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CO2-induced changes in the ERG of the fly, Sarcophaga bullata. A component analysis
J. Insect Physiol., 1975,Vol. 21,pp. 181to 197.Pergamon Press. Printed in Great Britain
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BULLATA. ELLIS
IN THE ERG OF THE FLY, A COMPONENT ANALYSIS* R. LOEWt
Department of Biology, University of California, Los Angeles, California, U.S.A. (Received 20 April 1974; revised 5 June 1974)
Abstract-The changes in the ERG of the fly, Sarcophaga btdlata, with increases in the ambient carbon dioxide concentration are described and analysed using a method of successive algebraic subtraction of normal&d responses. On the basis of the behaviour of the five identifiable waves of the ERG before, during, and after carbon dioxide exposure, it is concluded that four components are responsible for the complex ERG. One of these components, which is unaffected by carbon dioxide, apparently arises from the receptor cells, while the other three components have a ganglionic origin. The waveforms of the four proposed components are derived by substracting normalised responses differing with respect to only one of the five waves. The finding that the loss of the waves, through the action of carbon dioxide, proceeds in a sequential and regular manner makes this approach to a component analysis possible. It is suggested that this sequential loss of components does not reflect differences in the basic sensitivity of visual neurons to carbon dioxide, but results from factors such as diffusion barriers and/or chemical processes which establish a decreasing radial concentration gradient of carbon dioxide within the visual ganglia. INTRODUCTION THE COMPLETEresolution
of an ERG into components should yield the number, waveform (under specified conditions), and anatomical origin of each electrical process summing to produce the complex, cornea1 ERG. Having this information, one can attempt to interpret changes in the ERG in terms of alterations of one or more of these components. The ideal situation is to be able to examine each component in isolation from any other, utilizing a non-injurious and reversible isolation method. While no single technique yet described satisfies these criteria, the judicious use of carbon dioxide can, in certain instances, approximate this ideal situation. GOLDSMITH (1960) was the first to describe the effect of carbon dioxide on the insect ERG. He found that soon after blowing carbon dioxide gas around the body of a honey-bee (Apti tnellifera), the fast transients were lost from the ERG leaving a simple cornea-negative wave. Goldsmith suggested that this remaining potential * This investigation was aided by grant B-1509 from the Division of Research Grants and Fellowships, National Institutes of Health, U.S. Public Health Service. t Present address: M.R.C. Vision Unit, C.R.P.C., University of Sussex, Falmer, Brighton BNl 9QG, England. 181
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originated from the receptor cell layer. These findings were confirmed and expanded by LEUTSCHER-HAZELHOFF and KUIPER (1964) using the blowfly, Calliphora mythrocephala. Their electrode advancement data showed that the first effect of carbon dioxide was suppression of ganglionic spike activity followed by a loss of the slower, light-dependent ganglionic potentials. The negative wave remaining after these losses was localized to the base of the receptor cells adjacent to the basement membrane. Ganglionic activity was restored upon removal of the carbon dioxide and the ERG returned to its pre-treatment waveform. On the basis of these studies and those of WOLBARSHTet aZ. (1966), and EICHENBAUMand GOLDSMITH(1968), it is now generally accepted that carbon dioxide isolates the receptor cell component of the insect ERG through the reversible and noninjurious suppression of the ganglionic components. Carbon dioxide appears to have no effect on the electrical activity of the receptor cells. The differential sensitivity of the ganglionic and receptor cell components to carbon dioxide has been used to isolate the receptor cell component for further study (WOLBARSHTet al., 1966; EICHENBAUMand GOLDSMITH, 1968), and/or to localize an already identified component to the ganglionic or receptor layers (LEUTSCHER-HAZELHOFF and KUIPER, 1964; LOEW, 1973a, b; KOOPOWITZet al., 1974). Little attention has been paid to the waveform changes accompanying the loss of the ganglionic components. It is shown in this report that an analysis of these changes gives information on the number, waveform, and origin of the ganglionic components.
MATERIALS AND METHODS Flesh-flies, Sarcophaga bullata, were raised in the laboratory using the technique of DORMANet al. (1938) as modified by WILKINS (1967) and LOEW (1973b). The flies were bred and maintained, and all experiments were performed at 22°C. Only flies between 1 and 3 weeks after emergence were examined, since older flies may show age-related changes in the waveform of their ERG (LOEW, 1973b). A fly was lightly narcotized with carbon dioxide and placed, head foremost, into one end of a glass tube having an inside diameter slightly greater than that of the fly. Using a wooden stick applied to the thorax, the fly was advanced down the tube until its head protruded from the opposite end. A bead of beeswax melted between the dorsal surface of the thorax and the rim of the tube prevented the escape of the fly. The head was immobilized by fixing it to the anterior aspect of the thorax with some melted beeswax. One of the compound eyes, which served as the indifferent lead for electrical recording, was blackened with several coats of enamel paint as were the three ocelli. As an added precaution against light striking these photoreceptors, an aluminium shield was placed around the blackened eye and ocelli and secured with wax. The glass tube holding the fly was attached to a moveable platform and placed within the recording chamber so that the unpainted eye, from which the ERG was to be recorded, was at the focus of a condensing lens present within the chamber.
