
233
176
234
182
109
449
types of stimuli. These checks were repeated four times for each type of stimulus which replaced each background, taking care to vary continuously the order of the successive presentation of different types of stimuli. The mean of the normalized response times (i.e. the mean of the ratios between each response time after a single pulse of one type and the previously calculated mean period length obtained in the undisturbed condition upon which the pulse was applied) was calculated for the combination of each type of pulse with the background used. This protocol was then repeated for the other undisturbed background conditions, taking care to vary the order of successive presentations of each type of background for different animals. The complete sequence of an experiment requires about 8 hours to test a single animal; however, only few animals remained in suitable condition for this length of time, and usually the experiment had to be stopped before the end or was planned with shorter stimulation protocols. Table 2 shows the ratios between the mean normalized response times obtained under specific conditions and the same parameter obtained by varying the background illumination only: response time depends on the wavelength of background illumination.
312 TABLE 2 Ratio between the mean of the normalized response times after a pulse stimulus of a definite type (on the line) applied on a background condition (subscript) and the mean of the normalized response times after the same stimulus applied on a different background condition. Each column represents one animal. The last 3 columns refer to aposymbiotic H. magnipupillata
D,/DW D450/Dw D55o/D450 w550mD w450mD w55o/w,,o
0.34 1.08 0.31
0.37 0.86 0.43
2.98 3.84 0.78
2.2 4.6 0.48
10 23.8 0.42
5 13.64 0.27
3.08 3.35 0.92 0.55
w600/w450
0.45
0.68
550450/550D
4.4 4.35
1.47 1.9
1.75 5.42
5.8
450,“/450o 4505,/450,
0.8 0.19
1.01 0.47
0.85 0.23
0.36
554yl559D
4. Discussion Previously published data show that: (i) for a stimulus of fixed wavelength (the stimulus intensity, duration and background conditions remaining unchanged), the response is a function of the stimulus application phase during the behavioural period [6]; (ii) for a stimulus of fixed application phase (the other conditions being unchanged), the response is a function of the stimulus wavelength [9]; (iii) the period length of the behavioural sequence in undisturbed conditions varies with the wavelength of background illumination [7] (confirmed by Table 1). In the first instance, according to the simplest hypothesis, because the length of the total period varies according to the background illumination wavelength, in undisturbed conditions this variation should occur isotropically along all phases. If this is the case, then for a stimulus of fixed application phase (as well as fixed intensity, duration and wavelength), the response should not vary with the background illumination wavelength variation (since the amount of response is expressed in normalized units, as a fraction of the total behavioural period length). However, present experiments, performed in order to test this hypothesis, showed the reverse to be true. For a stimulus of fixed application phase (as well as fixed intensity, duration and wavelength), the response varies with the wavelength of the background illumination on which the pulse light stimulus is applied. The data in Table 2 lead to the conclusions listed below. (i) Variations in response time occur when the background wavelength is changed. (ii) Despite individual differences, such variations always occur in the one direction, i.e. as an increase or a decrease (except in the 8th row of the 1st column compared with the 9th row) for each animal tested, when the background wavelength is changed in the same manner, and all the stimulus conditions remain unchanged. (iii) In the case of a D stimulus, response times are low for a 550 background, high for a 450 and intermediate for a W background (which should include chromatic components of the two previous ones). Thus, the 450 and 550 backgrounds seem to
313
