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Dark adaptation of the ocellus in Periplaneta americana: A study of the electrical response to illumination
J. ins. Physiol.,
1958, Vol. 2, pp. 189
to 198.
Pergamon Press Ltd., London
DARK ADAPTATION OF THE OCELLUS IN PERIPLANETA AMERICANA : ‘A STUDY OF THE ELECTRICAL RESPONSE TO ILLUMINATION* PHILIP Department
RUCK
of Biology,
(Received
Tufts
17 December
University 1957)
Abstract-Cornea1 illumination at 12,000 ft-candles reduces the sensitivity of the cockroach ocellus by a factor of at least 50,000. At the end of the first minute of dark adaptation, sensitivity differs from that of the fully dark-adapted state by a factor of only 2-5.
INTRODUCTION
IN a previous paper (RUCK, 1957) the structure of the cockroach ocellus and the general nature of its electrical response to light stimulation were described. This ocellus contains a structurally uniform population of receptor cells, the axons of which synapse within the ocellus with second-order neurons; the postsynaptic units, on leaving the ocellus, comprise the ocellar nerve. The electrical response of the organ, recorded with a subcorneal macro-electrode, originates predominantly in the photoreceptor cells. The only contribution the postsynaptic units make to the total response consists of “off-spikes” which originate in the largest ocellar nerve fibres. Since the photoreceptor cell response can easily be recognized in the response of the whole organ, this ocellus is a useful preparation for studies directed at the elucidation of photoreceptor cell processes. The electrical responsiveness of the photoreceptor cells varies with the level of illumination. As illumination increases, responsiveness decreases; i.e. the photoreceptor cells become light-adapted. In darkness following a period of light adaptation, the responsiveness of the receptor cells increases and finally attains a level characteristic of the fully dark-adapted state. It is the purpose of this paper to describe in some detail the changes of electrical responsiveness of the receptor cells which are consequent upon exposure to a high level of illumination. EQUIPMENT
AND
METHODS
The animal preparation and the optical stimulator have been described more fully in a previous paper (RUCK, 1957). Adult male roaches (Periplaneta americana) were immobilized on a small platform with the aid of tacky wax. The head was covered with aluminium foil. through which a small hole was made just large * This work was made possible by grants from the National Science Foundation and the U.S. Public Health Service to Dr. K. D. ROEDER. Some of the apparatus used was obtained under a previous contract between the U.S. Chemical Corps and Tufts University. 189
190
PHILIP RUCK
enough to expose fully the cornea of 1 ocellus. An electrode was passed through the edge of the cornea, the tip coming to lie at the surface of the receptor cell layer. The other (reference) electrode was ensheathed in the flagellum of an antenna. Electrodes were electrolytically pointed, stainless steel wires. These were condenser-coupled to the cathode follower input stage of a Grass P6 amplifier which was operated at a frequency band-pass of 1 c/s-2 kc/s. Condenser-coupling, used to secure baseline stability, inevitably resulted in some distortion of the ocellar responses. From the calibration records of Figs. 1, 7, and 8 it can be seen that the time constant of the amplifier is similar to the time constant of the negative component of the ocellar responses. Therefore, amplitudes of the negative component are somewhat lower than they would have been if recorded with This discrepancy, however, most probably had a negligible DC amplification. effect on the dark-adaptation data in spite of the fact that these were derived from measurements of negative wave amplitudes. As will be explained more fully later in the paper, any given measurement of change in ocellar sensitivity depended on matching a response obtained following light adaptation with some member of a series of control responses obtained before light adaptation. Because any two responses which matched with respect to negative wave amplitude also matched in total wave form, it is probable that both responses were distorted in the same way by the recording circuit. This situation permits one to disregard the distortion as a source of error. The light source was a 6-V, 100-W, strip-filament tungsten lamp powered by a DC supply connected in parallel with a 6-V storage battery. The beam of light passed through a cell containing a 0.1 N CuCI, solution, 2.5 cm in thickness, which served to absorb infra-red radiation. Beyond the heat filter a lens produced an image of the filament on an opaque diaphragm. The centre of the image fell on a circular hole in the diaphragm. This illuminated aperture served as the source for a collimating lens. The last lens in the system received collimated light and brought it to a focus, thereby producing a circular spot of light a little less than 1 mm in diameter. This spot was centred on the experimental cornea. The maximum illumination produced at the cornea was about 12,000 ft-candles, designated log I = 0 throughout the paper. This value was arrived at in the following way. A hole O-9 mm in diameter was punched in a sheet of opaque, black paper. The hole was placed in the position of an experimental cornea. The spot of stimulating light filled this aperture, beyond which diverging light rays formed a ccne. A surface placed at right angles to the axis of the cone at any distance from the aperture intercepted a circular area of light. A General Electric Co. ft-candle meter (kindly lent by the Tufts Physics Dept.) was placed at an appropriate distance so that its surface intercepted part of such a circular area. The entire surface of the meter was illuminated; the area of the meter was always less than the section of the cone of light in which it was placed. A reading in ft-candles was taken. The diameter of the conic section was measured in the plane of the meter surface, and the area of the section was calculated. The meter
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DARK ADAPTATION
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AMERICANA
191