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The recording chamber was a double-walled, copper enclosure mounted on the same optical bench as the stimulating apparatus. Water, circulating between the walls, kept the inside temperature at 22°C. A glass window allowed for the entry of the stimulating and adapting lights, while two other openings were used to admit the various atmospheres employed in the study. The control atmosphere was either air, or a mixture of SO% nitrogen and 20% oxygen. Both of these atmospheres produced the same results. The concentration of carbon dioxide was adjusted by substituting it for nitrogen in the gas mixture entering the chamber. This was done using a series of Venturi flow meters which allowed changes in the concentrations of the three gases (i.e. oxygen, nitrogen, and carbon dioxide) to be made while keeping the flow rate constant a 1 l./min. At no time was the oxygen concentration allowed to fall below 20%. Photic stimulation was provided by a Grass PS2 xenon flashlamp set at its highest intensity, referred to throughout as unit intensity, and was equivalent to 9 x lo5 horizontal candle power as deduced from the figures given by Grass. The intensity was attenuated with Corning neutral density filters. The adapting light source was a tungsten ribbon-filament lamp operating at 6.3 V d.c. The intensity of the adapting light at the surface of the compound eye was measured using a YSI radiometer and found to be 10 mW cm-2. After passing through a glass heat filter (Edmund Scientific) and a 5 cm water cell, the adapting light was mixed with the stimulus by a beam splitter. This combined beam was directed onto the compound eye by a series of condensing lenses. To provide a uniform intensity across the surface of the eye, a diffusing screen was placed between the eye and the field lens. The ERG was recorded using stainless steel microelectrodes (tip dia. 10-20 pm) insulated down to the tip with Insul-X resin. One electrode was placed subcorneally in each compound eye using a micromanipulator. A Kiethley 103 preamplifier having a bandwidth of 0.1 to 30,000 Hz provided differential preamplification. This amplified signal was displayed on the upper beam of a Tektronix 502 oscilloscope and recorded on film for later study. A stimulus mark, derived from a photodiode, and time marks were displayed on the lower beam. RESULTS
Dark-adapted eyes
(1) Control atmosphere (0% carbon dioxide). A fly was prepared as previously described and allowed to dark adapt within the recording chamber for 20 min. Stimuli were then presented, allowing a minimum of 2 min between each flash for the eye to recover from the previous flash. Control experiments showed that recovery of the response was complete within 1 min after a flash; however, the 2 min protocol was adopted as a precautionary measure. The ERG elicited by stimuli of various intensities is seen in Fig. 1. These responses are typical of those recorded from more than 200 flies studied, although the response amplitudes in individual cases varied by several millivolts, depending upon the impedance of the recording circuit. The unit intensity response (0) is
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similar to that previously reported for Surcophuga (HASSENSTEIN,1957; FOUCHARD and CARRICABURU, 1970 ; LOEW, 1973a, b) and consists of a series of waves designated alpha, beta, gamma, delta, and epsilon in the order of their appearance in the response.
I50 msec. FIG. 1. Control conditions-the ERG of the dark-adapted eye with decreasing stimulus intensity. The numbers to the left of the records under the heading D indicate the value of the density filter used for attentuation. The arrow indicates the stimulus.
Tenfold attenuation of the stimulus (- 1) caused a loss of the epsilon wave and revealed wavelets on the descending limb of the negative beta wave. The amplitudes and time-to-peak (TTP) values of the remaining waves were only slightly affected by the intensity decrease, and the loss of the epsilon wave did not markedly affect the shape of these waves. Further attentuation ( - 2) decreased the amplitude of all waves as well as increasing their TTP values. At a still lower intensity (- 3) the beta wave was lost from the response, leaving the delta wave and a single positive wave resulting from a fusion of the alpha and gamma waves. The amplitude of the delta wave continued to decrease with decreasing stimulus intensity until it disappeared from the response altogether, leaving a positive wave of low amplitude
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5). It is important to note that this positive wave is associated with the alpha and gamma waves in the unit intensity response (0). (2) Eflects of carbon dioxide. It is known that carbon dioxide influences the behaviour of insects and is capable, in sufficient concentrations, of producing a narcosis. A preliminary test confirmed that carbon dioxide could alter the waveform of the ERG of Surcophuga at concentrations below those producing complete narcosis. To examine these alterations, the ERG was recorded from flies exposed to increasing concentrations of carbon dioxide. A fly was dark adapted at 0% carbon dioxide for 20 min following which a unit intensity flash was given and the ERG recorded. The concentration of carbon dioxide was then increased to 5% as previously described and a 5-min period allowed before presentation of the test flash. The carbon dioxide was next increased to 10% and, after another 5-min period, the ERG recorded. This procedure was repeated with 5% step increases until the 60% level was reached. The chamber was then flushed with 100% oxygen for 2 min and the atmosphere returned to 0% carbon dioxide. The ERG was monitored every 2 min until recovery was complete, which could take as long as 30 min. The results for a particular fly were not accepted unless its ERG had recovered within this 30-min period. The 