have effects which are opposite with respect to the effect of W. This is similar to the results observed with the different experimental protocols reported above. (iv) In the case of a 550 stimulus, increased response times are obtained by using a D background, followed by a W, and finally a 450. Thus, the 450 background exhibits the same time-response-lengthening effect observed in (iii), while when a W background is used, one could consider that a lower intensity 450 background exerts the same effect upon a lower intensity 550 stimulus applied on a constant 550 background. (v) Conversely, in the case of a 450 stimulus, decreased response times are observed when using a D background, followed by a W and finally a 550. The same considerations as in (iv), but reversed, can be applied. (vi) In the case of a W stimulus, on the basis of all the previous observations and the fact that such a stimulus on a monochromatic background could be considered as a “complementary” flash on a constant background, one might expect that a lower/ higher response time would be obtained with a 550 (or 600)/450 background respectively, when compared with that obtained using a D background. However, higher response times are obtained with both 550 (or 600) and 450 backgrounds than with a D, although lower with 550 (or 600) than with 450. This last observation suggests that, as far as the W stimulus is concerned, the total of time-response-shortening monochromatic components is less than the total of lengthening components. The results reported here give an insight into the complexity of the mechanism responsible for the photoresponse. The fact that the response to short and long wavelengths differs not only in its amount but also in its direction [9], parallels the antagonistic effects of light of two different wavelengths in bacteria [19,20], invertebrates [21-241 and vertebrates [25]. This could be interpreted by suggesting that either two photosensitive pigments or two reciprocally reversible wavelength-dependent states of one photoconvertible pigment are involved in the phototransduction. The time-constant of the photochromic process would be very slow. The reported interaction between the background and the pulse wavelength effects could be supposed to intervene either directly at the photosensitive molecular level or further along the chain of effects culminating in the photoresponse. While the site of such interaction could be tested by the use of appropriate inhibitors interacting at several different places in the effect chain, the characterization of the photopigment would require [26], as a first stage, the microspectrophotometric analysis of cells which are candidates for the photoreceptive function [27]. The electrophysiological analysis of such cells under ditferent photic conditions could also be useful in order to identity the cells involved in the phototransduction. As a last consideration, it has been observed that, due to the experimental protocol used, the background illumination is suppressed when the light pulses are applied, which means that two transients are actually applied. The experiments could be repeated without switching off the background illumination when Hydra undergoes light flash stimulation. However, the necessity of halving the background illumination intensity and of supplying stimuli of halved intensity, in order to maintain constant illumination intensity, could introduce an unwanted variable. Acknowledgments
We are indebted to Professor A. Gierer (Max-Planck-Institut fiir Virusforschung, Tubingen) for the supply of the Swiss strain of H. attenuata, to Professor M. Rihat and Dr. W. Reich (The Hebrew University, Jerusalem) for constructive comments, and for the supply of aposymbiotic H. magnipapillata, and the African strain of H.
314
atienuata, to Dr. C. Ascoli and Dr. D. Petracchi (Istituto di Biofisica, CNR, Pisa) for useful suggestions and criticism, and to Mr. M. Briggs-Smith for the correction of the English manuscript.
References
1 L. M. Passano and C. B. McCullough, 2
3 4 5 6 7
8 9
10
11
12
13 14
15 16 17 18
19