reading was multiplied by the ratio of this area to that of the aperture placed at the focus. This product gave the illumination of a surface placed in the aperture, i.e. at the cornea. Five determinations were made, 2 on one day, 3 on another, and each after freshly positioning the aperture and the meter. The average of these 5 was 12,000 ft-candles, the range lO,OOO-13,000 ft-candles. (The light reaching the cornea had a greenish hue, a fact which somewhat lowers the precision of specification in ft-candle units.) Lower levels of illumination were obtained with the aid of Wratten neutral tint filters. Test flashes, all of g set duration, but of variable intensity, were supplied by the above source. A rotating sector disk cut the light beam at a focus. Just beyond the rotary shutter was a manual shutter which could be opened to admit a single test flash to the ocellus. The rotary shutter, mounted on a hinged platform, was raised out of the light path during periods of light adaptation. In one series of experiments 2 light sources were used, 1 to produce a steady level of illumination, the other to supply test flashes which were superimposed upon the steady illumination. The latter source in this situation is the same one described above. The source producing the steady illumination was another tungsten lamp. Its output was also filtered through a 2.5 cm layer of O-1 N CuCI, solution, and illumination was controlled with Wratten filters. Ocellar responses appeared together with a signal marker (photocell) on the screen of a dual beam oscilloscope where they could be photographed. Negativity of the ocellar electrode is indicated in the text figures as an upward deflexion. All experiments were conducted at room temperature, 23”-25°C. RESULTS
Dark-adaptation data are usually presented in terms of the light energy required to produce an arbitrary but constant response as a function of time in the dark following a period of light adaptation. In work with human subjects a visual threshold is most frequently chosen as the constant response. It is determined directly during dark adaptation by use of test flashes of variable intensity. In studies on the graded electrical responses of photoreceptor cells it is more convenient to stimulate a dark-adapting eye with a constant test flash and measure changes in the response during the recovery period. Data obtained using the latter method are not directly comparable with those obtained using the constant response method, but by suitable treatment can be converted to a comparable form. The method used here to accomplish this end is in principle the same as that used by HARTLINE (1930) and RIGGS (1937). A complete dark-adaptation curve relates sensitivity to time in the dark from the instant marking the end of the light-adaptation period (time zero in the dark). It proved impossible in this study to obtain the sensitivity at time zero by extrapolation from values obtained later during dark adaptation. A special method had to be devised for determining sensitivity at time zero. In the order of presentation below the method for determining sensitivities at the later intervals will be described first.
192
PHILIP RUCK
After positioning an animal in the apparatus it was left for periods of 2 hr or longer in darkness. During this interval photic sensitivity approached its maximum. At the end of this period the responsiveness of the fully sensitive ocellus was determined. The upper row of Fig. 1 illustrates the responses of the fully sensitive ocellus; this particular ocellus had been in the dark for 3 hr previously. In Fig. 1, log I = -7.0 is a stimulus intensity of slightly higher than threshold value. A series of 3 or 4 responses were obtained at this stimulus intensity. Then the intensity was raised to log I = -6.0 and another series of
LOG I FIG. 2. Solid curve: amplitude of the negative wave of the control (DA) responses in the upper row of Fig. 1, vs log 1. Broken curve: amplitude of the negative wave measured after 1 min of dark adaptation; a 2-min exposure at 12,000 ft-candles preceded determination of each point.
3 or 4 responses was obtained. Similarly for log I = -5.5 and -5-O. There was very little variation among responses to a given stimulus. Throughout this experiment test flashes were admitted at a rate of l/min. This interval allowed more than sufficient time for complete recovery from the effects produced by the previous test flash. This was found to hold for test flashes within at least 3 log units of threshold intensity. The usual procedure was to select test flash intensities within this range. At high levels of stimulus intensity the effects of a single test flash persisted for longer than 1 min, as the experiment of Fig. 8 will illustrate. Of the 3 main components of the responses of Fig. l-the small positive on-wave, the negative wave which follows it, and the very short duration positive off-effect-the first two originate in the photoreceptor cell layer, the last in large fibres of the ocellar nerve (RUSK, 1957). Test flash intensity and degree of dark adaptation influence the amplitude of the negative wave more than other features of the response. Attention was focused on it. The solid curve of Fig. 2 was obtained by plotting the amplitudes of the negative wave (upper row of Fig. 1) elicited from the dark-adapted ocellus against log I. The ocellus was then light-adapted for 2 min. at a cornea1 illumination of 12,000 ft-candles. The responses of the middle row of Fig. 1 were obtained at the
DARK
ADAPTATION
OF
THE
OCELLUS
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AMERICANA
193
indicated times during subsequent dark adaptation using a test flash intensity of log I= -5-O. Another identical light-adaptation period followed immediately and the lower row of Fig. 1 was obtained. In this case, however, the test flash at one minute of dark adaptation had the value log I= -4.5. At 2 and 3 min it was reduced again to log I = -5.0. The 2-min responses of middle and lower rows are virtually identical and so are the 3-min responses. Therefore the states of adaptation produced by the 2 light-adaptation periods must have been the same, i.e. the second period added nothing to the state produced by the first.
FIG. 3. Dark-adaptation curve. The course of dark adaptation for the ocellus of Fig. 1 following a 2-min exposure at 12,000 ft-candles is plotted for the interval, 1 min-30 min. The point at time zero is an approximation and was determined in separate experiments (see text for explanation).
Following several successive identical light-adaptation periods the ocellus of Fig. 1 was tested at 1 min of dark adaptation with test flashes of log I = -5.5, - 5.0, and -4.5. The amplitudes of the resulting responses are plotted as the broken curve of Fig. 2. It and the solid curve are nearly parallel. Four similar experiments have been conducted and in each the l-min curve is parallel, or almost so, to the curve describing the dark-adapted (DA) state. The 1-min curve can therefore be very closely approximated by shifting the DA curve a certain distance to the right. The distance of this shift along the abscissa measures log decreased sensitivity at 1 min of dark adaptation induced by the specified period of light adaptation. As dark adaptation proceeds the curve shifts to the left and is complete when it coincides with the DA curve.