5-min lag between the gas increase and the stimulus was necessary to overcome the problems associated with mixing of each new atmosphere and to minimize the effects of any difference in carbon dioxide sensitivity between flies. The responses recorded using lag periods of up to 10 min were, in most cases, similar to those obtained using 5-min periods indicating a steady-state condition within the 5 to 10 min interval. However, after 10 min at a particular carbon dioxide concentration inconsistent changes were often seen in the response waveforms, particularly for those obtained below 15% and above 40% carbon dioxide. Responses typical of 18 flies exposed to different levels of carbon dioxide are seen in Fig. 2. The percentages to the right indicate the carbon dioxide concentration. At 0% the response was identical to that observed in air (Fig. 1). Between 0 and 25% carbon dioxide there was an increase in the amplitude of the epsilon wave and an increased TTP for the delta and epsilon waves. There was also a slight increase in beta wave amplitude. At 30% carbon dioxide the amplitude of the delta and epsilon waves started to decrease until, between 45 and SO%, they disappeared from the response. At 50% the alpha wave was reduced to a small transient on the ascending limb of the beta wave which, under these circumstances, had a faster time-course than the alpha wave. Oscillations with an average frequency of 150 Hz were present on the descending limb of the beta wave at 50% carbon dioxide. In addition, the 50% record shows a positive wave following the beta wave which is not comparable to the epsilon wave. The oscillatory activity extends onto this positive wave. Above the 50% carbon dioxide level, the response simplified to a monophasic negative wave (60%) apparently associated with the beta wave as suggested by the progression of waveform changes between 0 and 60% carbon dioxide. A return to 0% carbon dioxide led to recovery of the ERG indicating that the effects of carbon dioxide were reversible. (-
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50%
55%
25%
60%
0%
I
125MSEC.
FIG. 2. Effect of carbon dioxide on the ERG from dark-adapted eyes. The percentages to the right of each record indicate the carbon dioxide concentration (at atmospheric pressure) in the gas mixture. The remaining gases were 20% oxygen and nitrogen to make 100%. All stimuli were unit intensity. A striking feature of carbon dioxide narcosis in insects is the speed with which induction and recovery occur. Exposure of Sarcophaga to 80% carbon dioxide produces narcosis within 30 sec. Experiments were performed to determine the rate with which carbon dioxide affects the ERG. A fly was dark adapted for 15 min at 0% carbon dioxide and the response to a unit intensity flash recorded. The carbon dioxide concentration was then rapidly increased to 80% allowing 2 min between the flash and the increase for the eye to recover. At specific intervals, after the increase in carbon dioxide, a unit intensity stimulus was given and the response recorded. Fig. 3 shows typical records for some 30 flies. The time to the right of each record indicates the interval between the carbon dioxide increase and the test
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50 mwc.
FIG. 3. Effect of 80% carbon dioxide on the responses to test flashes given at various intervals after carbon dioxide-dark adapted eyes. Record 0 is the response to a flash before carbon dioxide, while the record 10 set indicates a flash was delivered 10 set after carbon dioxide, etc. In each case the carbon dioxide was preceded by a flash such as record 0 given 2 min before the start of the carbon dioxide treatment. The carbon dioxide was maintained at 80% from the start of the experiment until the second test flash (i.e. 10 set, 20 set, etc.).
flash except for record 0 which is the response to the initial test flash. It is seen that very soon after the carbon dioxide increase (10 set) the epsilon wave apparently disappeared. This was followed by the loss of the gamma and delta waves at 30 set and the alpha wave at 60 sec. After 120 set at 80% carbon dioxide only a monophasic negative wave remained. 6
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After 120 set at 80% carbon dioxide, flushing the chamber with 100% oxygen atmosphere (i.e. 80% nitrogen and 20% from narcosis are seen in Fig. 4. In this
the atmosphere was returned to 0% by for 30 set and re-introducing the control oxygen). The responses during recovery case the time of each record is measured
IO
60
FIG. 4. Recovery of the ERG after exposure to 80% carbon dioxide for 120 secdark adapted eyes. Record 0 is the response while the fly was still exposed to 80% carbon dioxide. The other records were taken at various intervals after the removal of the carbon dioxide. After the 0 set flash, the chamber was flushed with 100% oxygen for 30 set, following which an atmosphere of 20% oxygen and 80% nitrogen was admitted. from the return to the control atmosphere
except for record 0 which is the response recorded just prior to the oxygen flush. It is seen that recovery proceeded in a manner almost the reverse of induction. The first wave to recover was the alpha followed, in order, by the delta, gamma, and epsilon waves. Recovery was usually complete by 180 set after carbon dioxide removal. It is interesting that the time necessary for recovery depends upon the duration of narcosis. In cases where the fly was kept at 80% carbon dioxide for 30 min, recovery could take as long as 45 min.
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Light-adapted eyes (1) Con&o1 at-e
(0% carbondioxide). Flies were dark adapted for 15 mm, their ERG recorded, and then light adapted at maximum background intensity (10 mW . cm-“) for 20 min before testing. Typical responses to stimuli of various intensities superimposed on the background are seen in Fig. 5. The unit intensity
i
E
m
L50 msec.
FIG.
5.
