- Email: [email protected]
The electrical response of the planarian photoreceptor
Comp. Biochem. Physiol., 1962, Vol. 5, pp. 129 to 138. Pergamon Press Ltd., London. Printed in Great Britain
THE ELECTRICAL RESPONSE OF THE PLANARIAN PHOTORECEPTOR M. E. BEHRENS* Department of Zoology, Syracuse University, New York, U.S.A. (Recdved in revised form 24 September 1961) Abstract--1. Slow negative potential changes in response to illumination have been
recorded extracellularly through micropipette electrodes which penetrated the eyespots of the planarian, Dugesia tigrina. 2. As in other photoreeeptors the response amplitude increased and the latent period decreased with increasing stimulus intensity. Dark-adaptation was half complete in 3 or 4 rain. 3. A transient decrease in amplitude complicated the response of the darkadapted eye to a high-intensity stimulus. A small positive deflection or a notch was present at the beginning of some responses. 4. Occasionallya shift in baseline and polarity reversal were observed in association with vertical electrode displacement. These events might be related to penetration of the membrane covering the pigment cup.
INTRODUCTION ALTHOUGHelectricalchanges in responseto illumination have been recorded from the photoreceptorsof representativesof a number of phyla, to our knowledge such electrical activity has never been recorded from a Platyhelminth. In the present experiments the electrical activity of the photoreceptor of the planarian, D~esia tigrina, was recorded extracellularly and its characteristics as a function of some parameters of white light-stimuli were determined. T h e comparatively simple structure of the planarian photoreceptor makes it an attractive preparation from the viewpoint of receptor physiology. Light (Taliaferro, 1920) and electron (Press, 1959) microscopic studies show that the rhabdome and middle region, which make up the expanded distal portion of the planarian receptor cell, lie within the pigment cup. T h e nucleus-bearing portion lies just outside the membrane covering the aperture of the cup and the proximal processes continue as fibers of the optic nerve which passes into the brain. This separation of the receptor cells from second-order neurons may be of advantage in the interpretation of the data.
METHOD An eyespot, together with a small amount of the surrounding tissue (approximately 0.3 m m x 0.4 m m x 0.3 ram), was mounted on an agar base [(2.5 per cent agar in frog physiological salt solution (Prosser et al., 1950)] in a lucite chamber. Sufficient 0.5 per cent agar salt solution was introduced on top of the tissue to hold * Present address: Masonic Medical Research Laboratory, Utica, New York.
130
M.E. BEH~NS
it in place and frog physiological salt solution was used to keep the preparation moist. T h e eye was oriented so that the aperture of the pigment cup was directed toward the stimulating light. A 500 W projection lamp which supplied approximately 1260 ft-c (referred to below as unit intensity) at the aperture of the eye served as the light source. T h e light-beam passed through 5 cm of distilled water before entering the dark compartment containing the preparation. Wratten neutral filters were used to decrease the intensity of the light over a six lOgl0 unit range and the duration of the stimulating light-flashes was controlled by a photographic shutter. T h e electrical activity of the photoreceptor was detected with a glass micropipette (filled with physiological salt solution) in reference to a non-polarizable half-cell in contact with the agar in which the preparation was imbedded. T h e micropipette was connected to a second half-cell via a salt bridge and both half-cells were connected to a Grass high-impedance preamplifier. T h e output of the preamplifier was fed into Offner d.c. amplifiers and the electrical activity was monitored with a cathode ray oscilloscope and recorded with an inkwriter (frequency response flat to 100 c/s). A second channel recorded the output of a photocell activated by a portion of the light-beam admitted to the preparation. T h e deflection of the inkwriter pen was calibrated at each attenuation by the application of d.c. pulses of known voltage. T h e characteristics of the electrical response were determined as a function of the intensity of a light-stimulus of constant duration and as a function of a constant intensity, constant duration light-stimulus after a period of light-adaptation. T h e dark-intervals are indicated in the results. In preliminary experiments modifications of the above method were employed. These modifications included (1) the use of the intact animal, (2) the use of anesthetics (chlorobutanol or methane tricaine sulfonate) and the use of metal electrodes or 3 M KCl-filled glass micropipettes. The characteristics of the receptor response did not appear to be affected by the method used, and, therefore, responses recorded during preliminary experiments were included in the reported results. RESULTS Much of the difficulty with the planarian preparation stemmed from spontaneous movements. Immobilization of the whole animal did not prevent movement of the eye, which can move medially until it disappears under the pigment of the body surface, or laterally until it comes to lie in the center of the non-pigmented region. In addition the eye can rotate so that the aperture of the cup is directed upward. These movements were observed in relation to the tip of a stationary electrode and could not be attributed to movement of the overlying tissue relative to the eye. Satisfactory immobilization was not achieved unless small pieces (approximately 0.3 m m x 0.4 m m x 0.3 mm) containing the eye were used. Electrical activity in response to illumination could only be recorded from a restricted region. Such activity was recorded most frequently when the electrode
THE ELECTRICAL RESPONSE OF THE PLANARIAN PHOTORECEPTOR
131
penetrated near the center of the pigmented eyespot. Responses were obtained from the lateral edge of the eyespot but these invariably were of lower magnitude than those recorded from the center of the eye under similar stimulating conditions. Responses to light-stimuli were never recorded if the electrode penetrated outside the pigmented area. Furthermore, vertical displacement of the electrode by 10-15/x often resulted in a marked reduction in the amplitude of the response. The results reported are based on responses recorded extracellularly with micropipettes. Although microelectrodes filled with 3 M KC1 with resistances of 20-30 Mf~ were used, it was not possible to penetrate and hold single photoreceptor cells under the conditions of these experiments.