PHILIP
194
RUCK
Having determined this relationship the course of dark adaptation was followed using a single test flash intensity (log I = -5.0). Values of log decreased sensitivity for the ocellus of Fig. 1 are plotted at intervals from the first through to the thirtieth minutes of dark adaptation in Fig. 3. A solid line connects these values. It is apparent that, after only 1 min, sensitivity of this preparation differs from that of the fully dark-adapted state by a factor of approximately 2.1 (Zag deceased sensitivity = 0.32 at 1 min). After the first minute recovery is concerned with a rather trivial gain in sensitivity.
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The course of dark adaptation for 6 ocelli following a 2-min exposure at 12,000 ft-candles. The lowest cnrve is drawn from the Fig. 3 data replotted from the first through to the thirtieth minutes. FIG.
4.
Data of 6 dark-adaptation experiments conducted in the manner described are included in Fig. 4. All 6 ocelli were exposed to a cornea1 illumination of 12,000 ftcandles for 2 min. The lowest curve of Fig. 4 is the Fig. 3 curve replotted from the first through to the thirtieth minutes. Log decreased sensitivity measured at 1 min ranges from 0.32 to 0.67. Otherwise stated, sensitivity after 1 min of dark adaptation is lower by a factor of only 2.1 to 4.7 than in the fully dark-adapted state. Two of the curves rise slightly after the first minute; the reason for this is unknown. If recovery were complete all the curves would reach the zero ordinate. The last increment of sensitivity is recovered very slowly, requiring several hours. The course of this final phase of recovery was not followed systematically, but in a few cases preparations left overnight in the dark were found to be fully recovered the following morning. The total range of sensitivity change during dark adaptation requires a measure of log decreased sensitivity at time zero of dark adaptation. The uppermost point of Fig. 3 supplies this measure. Its value, 4.7, is an approximation based on experiments of a different sort described below. For time zero of dark adaptation a test flash might be delivered at the very instant marking the end -of the light-adaptation period, but this is a technically difficult thing to do. It is much simpler to superimpose the test flash upon the steady level of adapting illumination. One may reason that a given level of steady illumination produces a certain characteristic state of decreased sensitivity. If the interval between the end of light adaptation and the application of the test flash approaches zero more and more closely, the level of decreased sensitivity approaches
DARK
ADAPTATION
OF
THE
OCELLUS
IN
PERIPLANETA
AMERICANA
195
that obtaining in the presence of the adapting light more and more closely. When the interval becomes vanishingly small the level of decreased sensitivity differs negligibly from that obtaining in the presence of the adapting light. In other words the test flash at time zero may just as well be superimposed on the adapting illumination, and this is what has been done.
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5. Amplitude of the negative wave as a function of test flash log I (abscissa) determined at different levels of steady illumination (specified in ft-candles at the right). Values of log I required to produce a 3-mV response at the different levels of light adaptation are plotted in Fig. 6. FIG.
Separate light sources were required to produce the steady level of illumination and the test flashes. Ideally the optical paths of both should have coincided, but this was impossible with the equipment available. The axes of the 2 beams formed an angle of about 22” measured at the cornea. As nearly as possible the preparation was positioned so that a perpendicular to the cornea bisected the angle between the 2 beams. Differences in stimulating effectiveness of the 2 beams were minimized by the following anatomical considerations: (1) the lack of a cornea1 lens (the cornea is a flat, transparent disk); (2) the fact that the cornea is not recessed in the head; and (3) the absence of shielding pigments and the presence of a white tapetum. It is unlikely that the data obtained would have differed significantly if the 2 beams had traversed the same path. The amplitude of the negative wave was determined as a function of test-flash intensity for the dark-adapted state and for increasing levels of light adaptation. Results from 1 animal are plotted in Fig. 5. With increasing light adaptation the curves shift to the right and are approximately parallel. Responses of the same amplitude on the separate curves had the same, or very nearly the same, wave form. The test-flash intensity required to produce a 3 mV response at each level of light adaptation was read off from these curves. The values are plotted in Fig. 6 against the adapting intensity. Data of 2 other identical experiments are included in Fig. 6. The 3 sets of results were so similar that there was no need
196
PHILIP
RUCK
to convert ordinates to a relative scale; i.e. for all three, log I = 0 = 12,000 ft-candles. The highest level of adapting illumination in these experiments was 90 ftcandles, but the value sought is the test-flash intensity required to produce a 3-mV response in the presence of an adapting illumination of 12,000 ft-candles. This desired value was obtained by an extrapolation based on the following assumption, namely, that the curve of Fig. 6 would continually rise at increasing rate to be limited finally by an adaptation level so high that only a test flash of infinite intensity could produce a 3-mV response. The dashed portion of the curve is drawn so that its rate is always increasing, but very slightly. The testflash intensity required to produce the 3-mV response in the presence of a 12,000 ft-candle adapting illumination obtained by this extrapolation is about
:
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I I 0.09 0.9 ADAPTING
I 9 INTENSITY
I so
I I 900 9000 IN FOOT-CANDLES
FIG. 6. Curve used to determine log decreased sev_sitinity at time zero of dark adaptation. Solid portion of curve: data from Fig. 5 and 2 similar experiments. Broken portion of curve: an extrapolation to the 12,000 ft-candle abscissa. See text for explanation.