Control conditions-the ERG of the light-adapted eye with decreasing stimulus intensity (nomenclature as in Fig. 1).
response (0) consisted of the same five waves recorded from dark-adapted eyes. However, the slopes of the waves were much steeper in the light-adapted state which reduced the duration of the ERG to about 100 msec. A fast negative transient preceded the alpha wave at unit intensity, but disappeared with a slight intensity decrease (- 1). Light adaptation reduced the amplitude of all the waves, with the epsilon wave showing the greatest reduction. In fact, in about 40 per cent of the light-adapted eyes studied, an epsilon wave could not be detected in the unit intensity response. The sequence of changes in waveform with decreasing stimulus intensity for light-adapted eyes was similar to that seen for dark-adapted eyes (Fig. 1). There was a decrease in amplitude of the negative beta and delta waves, and the positive
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epsilon wave; and an increase of the positive alpha and gamma waves. As with the dark-adapted eye, only a positive-dominant wave remained at low stimulus intensities (Fig. 4). (2) Eflects of carbon da’oxide. Responses typical of 22 light-adapted flies at various intervals after rapidly increasing the carbon dioxide concentration to 80% are seen in Fig. 6. The first effect was a slight increase in epsilon wave amplitude 0
30
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FIG. 6. Effect of 80% carbon dioxide on the responses to test flashes given at various intervals after carbon dioxide-light adapted eyes. Experimental procedures were like those for Fig. 3.
reaching a maximum at 30 sec. During the next 45 set there was an increase in amplitude, TTP, and time-course of the delta wave which was never observed in similar experiments using dark-adapted flies (Fig. 3). During this same period (O-75 set) there was little change in the alpha and beta waves. After 75 set the amplitude of the delta wave decreased and the alpha wave was lost leaving a monophasic negative wave similar to that isolated from dark-adapted eyes (Fig. 3).
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Recovery of the response after removal of the carbon dioxide followed a course that was the reverse of that seen during induction. As seen in Fig. 7, the first wave to recover was the alpha, followed by the gamma, delta, and epsilon waves. Again, the recovered response (60 set) was identical to that recorded before carbon dioxide exposure.
40rrc.
FIG. 7. Recovery of the ERG after exposure to 80% carbon dioxide for 120 seclight adapted eyes. Experimental procedures were like those for Fig. 4. INTERPRETATION
OF RESULTS
(1) The number of components
The records obtained with both light and dark adapted eyes under control conditions (Figs. 1,s) indicate at least two components-a positive and a negative. In no other way can the positive alpha and epsilon waves, and the negative beta
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and delta waves be explained. Assuming only these two components, the beta and delta waves would be the product of the negative component, while the epsilon wave would be the tail end of the positive component producing the alpha wave. Differences in latency and waveform of the two components would account for the gamma wave. The positive component would have the lowest threshold since only a positive wave remains at low stimulus intensities (Figs. 1, 5). The similarities between the responses from dark- and light-adapted eyes suggest that light adaptation does not alter the waveform of the components, but only alters their amplitude and latency. While such a two-component scheme might be manipulated so as to explain the responses recorded under control conditions, it fails to explain those recorded at elevated carbon dioxide concentrations. In Fig. 2 it is clear that alterations in both the amplitude and TTP of the delta wave occur without any observable effect on the beta wave. This is particularly evident from the responses at 30, 35, and 40% carbon dioxide. This independence of the beta and delta waves is also seen in the records of Fig. 6 from light-adapted eyes. The marked changes in the delta wave between 30 and 90 set after the carbon dioxide increase have little effect on the beta wave. The records from the recovery period also demonstrate this independence (Figs. 4, 7). The most reasonable explanation for this behaviour is that a separate component produces each negative wave of the ERG. It can also be argued, though less convincingly, that there are two positive components. This separation of the positivity into two components is based on the behaviour of the epsilon wave. It is seen in Fig. 1 that the epsilon wave disappeared with a tenfold decrease from unit stimulus intensity. To explain this loss using a single positive component scheme would require either that the duration of the component producing the delta wave increases, or that the duration of the positive component decreases. Both of these changes would reduce the summed potential in the temporal region of the epsilon wave. However, such changes would be expected to affect the slopes of the delta wave. The loss of the epsilon wave does not, however, alter the slopes of the delta wave or, for that matter, the slopes of any of the remaining waves. Thus, the loss of the epsilon wave must result from an amplitude decrease of a second positive component in addition to that producing the alpha wave. The only other explanation consistent with a single positive component is one that assumes changes in the waveform and amplitude of the two negative components in synchrony with changes in the positive component resulting in a loss of the epsilon wave. The sum of such synchronous changes would then fortuitously yield the response seen at density 1 in Fig. 1 which is almost identical to the unit intensity response except for the absence of the epsilon wave. Such exact and specific compensation, though possible, seems improbable in view of the regular changes in response waveform with further decreases in stimulus intensity (Fig. 1). The responses with carbon dioxide also support two positive components. It is seen in Fig. 2 that alterations in amplitude and TTP of the epsilon wave are not attended by changes in either the alpha or gamma waves. This is particularly
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noticeable between 5 and 25% carbon dioxide. While it is possible to explain these changes in terms of complicated alterations of two negative and one positive component, the proposal of a second positive component explains the data in the simplest way. In summary, the data are best explained by a component scheme of two positive and two negative components. (2) Waveform and origin of thefour proposed components The waveform, relative amplitude, and temporal relationships of the four proposed components are seen in Fig. 8, as is their algebraic summation. I believe
FIG. 8. The four components proposed for the ERG of SarcqAaga based on the data presented here. The dashed line is zero potential with an upward deflection indicating positivity of the cornea with respect to the indifferent electrode. The waveform in the lower half of the figure is the algebraic summation of the four proposed components. that
three of these components correspond to those proposed by FOUCI-IARD and CARRICABURU (1972) and have, therefore, adopted their terminology for the components NR, P, and NS. The fourth component, from this study, has been labelled PL because it is positive and appears late in the response.