Co)
2my
I
I' s e c
L
FIC. 1. T y p i c a l electrical r e s p o n s e s to illumination of t h e planarian p h o t o r e c e p t o r s h o w i n g t h e effect o f t h e intensity o f t h e s t i m u l a t i n g light on t h e w a v e f o r m . U p w a r d deflection indicates negativity of t h e active electrode. (a) Log,0 1 = - 2.0. (b) Log10 1 = 0. P r e c e d i n g dark-interval of 5 rain in b o t h cases. Calibration m a r k s apply to b o t h responses.
The waveform of the response of the dark-adapted planarian photoreceptor changes with the intensity of the light-stimulus; at low intensities of stimulation the curve rises to a maximum and decays slowly toward the baseline, and at high intensities the curve rises rapidly to a peak and then decays rapidly to a lower level and may subsequently exhibit a second maximum (Fig. 1). A typical response to a 0.1 sec stimulus of moderate intensity (Fig. la) rose to a maximum of 3.1 mV in 200 msec following a latent period of 50 msec. The response gradually declined, to reach the baseline in 2.8 see. In the same preparation the response to a lightstimulus of an intensity 100 times that employed above rose to a maximum of 5.8 mV in 168 msec after a latent period of 38 msec (Fig. lb). It then decayed rapidly to 3.0 mV and rose again to reach a second maximum of 3.1 mV. The response declined very slowly and, even 5 seconds after the stimulating lightflash, was more than 1 mV above the baseline. In most preparations the polarity of the response indicated that the active electrode became negative to the indifferent one. In a few preparations, however, as the electrode was advanced stepwise through the tissue, a shift in baseline occurred which was immediately followed by light-initiated responses of reversed polarity. In some of these preparations, however, and at low light-intensities in all of them, the positive potential change had the same waveform as the previously
132
M.E. BEHRENS
recorded negative responses (Fig. 2a). In two of the preparations in which reversal occurred, it was noted that the electrode had penetrated the eyespot near the concave edge (aperture) of the pigment cup. Additional complexities were observed
I 2rnV (a)
/----L._
12 ~v (b)
r'-L__ I
I I 5ec
FIG. 2. Positive electrical responses to illumination of the planarian photoreceptor. Upward deflection indicates negativity of the active electrode. (a) Log10 I = - 4 . 0 . (b) Log10 1 - - - 2 . 0 .
in the responses of several preparations; in some a small positive deflection occurred at the beginning of an otherwise typical negative response (Fig. 3a) and in others a notch was present on the rising phase of the slow-potential change (Fig. 3b). As in other photoreeeptors the magnitude and the latency of the response were markedly dependent on the intensity of the stimulating light. The length of the mV (o)
I
I
I ~ mv
____J
(b) i
I sec
FIG. 3. Complex electrical responses to illumination of the planarian photoreceptor. Upward deflection indicates negativity of the active electrode. (a) Log10 I = - 5 . 0 . Previous d a r k - i n t e r v a l - - 5 min. A positive deflection occurs at the beginning of the response. (b) Log10 I = 0. Previous dark-interval -- 3 min. A notch is present on the rising phase of the slow-potential change.
latent period decreased and the amplitude of the response increased with increasing light-intensity. In a typical preparation the amplitude of the response increased from 0.024 mV to 2-3 mV and the Iatent period decreased from 400 msee to 35 msec over the six log10 unit range of light-intensities employed (Figs. 4 and 5). The amplitude vs logxo intensity relation was linear over a range of several log units.
133
THE ELECTRICAL RESPONSI~ OF THE PLANARIAN PHOTORECEPTOR 2"5
2"0
1"5
a; D
l'O
E 0"5
i
-6
-;5
I
-4
i
-3
-2
J
-I
0
log=o i n t e n s i t y
FIG. 4. Effect of the intensity of the stimulating light-flashes on the amplitude of the response of the planarian photoreceptor. Light-flashes of 0.5 sec duration were presented in order of increasing intensity. Chlorobutanol anesthesia was used in immobilizing this preparation.
500
400
300
(i)
200
3 I00
0
!
-6
I
-5
!
-4
I
-3
I
-2
!
-I
0
lOgiO intensity
FxG. 5. Effect o f t h e i n t e n s i t y of t h e s t i m u l a t i n g light-flashes o n t h e latency of t h e r e s p o n s e o f t h e planarian photoreceptor. C h l o r o b u t a n o l anesthesia.