4.7 log units higher than that required to produce the 3-mV response in the dark-adapted ocellus. The value, 4.7, was therefore introduced into Fig. 3 as Zag decreased sensitivity at time zero. It probably represents a near-minimal figure. Thus the course of dark adaptation in the cockroach ocellus is characterized by almost total recovery during the first minute. Subsequent increase in sensitivity is very slight indeed by comparison. The course of dark adaptation during the first minute was also studied. Responses of the dark-adapted ocellus were obtained in the manner previously described and are illustrated in the upper row of Fig. 7. The ocellus was then exposed to a cornea1 illumination of 12,000 ft-candles for 1 min. After 7 set of dark adaptation a test flash was presented at log I = -5.0 (second row of Fig. 7); this test flash failed to produce a visible response. Identical periods of light adaptation preceded the 12-, 20-, 30-set, and 1-min records. (Earlier it was stated that successive identical exposures produce the same level of adaptation. The possibility of test-flash interaction was avoided by delivering no more than 1 test
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Flo. 8. Effect of duration of the light-adaptation period o n s u b s e q u e n t dark adaptation. U p p e r row: control (DA) responses as a function of log I. Middle row: responses to a test flash at l o g / = - - 5 . 0 following a ]-sec exposure at 12,000 ft-candles. L o w e r row: responses to the same test flash following a l - r a i n exposure at 12,000 if-candles.
DARK ADAPTATION
OF THE OCELLUS
IN SERIPLANETA
AMEhXANA
197
flash during any 1-min interval of dark adaptation and this required repeated periods of light adaptation.) At 12 set there is still no response. Failure to elicit a response in this experiment with a test flash of log I = -5.0 signifies an undetermined, but at least hundredfold, decreased sensitivity. At 20 set a response appears and by comparison with the upper row of control responses it can be seen that sensitivity is lower than in the dark-adapted state by a factor of a little more than ten. This and similar experiments have established that all but the last lo-fold increase of sensitivity occurs within the first 15-20 set of dark adaptation. Finally, brief consideration was given to the effect of duration of light adaptation on subsequent recovery. In early experiments it was found that the course of recovery was the same following exposures to 12,000 ft-candles for durations of 30 see-5 min. Fig. 8 illustrates that a light-adaptation period of only +sec duration produces a decrease in sensitivity almost equal, to that produced by a I-min adaptation. In the upper row are the control responses. In the middle row are responses to test flashes of log I = -5.0 at 30 set and at 1 min following a &sec light adaptation. In the lower row are responses to the same test flash following a l-min adaptation. One must conclude that, at the high level of light adaptation used in this study, almost the maximum effect is produced during the first Q set of the exposure period. DISCUSSION The dark-adaptation curve of Fig. 3 may serve to summarize the principal findings of this study. Light adaptation at a cornea1 illumination of 12,000 ftcandles reduces the sensitivity of the photoreceptor cells by a factor of 50,000 or more below that of the fully dark-adapted state. The course of recovery is almost complete within the first minute after the preparation is returned to darkness. The amplitude of the negative wave of the photoreceptor cell response was used as a criterion of sensitivity because change in it was the easiest feature of the total response to measure. That it is a satisfactory criterion is indicated by the responses shown in Fig. 7 and Fig. 8. The early positive wave increases apace with the negative wave during the recovery period, and the off-spikes, which represent ocellar nerve-fibre impulses, are present whenever a photoreceptor cell response is visible. An argument that the off-spikes are efferent impulses was developed in a previous study (RUCK, 1957), but there is reason to doubt this conclusion now. (Further discussion of the direction of propagation of the offspikes will be deferred to a later paper.) There is no doubt, however, that the off-spikes are postsynaptic responses. Their appearance within the first 20 set of recovery (Fig. 7) indicates that the photoreceptor cells are then capable of carrying out their terminal role in photoreception, namely, that of exciting neurons of the ocellar nerve. Between the appearance of the electrical response to light of the photoreceptor cells and the primary event in photoreception-the absorption of light quanta by a visual pigment-lies a largely unknown series of events. The
198
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RUCK
dark-adaptation data collected in this study appear to supply no information regarding the nature of the event, or events, in this series which limit the rate of recovery. The overall rate of recovery of the roach ocellus during dark adaptation does not differ greatly from that of vertebrate cones following comparably high levels of light adaptation, but is far faster than that of vertebrate rods (HECHT, HAIG, and CHASE, 1937; RIGGS, 1937). But the roach ocellus, on grounds of structure (high degree of convergence of receptor cells on second-order nerve fibres, absence of shielding pigments and presence of a white tapetum) and high photic sensitivity (RUCK, 1957), would appear to play a functional role more like that of a vertebrate rod than a vertebrate cone system. Comparisons more likely to yield clues as to the nature of the dark-adaptation process in insects should, however, be made among insects, and to this end a comparative study of dark adaptation in both compound eyes and ocelli of several insects species has been conducted. The results of that study will be described in a separate paper. REFERENCES HARTLINE H. K. (1930) The dark adaptation of the eye of Limulus, as manifested by its electric response to illumination. J. gen. Physiol. 13, 379-389. HECHT S., HAIG C., and CHASE A. M. (1937) The influence of light adaptation on subsequent dark adaptation of the eye. J. gen. Physiol. 20, 831-850. RIGGS L. A. (1937) Dark adaptation in the frog eye as determined by the electrical response of the retina. J. cell. camp. Physiol. 9, 491-510. RUCK P. (1957) The electrical responses of dorsal ocelli in cockroaches and grasshoppers. J. ins. Physiol. 1, 109-123.