Pacemaker hierarchies controlling the behaviour of hydras, Nature, 199 (1963) 1174-1175. C. Taddei-Ferretti, L. Cordella and S. Chillemi, Analysis of Hydru contraction behaviour, in G. 0. Mackie (ed.), CoeZentemte Ecology and Behavior, Plenum Press, New York, 1976, pp. 685-694. L. M. Passano and C. B. McCullough, Co-ordinating systems and behaviour in Hydra. I. Pacemaker system of the periodic contractions, J. Exp. Biol., 41 (1964) 643-664. L. M. Passano and C. B. McCullough, The light response and the rhythmic potentials in Hydra, Pmt. NatL Acud. Sci, USA., 48 (1962) 13761382. L. M. Passano and C. B. McCullough, Co-ordinating systems and behaviour in Hydra. II. The rhythmic potential system, J. @. Biol., 42 (1965) 205-231. C. Taddei-Ferretti and L. Cordella, Modulation of Hydra uttenuutu rhythmic activity. II. Phase response curve, J. Erp. Biol., 65 (1976) 737-751. C. Taddei-Ferretti, V. Di Maio, A. Cotugno and M. Durante, Photoresponses of symbiotic and aposymbiotic Hydra, Abstr. 8th Nutl. Congr. So&t& Ituliunu Biofisicu Puru e Applicuta, lkeggio, It&y, 1987, p. 62. C. Taddei-Ferretti and L. Cordella, Modulation of Hydra uttenuutu rhythmic activity: Photic stimulation, Arch. Ital. Biol., 113 (1975) 107-121. C. Taddei-Ferretti, V. Di Maio, S. Ferraro and A. Cotugno, Hydru photoresponses to different wavelengths, in R. H. Douglas, J. Moan and F. Daii’Acqua (eds.), Light in Biology and Medicine, VoZ. I, Plenum Press, New York, 1988, pp. 411-416. C. Taddei-Ferretti, L. Cordella and S. Chiiiemi, Hydra simple nervous system and behaviour, in R. Trappi, J. Klir and L. Ricciardi (eds.), Progress in Cybernetics und Systems Research, Vol. III, Hemisphere, Washington, DC, 1978, pp. 643-646. C. Taddei-Ferretti and A. Cotugno, Interaction between the effects of light pulses of different chromatic content on Hydra uttenuutu and H. mugntIxzpillutu, Hydrobiologiu, 2161217 (1991) 589-593. C. Taddei-Ferretti, C. Musio and A. Cotugno, Different weights of the influence of different light pulse position inside a stimulation in Hydru, in C. Fred&i (ed.), Cybernetics and Biophysics Ituliun Conference, SIF: Conference Proceedings, Vol. 31, Compositori, Bologna, Italy, 1991, pp. 123-128. M. Frigg, Vorkommen und Bedeutung der Carotinoide bei Hydra, Z. Vet@. PhysioI., 69 (1970) 186-224. C. Taddei-Ferretti, Transduction of photic information by a microorganism (Hydru attenuutu) lacking photoreceptive organs, Abstr. XIII Ahuron Kc&-Katchals~ Conference on Sensing and Response in Microorgunisms, Ayelet Hasbahar, Israel, 1985, D4. R. G. Ham, D. C. Fitzgerald, Jr. and R. E. Eakin, Effect of lithium ion on regeneration of Hydra in a chemically defined environment, _r. @. Zool., 133 (1956) 559-572. V. L. Ellis, The spectral sensitivity of Hydra cumeu L. Agassiz (1850), L Zool., 48 (1970) 63-68. R. H. Singer, N. B. Rushforth and A. L. Bumet, Photodynamic action of light in Hydra, I. I%p. Zoo& 154 (1963) 169-173. P. Tardent, E. Frei and M. Bomer, The reaction of Hydru uttenuata Pail. to various photic stimuli, in G. 0. Mackie (ed.), Coelenterute Ecology and Behavior, Plenum Press, New York, 1976, pp. 671-683. E. Hildebrand and N. Dencher, Two photosystems controlling behaviourai responses of Hulobucterium hulobinm, Nature, 257 (1975) 46-48.
315 20 J. L. Spudich and W. Stoeckenius, Photosensory and chemosensory behavior of Halobacterium halobium, Photobiochem. Photobiophys., 1 (1979) 43-53. 21 R. Menzel, Spectral sensitivity and color vision in invertebrates, in H. Autrum (ed.), Handbook of Sensory Physiology, MZ/6 Vision in Invertebrates, A. Invertebrate photoreceptors, Springer, Berlin, 1979, pp. 503-580. 22 B. Minke, S. Ho&stein and P. HiIlmann, Antagonist process as source of visible-light suppression of afterpotential in Limulus UV photoreceptors, .I. Gen. Physiol., 62 (1972) 787-791. 23 J. Nolte and J. E. Brown, Ultraviolet-induced sensitivity to visible light in ultraviolet receptors in Limulus, J. Gen. Physiol., 59 (1972) 186-200. 24 K. Ohtsu, UV-visible antagonism in extraocular photosensitive neurons of the anthomedusa, Spirocodon saltatrix (Tilesius), J. NeurobioL, 14 (1983) 145-155. 25 W. D. Eldred and J. Nolte, Pineal photoreceptors: evidence for a vertebrate visual pigment with two physiologically active states, VCon Res., 18 (1978) 29-32. 26 G. Golombetti and F. Lenci, Identification and spectroscopic characterization of photoreceptor pigment, in F. Lenci and G. Colombetti (eds.), Photoreception and sensov transduction in aneural o~anisms, Plenum Press, New York, 1980, pp. 173-188. 27 P. Gualtieri, L. Barsanti and V. Passarelli, Absorption spectrum of a single isolated paraflagellar swelling of Euglena gracilis, Biochim Biophys. Acta, 993 (1989) 293-296.