134
M . E . BEHRENS
The course of dark-adaptation of the planarian photoreceptor was followed by measuring the electrical response to short flashes of light administered at varying time intervals following a prolonged exposure to light of unit intensity. The recorded response, which disappeared completely during exposure to light of unit intensity, increased in amplitude during the subsequent dark-period (Fig. 6). The rate at which the amplitude of the response increased was greatest during the first few minutes of the dark-period, reaching 1/2 maximum amplitude in 3"5 min
>
E 3 D
2
0
4
8
12
Minutes
I 16
I 20
I 24
t 28
32
in dork
FIG. 6. Data showing the relationship between the response magnitude of the planarian photoreceptor and time in the dark after a 5-min exposure to light of unit intensity. Test flashes: duration = 0-1 sec, log10 intensity = -2"0.
(average of eight preparations). The rate of increase in amplitude then declined and, even after an hour in the dark, the amplitude of the response to a test stimulus was usually less than that of a control response recorded just prior to lightadaptation. In some preparations the amplitude of the response :to test stimuli reached a maximum after about 30 rain in the dark and then declined slightly. The magnitude and waveform of the eIectrical responses recorded after highintensity light-flashes were affected by the dark-interval between the stimuli (Fig. 7). With dark-intervals longer than 1 or 2 rain the simple waveform was complicated by a transient decrease in amplitude resulting in a peak in the response.
THE E L E C T R I C A L RESPONSE OF T H E P L A N A R I A N P H O T O R E C E P T O R
135
The response often rose to a second maximum before a more gradual decline toward the baseline. These changes in waveform diminished as the intensity of the stimulating light was decreased, were not apparent at low intensities, and were independent of the duration of the stimulus.
(o) t~x
7--------
I 2mv
(b) !
I I $ec
FIG. 7. Responses elicited by light-flashes of unit intensity admitted to a planarian photoreceptor showing the effect of the length of the preceding dark-interval on the waveform. (a) One minute dark-interval. (b) Two minute dark-interval. Calibration marks apply to both responses.
DISCUSSION The expanded distal portion of the receptor cell, which presumably contains the photosensitive pigment, is located within the pigment cup (Taliaferro, 1920; Press, 1959; Wetzel, 1961). The restricted region of the eye from which responses were obtained suggests that the potential changes recorded extraceUularly in these experiments may be the sum of the potential changes occurring in the expanded portions of the cells. Intracellular studies are necessary to confirm this but the idea is given support by the results obtained in other photoreceptors (Bernhard, 1942; Naka, 1961; Ruck, 1961). The reversal in polarity which was observed in a few preparations in relation to vertical displacement of the micropipette is of considerable interest and deserves some comment, but any conclusions should be considered of a tentative nature in view of the limited number of observations. The reversal of polarity could be explained in the following manner. If the eye were tilted so that the aperture was pointed slightly downward, an electrode which penetrated through the dorsal rim into the cavity of the cup could, when advanced downward, penetrate through the membrane covering the aperture of the eye and record potentials just outside the eyecup proper. The shift in baseline (10-20 mV) which was observed with the vertical displacement of the electrode, as well as the reversal of the polarity of the electrical response to illumination, might well be related to penetration of the membrane covering the aperture of the eye. Since these events were quite reproducible upon repeated lowering and raising of the electrode, it is unlikely that they were produced by penetration of single cells by the microelectrode. From behavioral studies Taliaferro (1920) concluded that light must strike a rhabdome parallel to its longitudinal axis in order to stimulate. The present
136
M.E. BEHRENS
study supports this view. When light-stimuli of low intensity were employed it was necessaryto orient the eye so that the openingof the pigment cup was directed toward the light-source. From electron micrographs Wetzel (1961) concluded that the longitudinallyoriented structures present in the rhabdome of the receptor cells, previouslydescribed as lamellaewith distal bulb-like swellings (Press, 1959), are tubules and suggested that these may represent elongated microvilli arising from the expanded portion of the retinula cell process. Press (1959) observed that light directed parallel to the longitudinal axis of the rhabdome would not be favorably directed to strike the longitudinally oriented surfaces and suggested the "distal swellings" as significant sites of visual biochemical phenomena. During the course of these experiments spike potentials were never observed in association with the Mow-potential changes, even though electrodes of 10-20 Ml) resistance were employed in a number of experiments and the responses were always monitored on an oscilloscope. This suggests that the site of impulse initiation is probably not in the structures within the pigment cup, or that such electrical activity is too small or too rapid to be detected under the conditions of the experiments. It also suggests that, if spike activity is initiated elsewhere, it is not conducted antidromically into the region from which the slow-potential change was recorded. The small positive deflection and the notch occasionally seen at the beginning of the light-initiated responses (Fig. 3) are similar to those present in the responses from the dorsal