1958, Vol. 2, pp. 189
to 198.
Pergamon Press Ltd., London
DARK ADAPTATION OF THE OCELLUS IN PERIPLANETA AMERICANA : ‘A STUDY OF THE ELECTRICAL RESPONSE TO ILLUMINATION* PHILIP Department
RUCK
of Biology,
(Received
Tufts
17 December
University 1957)
Abstract-Cornea1 illumination at 12,000 ft-candles reduces the sensitivity of the cockroach ocellus by a factor of at least 50,000. At the end of the first minute of dark adaptation, sensitivity differs from that of the fully dark-adapted state by a factor of only 2-5.
INTRODUCTION
IN a previous paper (RUCK, 1957) the structure of the cockroach ocellus and the general nature of its electrical response to light stimulation were described. This ocellus contains a structurally uniform population of receptor cells, the axons of which synapse within the ocellus with second-order neurons; the postsynaptic units, on leaving the ocellus, comprise the ocellar nerve. The electrical response of the organ, recorded with a subcorneal macro-electrode, originates predominantly in the photoreceptor cells. The only contribution the postsynaptic units make to the total response consists of “off-spikes” which originate in the largest ocellar nerve fibres. Since the photoreceptor cell response can easily be recognized in the response of the whole organ, this ocellus is a useful preparation for studies directed at the elucidation of photoreceptor cell processes. The electrical responsiveness of the photoreceptor cells varies with the level of illumination. As illumination increases, responsiveness decreases; i.e. the photoreceptor cells become light-adapted. In darkness following a period of light adaptation, the responsiveness of the receptor cells increases and finally attains a level characteristic of the fully dark-adapted state. It is the purpose of this paper to describe in some detail the changes of electrical responsiveness of the receptor cells which are consequent upon exposure to a high level of illumination. EQUIPMENT
AND
METHODS
The animal preparation and the optical stimulator have been described more fully in a previous paper (RUCK, 1957). Adult male roaches (Periplaneta americana) were immobilized on a small platform with the aid of tacky wax. The head was covered with aluminium foil. through which a small hole was made just large * This work was made possible by grants from the National Science Foundation and the U.S. Public Health Service to Dr. K. D. ROEDER. Some of the apparatus used was obtained under a previous contract between the U.S. Chemical Corps and Tufts University. 189
190
PHILIP RUCK
enough to expose fully the cornea of 1 ocellus. An electrode was passed through the edge of the cornea, the tip coming to lie at the surface of the receptor cell layer. The other (reference) electrode was ensheathed in the flagellum of an antenna. Electrodes were electrolytically pointed, stainless steel wires. These were condenser-coupled to the cathode follower input stage of a Grass P6 amplifier which was operated at a frequency band-pass of 1 c/s-2 kc/s. Condenser-coupling, used to secure baseline stability, inevitably resulted in some distortion of the ocellar responses. From the calibration records of Figs. 1, 7, and 8 it can be seen that the time constant of the amplifier is similar to the time constant of the negative component of the ocellar responses. Therefore, amplitudes of the negative component are somewhat lower than they would have been if recorded with This discrepancy, however, most probably had a negligible DC amplification. effect on the dark-adaptation data in spite of the fact that these were derived from measurements of negative wave amplitudes. As will be explained more fully later in the paper, any given measurement of change in ocellar sensitivity depended on matching a response obtained following light adaptation with some member of a series of control responses obtained before light adaptation. Because any two responses which matched with respect to negative wave amplitude also matched in total wave form, it is probable that both responses were distorted in the same way by the recording circuit. This situation permits one to disregard the distortion as a source of error. The light source was a 6-V, 100-W, strip-filament tungsten lamp powered by a DC supply connected in parallel with a 6-V storage battery. The beam of light passed through a cell containing a 0.1 N CuCI, solution, 2.5 cm in thickness, which served to absorb infra-red radiation. Beyond the heat filter a lens produced an image of the filament on an opaque diaphragm. The centre of the image fell on a circular hole in the diaphragm. This illuminated aperture served as the source for a collimating lens. The last lens in the system received collimated light and brought it to a focus, thereby producing a circular spot of light a little less than 1 mm in diameter. This spot was centred on the experimental cornea. The maximum illumination produced at the cornea was about 12,000 ft-candles, designated log I = 0 throughout the paper. This value was arrived at in the following way. A hole O-9 mm in diameter was punched in a sheet of opaque, black paper. The hole was placed in the position of an experimental cornea. The spot of stimulating light filled this aperture, beyond which diverging light rays formed a ccne. A surface placed at right angles to the axis of the cone at any distance from the aperture intercepted a circular area of light. A General Electric Co. ft-candle meter (kindly lent by the Tufts Physics Dept.) was placed at an appropriate distance so that its surface intercepted part of such a circular area. The entire surface of the meter was illuminated; the area of the meter was always less than the section of the cone of light in which it was placed. A reading in ft-candles was taken. The diameter of the conic section was measured in the plane of the meter surface, and the area of the section was calculated. The meter
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DARK ADAPTATION
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IN PERIPLANETA
AMERICANA
191