Modulation of Hydra attenuata rhythmic activity VI. Combined effects of background and pulse light wavelength Cloe Taddei-Ferretti+,
V. Di Maio, C. Musio and A. Cotugno
Istituto di Cibernetica de1 CNR, via Toiano 6, I-80072 Arco Felice, Napoli (Italy) (Received
January
17, 1992, accepted
April 10, 1992)
Abstract Hydra is a photosensitive organism, although lacking identified photoreceptive organs. The behaviour of Hydra in undisturbed conditions is characterized by a repetitive body con-
traction-elongation activity. A transient alteration of the period length of such natural rhythmic activity is evoked by a light pulse stimulus and results in an immediate phase shift of the activity cycle. The sign and amplitude of such a photoresponse is a function of the light pulse intensity, application phase and wavelength. A persistent alteration in the period length of the rhythmic activity can be produced by a change in the background illumination wavelength. Experiments were performed to study the effects of modifications of the light wavelength of both the pulse and the background illumination. The results show that the persistent modification of the period length mentioned above does not occur isotropically along all phases of the cycle, giving an insight into the complexity of the mechanism responsible for the photoresponse.
Hydra, photoresponsiveness, periodic behaviour.
Keyworok
wavelength,
background-stimulus
interaction,
1. Introduction
The Coelenterate Hydra at rest is characterized by a behaviour consisting of an uninterrupted sequence of alternate body contractions and elongations, carried out by two antagonistic effector layers: this periodic behaviour results in a repetitive motor pattern sequence [l]. Three types of repetitive bioelectric event are correlated to the periodic body behaviour, in phase with it: (i) a big slow wave (a few millivolts, period length a few minutes, i.e. equal to that of the behavioural contraction-elongation sequence) [2]; (ii) a series of contraction pulses (a few tens of millivolts) [l, 31, organized as a contraction pulse train (about 30 s duration), arising as soon as the body contraction begins, lasting for the duration of such contractions and linked to a definite phase (with a definite trend) of the big slow wave; (iii) a series (a few minutes duration, i.e. the big slow wave duration minus the contraction pulse train duration) of rhythmic +Author to whom correspondence
loll-1344/92/$5.00
should be addressed.
0 1992 - Elsevier
Sequoia.
All rights reserved
308
Fig. 1. Example of phase advance of a contraction pulse train after a light stin~lus. A 10 s light pulse (indicated in the bottom trace) on a dark background, producing 3000 lx on a Hydra attenuafa, immediately interrupts a train in progress and anticipates the next one. Note the existence of a second pulse of the first train, which has not been recorded perfectly by the pen (AC recording). pulses (a few tenths of a millivolt) [4,5], arising at the beginning of the body elongation, lasting for the duration, and linked to the phase of the big slow wave, but exhibiting the opposite trend with respect to (ii). Although Hydra does not bear organized identified photoreceptive structures, it is photosensitive. The photosensitivity is revealed by a persistent variation in the length of the total contraction-elongation period evoked by a change in the background conditions, such as darkness, white light or wavelength variation of monochromatic light 16, 7j and also by a transient period length variation evoked by a light pulse stimulus, which results in an immediate positive or negative phase shift of the activity cycle to a new (delayed or advanced) phase (after which the amount of the phase alteration fades within a few - usually two - cycles and the previous behavioural period length is restored) (Fig. 1) [l, 3-6, 8, 91. Photosensitivity varies during the behavioural period in such a way that different phase response curves can be constructed for pulses of white light or darkness (i.e. pulses of opposite polarity) [6, lo]. For a fixed phase of stimulus application, the amount and polarity of the evoked phase shift depend on the stimulus duration, intensity, polarity [6], and