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Ideally, in order to analyse the waveform of each component of the ERG, it would be necessary to isolate each one from the others and to observe it in an uncomplicated form. Of the four proposed components, only the NR has been isolated in this manner. The waveform of this component was taken to be that of the negative wave remaining after increasing the carbon dioxide concentration (Fig. 2, 60%). In keeping with the known effects of carbon dioxide on the insect ERG, it is suggested that this negative potential arises from the receptor cells and is the receptor cell component (NR). The waveform of NR is similar to the receptor cell component isolated by selective recording techniques (AUTRUMand GALLWITZ, 1951; MOTE, 1968, 1970; HEISENBERG,1972), by physical isolation of the receptor cell layer (AUTRUM,1958; WOLBAR~HTet al., 1966; EICHENBAUMand GOLDSMITH, 1968; LOEW, 1973b), and by drugs (AUTRUMand HOFFMANN,1957; CARRICABURU, 1971). It is seen that the peak of the NR component corresponds to that of the beta wave in the control responses (Fig. 2). Knowing the waveform of the NR component, the waveforms and relative amplitudes of the remaining three components were derived by a process of successive subtraction. For this analysis each response was normalized by assigning to the unit intensity delta wave amplitude the value of 100 per cent and referring all other amplitudes to this level. It has previously been shown that responses normalized in this manner are congruent at all stimulus intensities (LOEW, 1973b). The rising phase of the P component must have a steep slope in order to account for the appearance of the alpha wave before the beta wave (Fig. 8). This slope and the relative amplitude of P were determined by subtracting the NR component from the unit intensity response and finding the magnitude of the positivity needed to account for the amplitude difference between the NR component and the beta wave (Figs. 1, 8). Two methods were used to derive the remainder of the P component (i.e. that part within the envelope of the NS component). The first involved extrapolation from the shape of the positive wave isolated by decreasing the stimulus intensity (Fig. l), assuming that only the amplitude and TTP of the P component changed with stimulus intensity. The second method was to subtract the 60% response from the 50% one of Fig. 2. Both of these operations yielded similar results which, when combined with the initial subtraction of the NR component, produced a positive wave similar in waveform to the potential recorded across the lamina ganglionaris in Sarcophaga (MOTE, 1968) and Drosophila ( HEISENBERG,1972). The analysis of the second negative component (NS) was made difficult by the interference of both the NR and P components which interact temporally with the NS (Fig. 8). Accordingly, the procedure was first to subtract the known components (i.e. NR and P) from the unit intensity response and thus to obtain the peak and a portion of the descending limb of NS. The first part of this component, from its origin at the baseline to within 75 per cent of the peak height, is purely speculative and could start somewhat later than is shown in Fig. 8. The terminal portion of the descending limb obtained from the above subtractions is obviously affected by the epsilon wave. This effect was abolished by applying the above subtractions to
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responses recorded after carbon dioxide exposure (Figs. 6,7) and to those recorded at low stimulus intensity (Fig. l), both of which are similar to the unit intensity response except for the presence of the epsilon wave. Using these procedures, the terminal portion of NS was established to fall to the baseline as shown in Fig. 8. The same procedure was used to derive the PL component. In this case responses comparable in all respects except for the presence of the epsilon wave were subtracted from each other. Thus, by subtracting record - 1 from record 0 of Fig. 1, after appropriate normalization, the waveform and relative amplitude of PL were ascertained. The initial portion of PL is uncertain and may be different from that seen in Fig. 8. That the P, NS, and PL components are of ganglionic origin can be assumed from their sensitivity to carbon dioxide, but by using carbon dioxide alone it is not possible to locate the site within the ganglionic layers from which each component arises. On the basis of developmental and surgical ablation studies on the compound eye of Sarcophaga, the P, NS, and PL components have been localized to the lamina ganglionaris, medulla, and lobula, respectively (LOEW, 1973a, b). Assuming these assignments to be correct, it appears that the order in which the components are affected by carbon dioxide is correlated with the position of its ganglionic layer of origin. That is, the effect of carbon dioxide seems to progress from the proximal ganglionic layers (e.g. lobular) distally, towards the receptor cell layer. This sequential effect of carbon dioxide on the ganglionic components is evident from the records of Figs. 2,3, and 6, in which first the PL, then NS, then the P component is affected and eventually lost from the response leaving the NR. The reversed order is observed during recovery of the components (Figs. 4, 7). It thus appears that the neuronal layers show a differential sensitivity to