reading was multiplied by the ratio of this area to that of the aperture placed at the focus. This product gave the illumination of a surface placed in the aperture, i.e. at the cornea. Five determinations were made, 2 on one day, 3 on another, and each after freshly positioning the aperture and the meter. The average of these 5 was 12,000 ft-candles, the range lO,OOO-13,000 ft-candles. (The light reaching the cornea had a greenish hue, a fact which somewhat lowers the precision of specification in ft-candle units.) Lower levels of illumination were obtained with the aid of Wratten neutral tint filters. Test flashes, all of g set duration, but of variable intensity, were supplied by the above source. A rotating sector disk cut the light beam at a focus. Just beyond the rotary shutter was a manual shutter which could be opened to admit a single test flash to the ocellus. The rotary shutter, mounted on a hinged platform, was raised out of the light path during periods of light adaptation. In one series of experiments 2 light sources were used, 1 to produce a steady level of illumination, the other to supply test flashes which were superimposed upon the steady illumination. The latter source in this situation is the same one described above. The source producing the steady illumination was another tungsten lamp. Its output was also filtered through a 2.5 cm layer of O-1 N CuCI, solution, and illumination was controlled with Wratten filters. Ocellar responses appeared together with a signal marker (photocell) on the screen of a dual beam oscilloscope where they could be photographed. Negativity of the ocellar electrode is indicated in the text figures as an upward deflexion. All experiments were conducted at room temperature, 23”-25°C. RESULTS
Dark-adaptation data are usually presented in terms of the light energy required to produce an arbitrary but constant response as a function of time in the dark following a period of light adaptation. In work with human subjects a visual threshold is most frequently chosen as the constant response. It is determined directly during dark adaptation by use of test flashes of variable intensity. In studies on the graded electrical responses of photoreceptor cells it is more convenient to stimulate a dark-adapting eye with a constant test flash and measure changes in the response during the recovery period. Data obtained using the latter method are not directly comparable with those obtained using the constant response method, but by suitable treatment can be converted to a comparable form. The method used here to accomplish this end is in principle the same as that used by HARTLINE (1930) and RIGGS (1937). A complete dark-adaptation curve relates sensitivity to time in the dark from the instant marking the end of the light-adaptation period (time zero in the dark). It proved impossible in this study to obtain the sensitivity at time zero by extrapolation from values obtained later during dark adaptation. A special method had to be devised for determining sensitivity at time zero. In the order of presentation below the method for determining sensitivities at the later intervals will be described first.
192
PHILIP RUCK
After positioning an animal in the apparatus it was left for periods of 2 hr or longer in darkness. During this interval photic sensitivity approached its maximum. At the end of this period the responsiveness of the fully sensitive ocellus was determined. The upper row of Fig. 1 illustrates the responses of the fully sensitive ocellus; this particular ocellus had been in the dark for 3 hr previously. In Fig. 1, log I = -7.0 is a stimulus intensity of slightly higher than threshold value. A series of 3 or 4 responses were obtained at this stimulus intensity. Then the intensity was raised to log I = -6.0 and another series of
LOG I FIG. 2. Solid curve: amplitude of the negative wave of the control (DA) responses in the upper row of Fig. 1, vs log 1. Broken curve: amplitude of the negative wave measured after 1 min of dark adaptation; a 2-min exposure at 12,000 ft-candles preceded determination of each point.
3 or 4 responses was obtained. Similarly for log I = -5.5 and -5-O. There was very little variation among responses to a given stimulus. Throughout this experiment test flashes were admitted at a rate of l/min. This interval allowed more than sufficient time for complete recovery from the effects produced by the previous test flash. This was found to hold for test flashes within at least 3 log units of threshold intensity. The usual procedure was to select test flash intensities within this range. At high levels of stimulus intensity the effects of a single test flash persisted for longer than 1 min, as the experiment of Fig. 8 will illustrate. Of the 3 main components of the responses of Fig. l-the small positive on-wave, the negative wave which follows it, and the very short duration positive off-effect-the first two originate in the photoreceptor cell layer, the last in large fibres of the ocellar nerve (RUSK, 1957). Test flash intensity and degree of dark adaptation influence the amplitude of the negative wave more than other features of the response. Attention was focused on it. The solid curve of Fig. 2 was obtained by plotting the amplitudes of the negative wave (upper row of Fig. 1) elicited from the dark-adapted ocellus against log I. The ocellus was then light-adapted for 2 min. at a cornea1 illumination of 12,000 ft-candles. The responses of the middle row of Fig. 1 were obtained at the
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indicated times during subsequent dark adaptation using a test flash intensity of log I= -5-O. Another identical light-adaptation period followed immediately and the lower row of Fig. 1 was obtained. In this case, however, the test flash at one minute of dark adaptation had the value log I= -4.5. At 2 and 3 min it was reduced again to log I = -5.0. The 2-min responses of middle and lower rows are virtually identical and so are the 3-min responses. Therefore the states of adaptation produced by the 2 light-adaptation periods must have been the same, i.e. the second period added nothing to the state produced by the first.
FIG. 3. Dark-adaptation curve. The course of dark adaptation for the ocellus of Fig. 1 following a 2-min exposure at 12,000 ft-candles is plotted for the interval, 1 min-30 min. The point at time zero is an approximation and was determined in separate experiments (see text for explanation).
Following several successive identical light-adaptation periods the ocellus of Fig. 1 was tested at 1 min of dark adaptation with test flashes of log I = -5.5, - 5.0, and -4.5. The amplitudes of the resulting responses are plotted as the broken curve of Fig. 2. It and the solid curve are nearly parallel. Four similar experiments have been conducted and in each the l-min curve is parallel, or almost so, to the curve describing the dark-adapted (DA) state. The 1-min curve can therefore be very closely approximated by shifting the DA curve a certain distance to the right. The distance of this shift along the abscissa measures log decreased sensitivity at 1 min of dark adaptation induced by the specified period of light adaptation. As dark adaptation proceeds the curve shifts to the left and is complete when it coincides with the DA curve.