ocelli of the dragonfly and of the cockroach (Ruck, 1961). The receptor axons of these ocelli synapse at the base of the pigment cup with dendritic terminals of the ocellar nerve fibers. Ruck interprets the small positive deflection as a depolarization of the receptor axon and the notch on the rising phase of the slow-potential change as a sign of a hyperpolarizing post-synaptic potential in the dendritic terminals of the ocellar nerve fiber. It should be mentioned that, although axo-dendritic synapses have not been observed in the optic nerve of the planarian, the existence of such synapses has not been ruled out. In some of the dark-adaptation experiments the curve of response amplitude vs. time in the dark rose to a maximum and then declined. At first this was attributed to deterioration of the preparation and, indeed, this may well have been an important factor in some cases. However, in one preparation the response to a unit intensity stimulus, administered after 41 min of relative darkness, was reduced in amphtude and greatly prolonged (7.5 see). A light-flash of equal intensity presented one minute later elicited a response of greater amphtude and shorter duration. In this preparation the reduction in response amplitude could not be attributed to deterioration and appeared to be associated with the change in waveform which occurred following high-intensity stimulation of the darkadapted eye (Figs. 1 and 7). The relatively complex waveform of the response elicited by high-intensity stimuli, consisting of a rapid rise to maximum followed by a rapid decay to a lower level with possibly a second maximum, resembles both the extra-cellularly and intra-cellularly recorded responses of the sense cells of the lateral eye of the
T H ~ E L E C T R I C A L RESPONSE OF T H E P L A N A R I A N PHOTORECEPTOR
137
horse shoe crab (Hartline & Graham, 1932; Fourtes, 1958; Tomita, Kikuchi & Tanaka, 1960). In the latter it has been suggested that this complex waveform is related to the resting membrane level and to the gradient of depolarization (Tanaka & Yamanaka, 1960). In the planarian eye, also, the complexity and the rate at which the slow-potential change rises to a maximum seem to be related (Figs. 1 and 7). Although some of the characteristics of the response of the planarian photoreceptor are similar to response characteristics of other photoreceptors, quite different mechanisms may be involved. SUMMARY (1) Extracellular responses to light-stimuli were recorded from the planarian eyespot. (2) W h e n the active electrode is located within the eyespot, a slow-potential change is recorded in which the active electrode becomes negative to the indifferent electrode. Spikes were never observed. (3) In some preparations a small positive deflection occurs at the beginning of the response. In others a notch is seen on the rising phase of the slow-potential change. (4) The response amplitude increases and the length of the latent period decreases as the intensity of the stimulating light is increased. (5) T h e amplitude of the response of the light-adapted eyespot increases during a subsequent period in the dark, dark-adaptation being half complete in 3 or 4 minutes. (6) In the dark-adapted eye, the response to a high-intensity stimulus decays slightly following the initial maximum and then rises slowly to a second maximum before it declines slowly toward the baseline. (7) Reversal in the polarity of the response to illumination, and a ~hift in baseline associated with the vertical displacement of the electrode, was observed in a few preparations. A possible explanation for this is presented. Acknowledgements--I am especially grateful for the unfailing encouragement and assistance of Dr. Verner J. Wulff under whose supervision I was privileged to work. I am indebted to him for his assistance in the preparation of this manuscript. I also wish to thank Dr. Ralph Adams and Dr. Donald Kennedy for their assistance and many helpful suggestions. This investigation was carried out during the tenure of a Special Fellowship from the Division of General Medical Sciences, United States Public Health Service. This research was supported in part by grant No. G4049 from the National Science Foundation. REFERENCES
BERNHARDC. (1942) Isolation of retinal and optic ganglion responses in the eye of Dytiscus. J. Neurophysiol. 5, 32-48. Folmaxs M. G. F. (1958) Electrical activity in the eye of Limulus. Amer. ]. Ophthal. 46, No. 5, Part II, 210-223.
138
M . E . BEHRENS
HARTLINlg H. K. & GRAHAM C. H. (1932) Nerve impulses from single receptors in the eye. ft. Cell. Comp. Physiol. 1, 277-295. NAKA K. (1961) Recording of retinal action potential from single cells in the insect compound eye. ft. Gen. Physiol. 44, 571-584. PRESS N. (1959) Electron microscope study of the distal portion of a planarian retinula cell. Biol. Bull., Woods Hole 117, 511-517. PROSSER C. L., BROWN F. A., BISHOP D. W., JAHN T. L. ~ WULFF V. J. (1950) Comparative Animal Physiology, p. 95. Saunders, Philadelphia. RUCK P. (1961) Electrophysiology of the insect dorsal ocellus. I. Origin of the components of the electroretinogram, ft. Gen. Physiol. 44, 605--627. TALIAFERRO W. H. (1920) Reactions to light of Planaria maculata, with special reference to the function and structure of the eyes. ft. Exp. Zool. 31, 59-116. TANAKA I. ~ YAMANAKAT. (1960) Effect of linearly increasing and decreasing current on the optic nerve discharge of lateral eye of horseshoe crab. ft. Cell. Comp. Physiol. 56, 161-164. TOMITA T., KIKUCHI R. & TANAKA I. (1960) Excitation and inhibition in lateral eye of horseshoe crab. Electrical Activity of Single Cells, pp. 11-23. Igakushoin, Hongo, Japan. W~.TZEL B. K. (1961) Sodium permanganate fixation for electron microscopy, ft. Biophys. Biochem. Cytol. 9, 711-716.