carbon dioxide. If this differential sensitivity is real it means that different neurons possess very different carbon dioxide sensitivities and that neurons with the same sensitivity are grouped within the same nervous layer. While this is possible, it seems unlikely since the neurons in the different layers are similar electrically and morphologically (TRUJILLO-CEN~Z, 1972). It is possible, however, that the differential sensitivity is only apparent and that all the neurons within the visual system have roughly the same carbon dioxide sensitivity. This follows if one assumes that within the ganglia a radial concentration gradient in equilibrium with the atmosphere is established such that the carbon dioxide concentration is greater proximally. This gradient could arise from physical barriers to diffusion of carbon dioxide such as that imposed by the basement membrane (KOOPOWITZ et al., 1974), or from differences in pH or buffering capacity within and around the neuronal layers. Such a gradient is consistent with the steady-state effects observed in Fig. 2 as well as the dynamic changes seen in Figs. 3 and 6. Unfortunately, no evidence for such a gradient is available. CONCLUSIONS From data obtained before, during, and after exposure to carbon dioxide, it is concluded that the ERG of Sarcophaga is resolvable into four components, one
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(NR) of receptor origin and three (P, NS, PL) arising from the ganglionic layers. In addition, all or part of the waveform of each component was derived by successive subtraction utilizing both control and experimental responses. The component scheme so obtained (Fig. 8) agrees with that previously proposed for Sarcophaga on the basis of records obtained during differentiation of the compound eye (LOEW, 1973a). The scheme is also consistent with the potentials recorded across individual visual layers of Sarcophqa (MOTE, 1968, 1970), and with generalized component schemes proposed for other dipterans (FOUCHARD and CARRICABURU, 1972). The amount of information which can be derived using carbon dioxide will generally depend on the number of carbon dioxide-sensitive components present. If, for example, the ERG under study is dominated by the receptor potential or consists of more than one receptor cell component, carbon dioxide will be of limited value. Such was the case in a recent analysis of the ERG of the beeswax moth, Galleria trzdonella, which was found to have three components only one of which was carbon dioxide-sensitive (KOOPOWITZ et al., 1974). However, even in this study carbon dioxide provided data on the site of origin of the components. It remains for carbon dioxide to be adopted as a routine tool in the resolution of the insect ERG. Acknowle&ments-I should like to thank Professor F. CRIBCITELLI for hia aid and support during the course of this investigation, and Professor H. J. A. DARTNALLand Dr. J. K. BOWMAKER for critically reading the manuscript. REFERENCES AUTRUMH. (1958) Electrophysiological analysis of the visual systems in insects. Exp. Cell Res. (Sup~l.) 5,426-439. AUTRUMH. and GALLWITZU. (1951) Zur Analysis der Belichtungspotentiale des Insektenauges. Z. vergl. Physiol. 33,407-435. AUTRUMH. and HOFFMANNE. (1957) Die Wirkung von P&rotor& und Nikotin auf das Retinogramm von Insekten. 2. Natutforsch 12b, 752-757. CARRICABURU P. (1971) Action du parathion sur 1’6lectror&inogramme de la mouche Musca domestica. C. R. Ad. Sci., Paris 273D, 2576-2578. DORMANS. C., HALE W. C., and HOPKINSV. M. (1938) Laboratory rearing of flesh&s. J. econ. Ent. 31, 44-51. EICHENBAUMD. and GOLDSMITHT. H. (1968) Properties of intact photoreceptor cells lacking synapses. J. exp. Zool.l69,15-32. FOUCH~~RD R. and CARRICABURU P. (1970) La response electroretinographique oscillante chez cinq especb de mouche. Vision Res. 10,655-667. FOUCHARDR. and CARRICABURU P. (1972) Analyse de l’Clectror&inogramme de l’insecte. Vision Res. 12, 1-15. GOLDSMITHT. H. (1960) The nature of the retinal action potential, and the spectral sensitivities of ultraviolet and green receptor systems of the compound eye of the worker honeybee. g. gen. Ph siol. 43,775-800. I-&&ENSTBINB. (1957) i; ber Belichtungspotentiale in den Augen der Fliegen Sarcophaga und Eristalis. J. Insect Physiol. 1, 124-130. HEISBNBERG M. (1971) Separation of receptor and lamina potentials in the electroretinogram of normal and mutant Drosophila. J. exp. Biol. 55, 85-100. KOOPOWIT~H., STONEG., and MARTINEZ D. (1974) Components of the ERG of the moth, Galleria mellonella. J. Insect Physiol. 20, 9-20.
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LEUTSCHEI+HAZELHOFF J. T. and KUIPER J. (1964) Response of the blowfly (CaUiphora erythroce&Zu) to light flashes and to sinusoidally modulated light. Docum. Ophthul. 18,
275-283.
LOEW E. R. (1973a) Analysis of the visual response of the tly
Sarcophaga bullata during pupal development. In Docum. Ophthd. PYOC.(Ed. by HENKES H. E.), 2, 281-292. LOEW E. R. (1973b) Component analysis of the mass electrical response (ERG) of the fly Sanxphuga bullatu. Ph.D. Thesis, U.C.L.A. Department of Biology, Los Angeles. MOTE M. (1968) Integrative aspects of the lamina ganglionaria in the compound eye of the fly Sarcophuga bullata. Ph.D. Thesis, U.C.L.A. Department of Zoology, Los Angeles. MOTE M. (1970) Electrical correlates of neural superposition in the eye of the fly Smcophuga bullata. J. exp. Zool. 175,159-168.