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RUCK
Having determined this relationship the course of dark adaptation was followed using a single test flash intensity (log I = -5.0). Values of log decreased sensitivity for the ocellus of Fig. 1 are plotted at intervals from the first through to the thirtieth minutes of dark adaptation in Fig. 3. A solid line connects these values. It is apparent that, after only 1 min, sensitivity of this preparation differs from that of the fully dark-adapted state by a factor of approximately 2.1 (Zag deceased sensitivity = 0.32 at 1 min). After the first minute recovery is concerned with a rather trivial gain in sensitivity.
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IO TIME
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I
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The course of dark adaptation for 6 ocelli following a 2-min exposure at 12,000 ft-candles. The lowest cnrve is drawn from the Fig. 3 data replotted from the first through to the thirtieth minutes. FIG.
4.
Data of 6 dark-adaptation experiments conducted in the manner described are included in Fig. 4. All 6 ocelli were exposed to a cornea1 illumination of 12,000 ftcandles for 2 min. The lowest curve of Fig. 4 is the Fig. 3 curve replotted from the first through to the thirtieth minutes. Log decreased sensitivity measured at 1 min ranges from 0.32 to 0.67. Otherwise stated, sensitivity after 1 min of dark adaptation is lower by a factor of only 2.1 to 4.7 than in the fully dark-adapted state. Two of the curves rise slightly after the first minute; the reason for this is unknown. If recovery were complete all the curves would reach the zero ordinate. The last increment of sensitivity is recovered very slowly, requiring several hours. The course of this final phase of recovery was not followed systematically, but in a few cases preparations left overnight in the dark were found to be fully recovered the following morning. The total range of sensitivity change during dark adaptation requires a measure of log decreased sensitivity at time zero of dark adaptation. The uppermost point of Fig. 3 supplies this measure. Its value, 4.7, is an approximation based on experiments of a different sort described below. For time zero of dark adaptation a test flash might be delivered at the very instant marking the end -of the light-adaptation period, but this is a technically difficult thing to do. It is much simpler to superimpose the test flash upon the steady level of adapting illumination. One may reason that a given level of steady illumination produces a certain characteristic state of decreased sensitivity. If the interval between the end of light adaptation and the application of the test flash approaches zero more and more closely, the level of decreased sensitivity approaches
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that obtaining in the presence of the adapting light more and more closely. When the interval becomes vanishingly small the level of decreased sensitivity differs negligibly from that obtaining in the presence of the adapting light. In other words the test flash at time zero may just as well be superimposed on the adapting illumination, and this is what has been done.
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5. Amplitude of the negative wave as a function of test flash log I (abscissa) determined at different levels of steady illumination (specified in ft-candles at the right). Values of log I required to produce a 3-mV response at the different levels of light adaptation are plotted in Fig. 6. FIG.
Separate light sources were required to produce the steady level of illumination and the test flashes. Ideally the optical paths of both should have coincided, but this was impossible with the equipment available. The axes of the 2 beams formed an angle of about 22” measured at the cornea. As nearly as possible the preparation was positioned so that a perpendicular to the cornea bisected the angle between the 2 beams. Differences in stimulating effectiveness of the 2 beams were minimized by the following anatomical considerations: (1) the lack of a cornea1 lens (the cornea is a flat, transparent disk); (2) the fact that the cornea is not recessed in the head; and (3) the absence of shielding pigments and the presence of a white tapetum. It is unlikely that the data obtained would have differed significantly if the 2 beams had traversed the same path. The amplitude of the negative wave was determined as a function of test-flash intensity for the dark-adapted state and for increasing levels of light adaptation. Results from 1 animal are plotted in Fig. 5. With increasing light adaptation the curves shift to the right and are approximately parallel. Responses of the same amplitude on the separate curves had the same, or very nearly the same, wave form. The test-flash intensity required to produce a 3 mV response at each level of light adaptation was read off from these curves. The values are plotted in Fig. 6 against the adapting intensity. Data of 2 other identical experiments are included in Fig. 6. The 3 sets of results were so similar that there was no need
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to convert ordinates to a relative scale; i.e. for all three, log I = 0 = 12,000 ft-candles. The highest level of adapting illumination in these experiments was 90 ftcandles, but the value sought is the test-flash intensity required to produce a 3-mV response in the presence of an adapting illumination of 12,000 ft-candles. This desired value was obtained by an extrapolation based on the following assumption, namely, that the curve of Fig. 6 would continually rise at increasing rate to be limited finally by an adaptation level so high that only a test flash of infinite intensity could produce a 3-mV response. The dashed portion of the curve is drawn so that its rate is always increasing, but very slightly. The testflash intensity required to produce the 3-mV response in the presence of a 12,000 ft-candle adapting illumination obtained by this extrapolation is about
:
L
DA LOG
I I 0.09 0.9 ADAPTING
I 9 INTENSITY
I so
I I 900 9000 IN FOOT-CANDLES
FIG. 6. Curve used to determine log decreased sev_sitinity at time zero of dark adaptation. Solid portion of curve: data from Fig. 5 and 2 similar experiments. Broken portion of curve: an extrapolation to the 12,000 ft-candle abscissa. See text for explanation.