wavelength [9]. Furthermore, all other conditions remaining unchanged, phase shifts of opposite directions (i.e. response times of different amounts) are obtained with short (450 run) and long (550-600 nm) wavelengths of the stimulus [9]. Combined effects of pulse light stimuli of different wavelengths, applied either simultaneously or in immediate sequence, have been reported [ll, 121. Carotenoids, taken in with food, have been found in epitheliomuscular cells of Hydra and their concentration depends on the illumination level [13]. Nevertheless, the molecular mechanism of Hydra photosensitivity remains unknown [14]. This paper reports an investigation into the possible interaction between the influence of the background light stimulation and the light pulse stimulation. 2. Materials
and methods
A Swiss strain of Hydra attenrrata was used. Tests were also performed using an African strain of H. attenuata and a strain of aposymbiotic H. magnipapillata, in order to compare the results with those obtained from the Swiss strain. The animals were
309
cultured in a solution described by Ham et al. [15], at 18 “C, under a circadian 600 Ix light-dark cycle. They were fed twice a week with Artemia salina nauplii, washed 2 h after the meal and used for experiments from the day after feeding. Before the start of an experiment, each animal was kept in total darkness for 1 h and the period length of the behavioural cycle was recorded. If the temporal distribution of the contraction pulse trains was so even that evaluation of the intervals between the trains was meaningless (Fig. 2(a)), the animal was not tested further. However, if the evaluation was meaningful (Fig. 2(b)), the animal was used in the experiment. All three types of bioelectric event correlated to periodic behaviour can be a useful monitor of the body behaviour itself. The first contraction pulse of a contraction pulse train was arbitrarily chosen by us as a recognizable point of the behavioural pattern appearing repetitively. A phase value of “0” was assigned at this reference point. The potentials were picked up using a polyethylene suction Ag/AgCl electrode, an Ag/AgCl indifferent electrode and a PAR 113 pre-amplifier and displayed on a Tektronix 5031 oscilloscope and a YEW 3047 pen recorder. The parameter measured was either the period length of the behavioural activity cycle in undisturbed conditions, or the elapsed response time between the end of an applied light pulse and the first contraction pulse of the next train. In the second case, two undisturbed contraction pulse trains were allowed to pass before the application of a further pulse. The light source was a Philips 6824, 12 volt, 100 watt lamp. The wavelength was varied by means of Balzers interference filters (5 cm diameter, 5 nm band, maximum transmittance at 400, 450, 500, 550, 600, 650 and 700 nm). An output of 7 X lo-’ J s-i cmw2, measured with a digital photometer Tektronix 516 with radiometric head 56502, was provided in all cases by using suitable neutral Balzers filters.
5
6
7
8
91011
8
910
._ IT :
f *
60-
40-
20-
0 @I
.liLIsj 1
2 TIME
3
4
5
INTERVALS
6
7
(min)
Fig. 2. Distribution of the time intervals between the beginning of successive contraction pulse trains. Two Hydra otienuatu were tested in continuous darkness. Duration of the experiment: top, 340 min; bottom, 432 min.
310
The different background conditions were D, W, 400, 450, 500, 550, 600, 650 or 700 and the pulse conditions were either D, W, 450 or 550 where: D= darkness; W =white light; 400=400 nm light; 450=450 nm light; 500=500 nm light; 550=550 nm light; 600 = 600 nm light; 650 = 650 nm light; 700 = 700 nm light. Each light pulse was always 7.5 s in duration, and administered immediately after the second contraction pulse of a train, i.e. 7.5 s ( f 2.5 s, depending on the reaction time of the operator) after the start of the first contraction pulse (always roughly at the same phase, notwithstanding the period length variability). The second contraction pulse was used instead of the first in order to avoid rare isolated single-contraction pulses, but it was necessary to apply the stimulus at an early phase, where the elicited effect of a D or W pulse is greatest [6].