THE ELECTRICAL RESPONSE OF THE PLANARIAN PHOTORECEPTOR M. E. BEHRENS* Department of Zoology, Syracuse University, New York, U.S.A. (Recdved in revised form 24 September 1961) Abstract--1. Slow negative potential changes in response to illumination have been
recorded extracellularly through micropipette electrodes which penetrated the eyespots of the planarian, Dugesia tigrina. 2. As in other photoreeeptors the response amplitude increased and the latent period decreased with increasing stimulus intensity. Dark-adaptation was half complete in 3 or 4 rain. 3. A transient decrease in amplitude complicated the response of the darkadapted eye to a high-intensity stimulus. A small positive deflection or a notch was present at the beginning of some responses. 4. Occasionallya shift in baseline and polarity reversal were observed in association with vertical electrode displacement. These events might be related to penetration of the membrane covering the pigment cup.
INTRODUCTION ALTHOUGHelectricalchanges in responseto illumination have been recorded from the photoreceptorsof representativesof a number of phyla, to our knowledge such electrical activity has never been recorded from a Platyhelminth. In the present experiments the electrical activity of the photoreceptor of the planarian, D~esia tigrina, was recorded extracellularly and its characteristics as a function of some parameters of white light-stimuli were determined. T h e comparatively simple structure of the planarian photoreceptor makes it an attractive preparation from the viewpoint of receptor physiology. Light (Taliaferro, 1920) and electron (Press, 1959) microscopic studies show that the rhabdome and middle region, which make up the expanded distal portion of the planarian receptor cell, lie within the pigment cup. T h e nucleus-bearing portion lies just outside the membrane covering the aperture of the cup and the proximal processes continue as fibers of the optic nerve which passes into the brain. This separation of the receptor cells from second-order neurons may be of advantage in the interpretation of the data.
METHOD An eyespot, together with a small amount of the surrounding tissue (approximately 0.3 m m x 0.4 m m x 0.3 ram), was mounted on an agar base [(2.5 per cent agar in frog physiological salt solution (Prosser et al., 1950)] in a lucite chamber. Sufficient 0.5 per cent agar salt solution was introduced on top of the tissue to hold * Present address: Masonic Medical Research Laboratory, Utica, New York.
130
M.E. BEH~NS
it in place and frog physiological salt solution was used to keep the preparation moist. T h e eye was oriented so that the aperture of the pigment cup was directed toward the stimulating light. A 500 W projection lamp which supplied approximately 1260 ft-c (referred to below as unit intensity) at the aperture of the eye served as the light source. T h e light-beam passed through 5 cm of distilled water before entering the dark compartment containing the preparation. Wratten neutral filters were used to decrease the intensity of the light over a six lOgl0 unit range and the duration of the stimulating light-flashes was controlled by a photographic shutter. T h e electrical activity of the photoreceptor was detected with a glass micropipette (filled with physiological salt solution) in reference to a non-polarizable half-cell in contact with the agar in which the preparation was imbedded. T h e micropipette was connected to a second half-cell via a salt bridge and both half-cells were connected to a Grass high-impedance preamplifier. T h e output of the preamplifier was fed into Offner d.c. amplifiers and the electrical activity was monitored with a cathode ray oscilloscope and recorded with an inkwriter (frequency response flat to 100 c/s). A second channel recorded the output of a photocell activated by a portion of the light-beam admitted to the preparation. T h e deflection of the inkwriter pen was calibrated at each attenuation by the application of d.c. pulses of known voltage. T h e characteristics of the electrical response were determined as a function of the intensity of a light-stimulus of constant duration and as a function of a constant intensity, constant duration light-stimulus after a period of light-adaptation. T h e dark-intervals are indicated in the results. In preliminary experiments modifications of the above method were employed. These modifications included (1) the use of the intact animal, (2) the use of anesthetics (chlorobutanol or methane tricaine sulfonate) and the use of metal electrodes or 3 M KCl-filled glass micropipettes. The characteristics of the receptor response did not appear to be affected by the method used, and, therefore, responses recorded during preliminary experiments were included in the reported results. RESULTS Much of the difficulty with the planarian preparation stemmed from spontaneous movements. Immobilization of the whole animal did not prevent movement of the eye, which can move medially until it disappears under the pigment of the body surface, or laterally until it comes to lie in the center of the non-pigmented region. In addition the eye can rotate so that the aperture of the cup is directed upward. These movements were observed in relation to the tip of a stationary electrode and could not be attributed to movement of the overlying tissue relative to the eye. Satisfactory immobilization was not achieved unless small pieces (approximately 0.3 m m x 0.4 m m x 0.3 mm) containing the eye were used. Electrical activity in response to illumination could only be recorded from a restricted region. Such activity was recorded most frequently when the electrode
THE ELECTRICAL RESPONSE OF THE PLANARIAN PHOTORECEPTOR
131
penetrated near the center of the pigmented eyespot. Responses were obtained from the lateral edge of the eyespot but these invariably were of lower magnitude than those recorded from the center of the eye under similar stimulating conditions. Responses to light-stimuli were never recorded if the electrode penetrated outside the pigmented area. Furthermore, vertical displacement of the electrode by 10-15/x often resulted in a marked reduction in the amplitude of the response. The results reported are based on responses recorded extracellularly with micropipettes. Although microelectrodes filled with 3 M KC1 with resistances of 20-30 Mf~ were used, it was not possible to penetrate and hold single photoreceptor cells under the conditions of these experiments.