4.7 log units higher than that required to produce the 3-mV response in the dark-adapted ocellus. The value, 4.7, was therefore introduced into Fig. 3 as Zag decreased sensitivity at time zero. It probably represents a near-minimal figure. Thus the course of dark adaptation in the cockroach ocellus is characterized by almost total recovery during the first minute. Subsequent increase in sensitivity is very slight indeed by comparison. The course of dark adaptation during the first minute was also studied. Responses of the dark-adapted ocellus were obtained in the manner previously described and are illustrated in the upper row of Fig. 7. The ocellus was then exposed to a cornea1 illumination of 12,000 ft-candles for 1 min. After 7 set of dark adaptation a test flash was presented at log I = -5.0 (second row of Fig. 7); this test flash failed to produce a visible response. Identical periods of light adaptation preceded the 12-, 20-, 30-set, and 1-min records. (Earlier it was stated that successive identical exposures produce the same level of adaptation. The possibility of test-flash interaction was avoided by delivering no more than 1 test
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Flo. 8. Effect of duration of the light-adaptation period o n s u b s e q u e n t dark adaptation. U p p e r row: control (DA) responses as a function of log I. Middle row: responses to a test flash at l o g / = - - 5 . 0 following a ]-sec exposure at 12,000 ft-candles. L o w e r row: responses to the same test flash following a l - r a i n exposure at 12,000 if-candles.
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flash during any 1-min interval of dark adaptation and this required repeated periods of light adaptation.) At 12 set there is still no response. Failure to elicit a response in this experiment with a test flash of log I = -5.0 signifies an undetermined, but at least hundredfold, decreased sensitivity. At 20 set a response appears and by comparison with the upper row of control responses it can be seen that sensitivity is lower than in the dark-adapted state by a factor of a little more than ten. This and similar experiments have established that all but the last lo-fold increase of sensitivity occurs within the first 15-20 set of dark adaptation. Finally, brief consideration was given to the effect of duration of light adaptation on subsequent recovery. In early experiments it was found that the course of recovery was the same following exposures to 12,000 ft-candles for durations of 30 see-5 min. Fig. 8 illustrates that a light-adaptation period of only +sec duration produces a decrease in sensitivity almost equal, to that produced by a I-min adaptation. In the upper row are the control responses. In the middle row are responses to test flashes of log I = -5.0 at 30 set and at 1 min following a &sec light adaptation. In the lower row are responses to the same test flash following a l-min adaptation. One must conclude that, at the high level of light adaptation used in this study, almost the maximum effect is produced during the first Q set of the exposure period. DISCUSSION The dark-adaptation curve of Fig. 3 may serve to summarize the principal findings of this study. Light adaptation at a cornea1 illumination of 12,000 ftcandles reduces the sensitivity of the photoreceptor cells by a factor of 50,000 or more below that of the fully dark-adapted state. The course of recovery is almost complete within the first minute after the preparation is returned to darkness. The amplitude of the negative wave of the photoreceptor cell response was used as a criterion of sensitivity because change in it was the easiest feature of the total response to measure. That it is a satisfactory criterion is indicated by the responses shown in Fig. 7 and Fig. 8. The early positive wave increases apace with the negative wave during the recovery period, and the off-spikes, which represent ocellar nerve-fibre impulses, are present whenever a photoreceptor cell response is visible. An argument that the off-spikes are efferent impulses was developed in a previous study (RUCK, 1957), but there is reason to doubt this conclusion now. (Further discussion of the direction of propagation of the offspikes will be deferred to a later paper.) There is no doubt, however, that the off-spikes are postsynaptic responses. Their appearance within the first 20 set of recovery (Fig. 7) indicates that the photoreceptor cells are then capable of carrying out their terminal role in photoreception, namely, that of exciting neurons of the ocellar nerve. Between the appearance of the electrical response to light of the photoreceptor cells and the primary event in photoreception-the absorption of light quanta by a visual pigment-lies a largely unknown series of events. The
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dark-adaptation data collected in this study appear to supply no information regarding the nature of the event, or events, in this series which limit the rate of recovery. The overall rate of recovery of the roach ocellus during dark adaptation does not differ greatly from that of vertebrate cones following comparably high levels of light adaptation, but is far faster than that of vertebrate rods (HECHT, HAIG, and CHASE, 1937; RIGGS, 1937). But the roach ocellus, on grounds of structure (high degree of convergence of receptor cells on second-order nerve fibres, absence of shielding pigments and presence of a white tapetum) and high photic sensitivity (RUCK, 1957), would appear to play a functional role more like that of a vertebrate rod than a vertebrate cone system. Comparisons more likely to yield clues as to the nature of the dark-adaptation process in insects should, however, be made among insects, and to this end a comparative study of dark adaptation in both compound eyes and ocelli of several insects species has been conducted. The results of that study will be described in a separate paper. REFERENCES HARTLINE H. K. (1930) The dark adaptation of the eye of Limulus, as manifested by its electric response to illumination. J. gen. Physiol. 13, 379-389. HECHT S., HAIG C., and CHASE A. M. (1937) The influence of light adaptation on subsequent dark adaptation of the eye. J. gen. Physiol. 20, 831-850. RIGGS L. A. (1937) Dark adaptation in the frog eye as determined by the electrical response of the retina. J. cell. camp. Physiol. 9, 491-510. RUCK P. (1957) The electrical responses of dorsal ocelli in cockroaches and grasshoppers. J. ins. Physiol. 1, 109-123.