3. Results A preliminary set of experiments was carried out in order to test, on a greater number of animals and also using new Hydra strains, the preliminary results of TaddeiFerretti et al. [7], which described the influence of background illumination wavelength on the period length of the behavioural cycle, using the background conditions described above. It was ascertained that the period length of the behavioural sequence in undisturbed conditions varies with the wavelength of the background illumination (Table 1). Longer periods are found with 450 background illumination and shorter ones with 600. The data obtained in uninterrupted darkness and in continuous white light illumination are reported for comparison: they confirm the earlier results of TaddeiFerretti and Cordella [6], showing that in darkness the period is shorter. A second set of experiments was then performed for each animal by testing the influence of the background illumination on the response evoked by a pulse stimulus, which always maintained the same characteristics (of intensity, duration, application phase and - for each single test series - wavelength) and briefly replaced the background conditions in a step-wise manner. The background conditions were chosen a D, W, 450, 550 and in two cases 600. The pulse conditions were D, W, 450 and 550. The 450 and 550 (or 600) conditions, rather than any of the other possible monochromatic wavelength conditions were chosen, (i) for the background during pulse stimulation, and (ii) for the pulse stimuli, for different reasons. In (i), these two conditions produce two peaks in the variation of the behavioural cycle period length (high persistent period increase/low persistent period increase respectively) with respect to the length produced with D, linked to the background wavelength change from 450 to 550 (or 600) [7]. In (ii), these conditions produce two peaks in response in opposite directions (phase delay, i.e. transient behavioural period increase/phase advance, i.e. transient behavioural period decrease respectively), linked to the same pulse wavelength variation, where stimulus intensity, duration, early application phase and D background remain unchanged [9]. Comparable results have also been obtained by Ellis [16], Singer et al. [17] and Tardent et al. [Ml, with different protocols. In each experiment with a single animal, the period lengths of the contraction-elongation sequence in undisturbed background conditions were checked for 30 min and the mean period length was calculated. The response time (i.e. between the end of each pulse stimulus and the first contraction pulse of the subsequent contraction pulse train) was then checked for all
311 TABLE 1 Influence of background illumination on the period length of the behaviour cycle. Mean length (s) of the behavioural period in different undisturbed conditions (indicated by the column headings); each row represents a single animal Strain
Background illumination D
W
315 242 135 121 165 120 111 122 115 150 77 115
277 283 195 288
Mean
149
246
H. attenuata (African strain)
276
739
Aposymbiotic H. magnipapillata
220 135 177
H. attenwta (Swiss strain)
Mean
400
109
450
500
550
600
6.50
228 184
150
71
165
157
700
337 187 160 232
604 540 315 451 442 465
454
319
270
199 230 82
187
362
204
161
157
315 330 346 283 337
334 357
286 295 295 130 159
158 194
234
182
193
391
345
233
176
234
182
109
449
types of stimuli. These checks were repeated four times for each type of stimulus which replaced each background, taking care to vary continuously the order of the successive presentation of different types of stimuli. The mean of the normalized response times (i.e. the mean of the ratios between each response time after a single pulse of one type and the previously calculated mean period length obtained in the undisturbed condition upon which the pulse was applied) was calculated for the combination of each type of pulse with the background used. This protocol was then repeated for the other undisturbed background conditions, taking care to vary the order of successive presentations of each type of background for different animals. The complete sequence of an experiment requires about 8 hours to test a single animal; however, only few animals remained in suitable condition for this length of time, and usually the experiment had to be stopped before the end or was planned with shorter stimulation protocols. Table 2 shows the ratios between the mean normalized response times obtained under specific conditions and the same parameter obtained by varying the background illumination only: response time depends on the wavelength of background illumination.
312 TABLE 2 Ratio between the mean of the normalized response times after a pulse stimulus of a definite type (on the line) applied on a background condition (subscript) and the mean of the normalized response times after the same stimulus applied on a different background condition. Each column represents one animal. The last 3 columns refer to aposymbiotic H. magnipupillata
D,/DW D450/Dw D55o/D450 w550mD w450mD w55o/w,,o
0.34 1.08 0.31
0.37 0.86 0.43
2.98 3.84 0.78
2.2 4.6 0.48
10 23.8 0.42
5 13.64 0.27
3.08 3.35 0.92 0.55
w600/w450
0.45
0.68
550450/550D
4.4 4.35
1.47 1.9
1.75 5.42
5.8
450,“/450o 4505,/450,
0.8 0.19
1.01 0.47
0.85 0.23
0.36
554yl559D