Co)
2my
I
I' s e c
L
FIC. 1. T y p i c a l electrical r e s p o n s e s to illumination of t h e planarian p h o t o r e c e p t o r s h o w i n g t h e effect o f t h e intensity o f t h e s t i m u l a t i n g light on t h e w a v e f o r m . U p w a r d deflection indicates negativity of t h e active electrode. (a) Log,0 1 = - 2.0. (b) Log10 1 = 0. P r e c e d i n g dark-interval of 5 rain in b o t h cases. Calibration m a r k s apply to b o t h responses.
The waveform of the response of the dark-adapted planarian photoreceptor changes with the intensity of the light-stimulus; at low intensities of stimulation the curve rises to a maximum and decays slowly toward the baseline, and at high intensities the curve rises rapidly to a peak and then decays rapidly to a lower level and may subsequently exhibit a second maximum (Fig. 1). A typical response to a 0.1 sec stimulus of moderate intensity (Fig. la) rose to a maximum of 3.1 mV in 200 msec following a latent period of 50 msec. The response gradually declined, to reach the baseline in 2.8 see. In the same preparation the response to a lightstimulus of an intensity 100 times that employed above rose to a maximum of 5.8 mV in 168 msec after a latent period of 38 msec (Fig. lb). It then decayed rapidly to 3.0 mV and rose again to reach a second maximum of 3.1 mV. The response declined very slowly and, even 5 seconds after the stimulating lightflash, was more than 1 mV above the baseline. In most preparations the polarity of the response indicated that the active electrode became negative to the indifferent one. In a few preparations, however, as the electrode was advanced stepwise through the tissue, a shift in baseline occurred which was immediately followed by light-initiated responses of reversed polarity. In some of these preparations, however, and at low light-intensities in all of them, the positive potential change had the same waveform as the previously
132
M.E. BEHRENS
recorded negative responses (Fig. 2a). In two of the preparations in which reversal occurred, it was noted that the electrode had penetrated the eyespot near the concave edge (aperture) of the pigment cup. Additional complexities were observed
I 2rnV (a)
/----L._
12 ~v (b)
r'-L__ I
I I 5ec
FIG. 2. Positive electrical responses to illumination of the planarian photoreceptor. Upward deflection indicates negativity of the active electrode. (a) Log10 I = - 4 . 0 . (b) Log10 1 - - - 2 . 0 .
in the responses of several preparations; in some a small positive deflection occurred at the beginning of an otherwise typical negative response (Fig. 3a) and in others a notch was present on the rising phase of the slow-potential change (Fig. 3b). As in other photoreeeptors the magnitude and the latency of the response were markedly dependent on the intensity of the stimulating light. The length of the mV (o)
I
I
I ~ mv
____J
(b) i
I sec
FIG. 3. Complex electrical responses to illumination of the planarian photoreceptor. Upward deflection indicates negativity of the active electrode. (a) Log10 I = - 5 . 0 . Previous d a r k - i n t e r v a l - - 5 min. A positive deflection occurs at the beginning of the response. (b) Log10 I = 0. Previous dark-interval -- 3 min. A notch is present on the rising phase of the slow-potential change.
latent period decreased and the amplitude of the response increased with increasing light-intensity. In a typical preparation the amplitude of the response increased from 0.024 mV to 2-3 mV and the Iatent period decreased from 400 msee to 35 msec over the six log10 unit range of light-intensities employed (Figs. 4 and 5). The amplitude vs logxo intensity relation was linear over a range of several log units.
133
THE ELECTRICAL RESPONSI~ OF THE PLANARIAN PHOTORECEPTOR 2"5
2"0
1"5
a; D
l'O
E 0"5
i
-6
-;5
I
-4
i
-3
-2
J
-I
0
log=o i n t e n s i t y
FIG. 4. Effect of the intensity of the stimulating light-flashes on the amplitude of the response of the planarian photoreceptor. Light-flashes of 0.5 sec duration were presented in order of increasing intensity. Chlorobutanol anesthesia was used in immobilizing this preparation.
500
400
300
(i)
200
3 I00
0
!
-6
I
-5
!
-4
I
-3
I
-2
!
-I
0
lOgiO intensity
FxG. 5. Effect o f t h e i n t e n s i t y of t h e s t i m u l a t i n g light-flashes o n t h e latency of t h e r e s p o n s e o f t h e planarian photoreceptor. C h l o r o b u t a n o l anesthesia.