TRUJILLO-CIIN~Z 0. (1972) The structural organization of the compound eye in insects. In The Handbook of Sensory Physiology (Ed. by FUORTB~M. G. F.), 7 (2). Springer-Verlag,
Berlin. WILKINS
J. L. (1967) Reproduction in the fleshfly Sarcophaga bullata and its endocrine
control. Ph.D. Thesis, U.C.L.A.
Department of Zoology, Los Angeles.
WOLBARSIIT M. L., WAGNER H. G., and BODBNSISIN D. (1966) Origin of the electrical response in the eye of Periplaneta americana. In The Functional Organizration of the Compound Eye (Ed. by BERNHARDC. G.). Pergamon Press, Oxford.
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BULLATA. ELLIS
IN THE ERG OF THE FLY, A COMPONENT ANALYSIS* R. LOEWt
Department of Biology, University of California, Los Angeles, California, U.S.A. (Received 20 April 1974; revised 5 June 1974)
Abstract-The changes in the ERG of the fly, Sarcophaga btdlata, with increases in the ambient carbon dioxide concentration are described and analysed using a method of successive algebraic subtraction of normal&d responses. On the basis of the behaviour of the five identifiable waves of the ERG before, during, and after carbon dioxide exposure, it is concluded that four components are responsible for the complex ERG. One of these components, which is unaffected by carbon dioxide, apparently arises from the receptor cells, while the other three components have a ganglionic origin. The waveforms of the four proposed components are derived by substracting normalised responses differing with respect to only one of the five waves. The finding that the loss of the waves, through the action of carbon dioxide, proceeds in a sequential and regular manner makes this approach to a component analysis possible. It is suggested that this sequential loss of components does not reflect differences in the basic sensitivity of visual neurons to carbon dioxide, but results from factors such as diffusion barriers and/or chemical processes which establish a decreasing radial concentration gradient of carbon dioxide within the visual ganglia. INTRODUCTION THE COMPLETEresolution
of an ERG into components should yield the number, waveform (under specified conditions), and anatomical origin of each electrical process summing to produce the complex, cornea1 ERG. Having this information, one can attempt to interpret changes in the ERG in terms of alterations of one or more of these components. The ideal situation is to be able to examine each component in isolation from any other, utilizing a non-injurious and reversible isolation method. While no single technique yet described satisfies these criteria, the judicious use of carbon dioxide can, in certain instances, approximate this ideal situation. GOLDSMITH (1960) was the first to describe the effect of carbon dioxide on the insect ERG. He found that soon after blowing carbon dioxide gas around the body of a honey-bee (Apti tnellifera), the fast transients were lost from the ERG leaving a simple cornea-negative wave. Goldsmith suggested that this remaining potential * This investigation was aided by grant B-1509 from the Division of Research Grants and Fellowships, National Institutes of Health, U.S. Public Health Service. t Present address: M.R.C. Vision Unit, C.R.P.C., University of Sussex, Falmer, Brighton BNl 9QG, England. 181
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originated from the receptor cell layer. These findings were confirmed and expanded by LEUTSCHER-HAZELHOFF and KUIPER (1964) using the blowfly, Calliphora mythrocephala. Their electrode advancement data showed that the first effect of carbon dioxide was suppression of ganglionic spike activity followed by a loss of the slower, light-dependent ganglionic potentials. The negative wave remaining after these losses was localized to the base of the receptor cells adjacent to the basement membrane. Ganglionic activity was restored upon removal of the carbon dioxide and the ERG returned to its pre-treatment waveform. On the basis of these studies and those of WOLBARSHTet aZ. (1966), and EICHENBAUMand GOLDSMITH(1968), it is now generally accepted that carbon dioxide isolates the receptor cell component of the insect ERG through the reversible and noninjurious suppression of the ganglionic components. Carbon dioxide appears to have no effect on the electrical activity of the receptor cells. The differential sensitivity of the ganglionic and receptor cell components to carbon dioxide has been used to isolate the receptor cell component for further study (WOLBARSHTet al., 1966; EICHENBAUMand GOLDSMITH, 1968), and/or to localize an already identified component to the ganglionic or receptor layers (LEUTSCHER-HAZELHOFF and KUIPER, 1964; LOEW, 1973a, b; KOOPOWITZet al., 1974). Little attention has been paid to the waveform changes accompanying the loss of the ganglionic components. It is shown in this report that an analysis of these changes gives information on the number, waveform, and origin of the ganglionic components.