4. Discussion Previously published data show that: (i) for a stimulus of fixed wavelength (the stimulus intensity, duration and background conditions remaining unchanged), the response is a function of the stimulus application phase during the behavioural period [6]; (ii) for a stimulus of fixed application phase (the other conditions being unchanged), the response is a function of the stimulus wavelength [9]; (iii) the period length of the behavioural sequence in undisturbed conditions varies with the wavelength of background illumination [7] (confirmed by Table 1). In the first instance, according to the simplest hypothesis, because the length of the total period varies according to the background illumination wavelength, in undisturbed conditions this variation should occur isotropically along all phases. If this is the case, then for a stimulus of fixed application phase (as well as fixed intensity, duration and wavelength), the response should not vary with the background illumination wavelength variation (since the amount of response is expressed in normalized units, as a fraction of the total behavioural period length). However, present experiments, performed in order to test this hypothesis, showed the reverse to be true. For a stimulus of fixed application phase (as well as fixed intensity, duration and wavelength), the response varies with the wavelength of the background illumination on which the pulse light stimulus is applied. The data in Table 2 lead to the conclusions listed below. (i) Variations in response time occur when the background wavelength is changed. (ii) Despite individual differences, such variations always occur in the one direction, i.e. as an increase or a decrease (except in the 8th row of the 1st column compared with the 9th row) for each animal tested, when the background wavelength is changed in the same manner, and all the stimulus conditions remain unchanged. (iii) In the case of a D stimulus, response times are low for a 550 background, high for a 450 and intermediate for a W background (which should include chromatic components of the two previous ones). Thus, the 450 and 550 backgrounds seem to
313
have effects which are opposite with respect to the effect of W. This is similar to the results observed with the different experimental protocols reported above. (iv) In the case of a 550 stimulus, increased response times are obtained by using a D background, followed by a W, and finally a 450. Thus, the 450 background exhibits the same time-response-lengthening effect observed in (iii), while when a W background is used, one could consider that a lower intensity 450 background exerts the same effect upon a lower intensity 550 stimulus applied on a constant 550 background. (v) Conversely, in the case of a 450 stimulus, decreased response times are observed when using a D background, followed by a W and finally a 550. The same considerations as in (iv), but reversed, can be applied. (vi) In the case of a W stimulus, on the basis of all the previous observations and the fact that such a stimulus on a monochromatic background could be considered as a “complementary” flash on a constant background, one might expect that a lower/ higher response time would be obtained with a 550 (or 600)/450 background respectively, when compared with that obtained using a D background. However, higher response times are obtained with both 550 (or 600) and 450 backgrounds than with a D, although lower with 550 (or 600) than with 450. This last observation suggests that, as far as the W stimulus is concerned, the total of time-response-shortening monochromatic components is less than the total of lengthening components. The results reported here give an insight into the complexity of the mechanism responsible for the photoresponse. The fact that the response to short and long wavelengths differs not only in its amount but also in its direction [9], parallels the antagonistic effects of light of two different wavelengths in bacteria [19,20], invertebrates [21-241 and vertebrates [25]. This could be interpreted by suggesting that either two photosensitive pigments or two reciprocally reversible wavelength-dependent states of one photoconvertible pigment are involved in the phototransduction. The time-constant of the photochromic process would be very slow. The reported interaction between the background and the pulse wavelength effects could be supposed to intervene either directly at the photosensitive molecular level or further along the chain of effects culminating in the photoresponse. While the site of such interaction could be tested by the use of appropriate inhibitors interacting at several different places in the effect chain, the characterization of the photopigment would require [26], as a first stage, the microspectrophotometric analysis of cells which are candidates for the photoreceptive function [27]. The electrophysiological analysis of such cells under ditferent photic conditions could also be useful in order to identity the cells involved in the phototransduction. As a last consideration, it has been observed that, due to the experimental protocol used, the background illumination is suppressed when the light pulses are applied, which means that two transients are actually applied. The experiments could be repeated without switching off the background illumination when Hydra undergoes light flash stimulation. However, the necessity of halving the background illumination intensity and of supplying stimuli of halved intensity, in order to maintain constant illumination intensity, could introduce an unwanted variable. Acknowledgments
We are indebted to Professor A. Gierer (Max-Planck-Institut fiir Virusforschung, Tubingen) for the supply of the Swiss strain of H. attenuata, to Professor M. Rihat and Dr. W. Reich (The Hebrew University, Jerusalem) for constructive comments, and for the supply of aposymbiotic H. magnipapillata, and the African strain of H.
314
atienuata, to Dr. C. Ascoli and Dr. D. Petracchi (Istituto di Biofisica, CNR, Pisa) for useful suggestions and criticism, and to Mr. M. Briggs-Smith for the correction of the English manuscript.
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