134
M . E . BEHRENS
The course of dark-adaptation of the planarian photoreceptor was followed by measuring the electrical response to short flashes of light administered at varying time intervals following a prolonged exposure to light of unit intensity. The recorded response, which disappeared completely during exposure to light of unit intensity, increased in amplitude during the subsequent dark-period (Fig. 6). The rate at which the amplitude of the response increased was greatest during the first few minutes of the dark-period, reaching 1/2 maximum amplitude in 3"5 min
>
E 3 D
2
0
4
8
12
Minutes
I 16
I 20
I 24
t 28
32
in dork
FIG. 6. Data showing the relationship between the response magnitude of the planarian photoreceptor and time in the dark after a 5-min exposure to light of unit intensity. Test flashes: duration = 0-1 sec, log10 intensity = -2"0.
(average of eight preparations). The rate of increase in amplitude then declined and, even after an hour in the dark, the amplitude of the response to a test stimulus was usually less than that of a control response recorded just prior to lightadaptation. In some preparations the amplitude of the response :to test stimuli reached a maximum after about 30 rain in the dark and then declined slightly. The magnitude and waveform of the eIectrical responses recorded after highintensity light-flashes were affected by the dark-interval between the stimuli (Fig. 7). With dark-intervals longer than 1 or 2 rain the simple waveform was complicated by a transient decrease in amplitude resulting in a peak in the response.
THE E L E C T R I C A L RESPONSE OF T H E P L A N A R I A N P H O T O R E C E P T O R
135
The response often rose to a second maximum before a more gradual decline toward the baseline. These changes in waveform diminished as the intensity of the stimulating light was decreased, were not apparent at low intensities, and were independent of the duration of the stimulus.
(o) t~x
7--------
I 2mv
(b) !
I I $ec
FIG. 7. Responses elicited by light-flashes of unit intensity admitted to a planarian photoreceptor showing the effect of the length of the preceding dark-interval on the waveform. (a) One minute dark-interval. (b) Two minute dark-interval. Calibration marks apply to both responses.
DISCUSSION The expanded distal portion of the receptor cell, which presumably contains the photosensitive pigment, is located within the pigment cup (Taliaferro, 1920; Press, 1959; Wetzel, 1961). The restricted region of the eye from which responses were obtained suggests that the potential changes recorded extraceUularly in these experiments may be the sum of the potential changes occurring in the expanded portions of the cells. Intracellular studies are necessary to confirm this but the idea is given support by the results obtained in other photoreceptors (Bernhard, 1942; Naka, 1961; Ruck, 1961). The reversal in polarity which was observed in a few preparations in relation to vertical displacement of the micropipette is of considerable interest and deserves some comment, but any conclusions should be considered of a tentative nature in view of the limited number of observations. The reversal of polarity could be explained in the following manner. If the eye were tilted so that the aperture was pointed slightly downward, an electrode which penetrated through the dorsal rim into the cavity of the cup could, when advanced downward, penetrate through the membrane covering the aperture of the eye and record potentials just outside the eyecup proper. The shift in baseline (10-20 mV) which was observed with the vertical displacement of the electrode, as well as the reversal of the polarity of the electrical response to illumination, might well be related to penetration of the membrane covering the aperture of the eye. Since these events were quite reproducible upon repeated lowering and raising of the electrode, it is unlikely that they were produced by penetration of single cells by the microelectrode. From behavioral studies Taliaferro (1920) concluded that light must strike a rhabdome parallel to its longitudinal axis in order to stimulate. The present
136
M.E. BEHRENS
study supports this view. When light-stimuli of low intensity were employed it was necessaryto orient the eye so that the openingof the pigment cup was directed toward the light-source. From electron micrographs Wetzel (1961) concluded that the longitudinallyoriented structures present in the rhabdome of the receptor cells, previouslydescribed as lamellaewith distal bulb-like swellings (Press, 1959), are tubules and suggested that these may represent elongated microvilli arising from the expanded portion of the retinula cell process. Press (1959) observed that light directed parallel to the longitudinal axis of the rhabdome would not be favorably directed to strike the longitudinally oriented surfaces and suggested the "distal swellings" as significant sites of visual biochemical phenomena. During the course of these experiments spike potentials were never observed in association with the Mow-potential changes, even though electrodes of 10-20 Ml) resistance were employed in a number of experiments and the responses were always monitored on an oscilloscope. This suggests that the site of impulse initiation is probably not in the structures within the pigment cup, or that such electrical activity is too small or too rapid to be detected under the conditions of the experiments. It also suggests that, if spike activity is initiated elsewhere, it is not conducted antidromically into the region from which the slow-potential change was recorded. The small positive deflection and the notch occasionally seen at the beginning of the light-initiated responses (Fig. 3) are similar to those present in the responses from the dorsal ocelli of the dragonfly and of the cockroach (Ruck, 1961). The receptor axons of these ocelli synapse at the base of the pigment cup with dendritic terminals of the ocellar nerve fibers. Ruck interprets the small positive deflection as a depolarization of the receptor axon and the notch on the rising phase of the slow-potential change as a sign of a hyperpolarizing post-synaptic potential in the dendritic terminals of the ocellar nerve fiber. It should be mentioned that, although axo-dendritic synapses have not been observed in the optic nerve of the planarian, the existence of such synapses has not been ruled out. In some of the dark-adaptation experiments the curve of response amplitude vs. time in the dark rose to a maximum and then declined. At first this was attributed to deterioration of the preparation and, indeed, this may well have been an important factor in some cases. However, in one preparation the response to a unit intensity stimulus, administered after 41 min of relative darkness, was reduced in amphtude and greatly prolonged (7.5 see). A light-flash of equal intensity presented one minute later elicited a response of greater amphtude and shorter duration. In this preparation the reduction in response amplitude could not be attributed to deterioration and appeared to be associated with the change in waveform which occurred following high-intensity stimulation of the darkadapted eye (Figs. 1 and 7). The relatively complex waveform of the response elicited by high-intensity stimuli, consisting of a rapid rise to maximum followed by a rapid decay to a lower level with possibly a second maximum, resembles both the extra-cellularly and intra-cellularly recorded responses of the sense cells of the lateral eye of the
T H ~ E L E C T R I C A L RESPONSE OF T H E P L A N A R I A N PHOTORECEPTOR
137
horse shoe crab (Hartline & Graham, 1932; Fourtes, 1958; Tomita, Kikuchi & Tanaka, 1960). In the latter it has been suggested that this complex waveform is related to the resting membrane level and to the gradient of depolarization (Tanaka & Yamanaka, 1960). In the planarian eye, also, the complexity and the rate at which the slow-potential change rises to a maximum seem to be related (Figs. 1 and 7). Although some of the characteristics of the response of the planarian photoreceptor are similar to response characteristics of other photoreceptors, quite different mechanisms may be involved. SUMMARY (1) Extracellular responses to light-stimuli were recorded from the planarian eyespot. (2) W h e n the active electrode is located within the eyespot, a slow-potential change is recorded in which the active electrode becomes negative to the indifferent electrode. Spikes were never observed. (3) In some preparations a small positive deflection occurs at the beginning of the response. In others a notch is seen on the rising phase of the slow-potential change. (4) The response amplitude increases and the length of the latent period decreases as the intensity of the stimulating light is increased. (5) T h e amplitude of the response of the light-adapted eyespot increases during a subsequent period in the dark, dark-adaptation being half complete in 3 or 4 minutes. (6) In the dark-adapted eye, the response to a high-intensity stimulus decays slightly following the initial maximum and then rises slowly to a second maximum before it declines slowly toward the baseline. (7) Reversal in the polarity of the response to illumination, and a ~hift in baseline associated with the vertical displacement of the electrode, was observed in a few preparations. A possible explanation for this is presented. Acknowledgements--I am especially grateful for the unfailing encouragement and assistance of Dr. Verner J. Wulff under whose supervision I was privileged to work. I am indebted to him for his assistance in the preparation of this manuscript. I also wish to thank Dr. Ralph Adams and Dr. Donald Kennedy for their assistance and many helpful suggestions. This investigation was carried out during the tenure of a Special Fellowship from the Division of General Medical Sciences, United States Public Health Service. This research was supported in part by grant No. G4049 from the National Science Foundation. REFERENCES
BERNHARDC. (1942) Isolation of retinal and optic ganglion responses in the eye of Dytiscus. J. Neurophysiol. 5, 32-48. Folmaxs M. G. F. (1958) Electrical activity in the eye of Limulus. Amer. ]. Ophthal. 46, No. 5, Part II, 210-223.
138
M . E . BEHRENS
HARTLINlg H. K. & GRAHAM C. H. (1932) Nerve impulses from single receptors in the eye. ft. Cell. Comp. Physiol. 1, 277-295. NAKA K. (1961) Recording of retinal action potential from single cells in the insect compound eye. ft. Gen. Physiol. 44, 571-584. PRESS N. (1959) Electron microscope study of the distal portion of a planarian retinula cell. Biol. Bull., Woods Hole 117, 511-517. PROSSER C. L., BROWN F. A., BISHOP D. W., JAHN T. L. ~ WULFF V. J. (1950) Comparative Animal Physiology, p. 95. Saunders, Philadelphia. RUCK P. (1961) Electrophysiology of the insect dorsal ocellus. I. Origin of the components of the electroretinogram, ft. Gen. Physiol. 44, 605--627. TALIAFERRO W. H. (1920) Reactions to light of Planaria maculata, with special reference to the function and structure of the eyes. ft. Exp. Zool. 31, 59-116. TANAKA I. ~ YAMANAKAT. (1960) Effect of linearly increasing and decreasing current on the optic nerve discharge of lateral eye of horseshoe crab. ft. Cell. Comp. Physiol. 56, 161-164. TOMITA T., KIKUCHI R. & TANAKA I. (1960) Excitation and inhibition in lateral eye of horseshoe crab. Electrical Activity of Single Cells, pp. 11-23. Igakushoin, Hongo, Japan. W~.TZEL B. K. (1961) Sodium permanganate fixation for electron microscopy, ft. Biophys. Biochem. Cytol. 9, 711-716.