MATERIALS AND METHODS Flesh-flies, Sarcophaga bullata, were raised in the laboratory using the technique of DORMANet al. (1938) as modified by WILKINS (1967) and LOEW (1973b). The flies were bred and maintained, and all experiments were performed at 22°C. Only flies between 1 and 3 weeks after emergence were examined, since older flies may show age-related changes in the waveform of their ERG (LOEW, 1973b). A fly was lightly narcotized with carbon dioxide and placed, head foremost, into one end of a glass tube having an inside diameter slightly greater than that of the fly. Using a wooden stick applied to the thorax, the fly was advanced down the tube until its head protruded from the opposite end. A bead of beeswax melted between the dorsal surface of the thorax and the rim of the tube prevented the escape of the fly. The head was immobilized by fixing it to the anterior aspect of the thorax with some melted beeswax. One of the compound eyes, which served as the indifferent lead for electrical recording, was blackened with several coats of enamel paint as were the three ocelli. As an added precaution against light striking these photoreceptors, an aluminium shield was placed around the blackened eye and ocelli and secured with wax. The glass tube holding the fly was attached to a moveable platform and placed within the recording chamber so that the unpainted eye, from which the ERG was to be recorded, was at the focus of a condensing lens present within the chamber.
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183
The recording chamber was a double-walled, copper enclosure mounted on the same optical bench as the stimulating apparatus. Water, circulating between the walls, kept the inside temperature at 22°C. A glass window allowed for the entry of the stimulating and adapting lights, while two other openings were used to admit the various atmospheres employed in the study. The control atmosphere was either air, or a mixture of SO% nitrogen and 20% oxygen. Both of these atmospheres produced the same results. The concentration of carbon dioxide was adjusted by substituting it for nitrogen in the gas mixture entering the chamber. This was done using a series of Venturi flow meters which allowed changes in the concentrations of the three gases (i.e. oxygen, nitrogen, and carbon dioxide) to be made while keeping the flow rate constant a 1 l./min. At no time was the oxygen concentration allowed to fall below 20%. Photic stimulation was provided by a Grass PS2 xenon flashlamp set at its highest intensity, referred to throughout as unit intensity, and was equivalent to 9 x lo5 horizontal candle power as deduced from the figures given by Grass. The intensity was attenuated with Corning neutral density filters. The adapting light source was a tungsten ribbon-filament lamp operating at 6.3 V d.c. The intensity of the adapting light at the surface of the compound eye was measured using a YSI radiometer and found to be 10 mW cm-2. After passing through a glass heat filter (Edmund Scientific) and a 5 cm water cell, the adapting light was mixed with the stimulus by a beam splitter. This combined beam was directed onto the compound eye by a series of condensing lenses. To provide a uniform intensity across the surface of the eye, a diffusing screen was placed between the eye and the field lens. The ERG was recorded using stainless steel microelectrodes (tip dia. 10-20 pm) insulated down to the tip with Insul-X resin. One electrode was placed subcorneally in each compound eye using a micromanipulator. A Kiethley 103 preamplifier having a bandwidth of 0.1 to 30,000 Hz provided differential preamplification. This amplified signal was displayed on the upper beam of a Tektronix 502 oscilloscope and recorded on film for later study. A stimulus mark, derived from a photodiode, and time marks were displayed on the lower beam. RESULTS
Dark-adapted eyes
(1) Control atmosphere (0% carbon dioxide). A fly was prepared as previously described and allowed to dark adapt within the recording chamber for 20 min. Stimuli were then presented, allowing a minimum of 2 min between each flash for the eye to recover from the previous flash. Control experiments showed that recovery of the response was complete within 1 min after a flash; however, the 2 min protocol was adopted as a precautionary measure. The ERG elicited by stimuli of various intensities is seen in Fig. 1. These responses are typical of those recorded from more than 200 flies studied, although the response amplitudes in individual cases varied by several millivolts, depending upon the impedance of the recording circuit. The unit intensity response (0) is
184
ELLIS R. LOEW
similar to that previously reported for Surcophuga (HASSENSTEIN,1957; FOUCHARD and CARRICABURU, 1970 ; LOEW, 1973a, b) and consists of a series of waves designated alpha, beta, gamma, delta, and epsilon in the order of their appearance in the response.
I50 msec. FIG. 1. Control conditions-the ERG of the dark-adapted eye with decreasing stimulus intensity. The numbers to the left of the records under the heading D indicate the value of the density filter used for attentuation. The arrow indicates the stimulus.
Tenfold attenuation of the stimulus (- 1) caused a loss of the epsilon wave and revealed wavelets on the descending limb of the negative beta wave. The amplitudes and time-to-peak (TTP) values of the remaining waves were only slightly affected by the intensity decrease, and the loss of the epsilon wave did not markedly affect the shape of these waves. Further attentuation ( - 2) decreased the amplitude of all waves as well as increasing their TTP values. At a still lower intensity (- 3) the beta wave was lost from the response, leaving the delta wave and a single positive wave resulting from a fusion of the alpha and gamma waves. The amplitude of the delta wave continued to decrease with decreasing stimulus intensity until it disappeared from the response altogether, leaving a positive wave of low amplitude
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ERG OF
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185
5). It is important to note that this positive wave is associated with the alpha and gamma waves in the unit intensity response (0). (2) Eflects of carbon dioxide. It is known that carbon dioxide influences the behaviour of insects and is capable, in sufficient concentrations, of producing a narcosis. A preliminary test confirmed that carbon dioxide could alter the waveform of the ERG of Surcophuga at concentrations below those producing complete narcosis. To examine these alterations, the ERG was recorded from flies exposed to increasing concentrations of carbon dioxide. A fly was dark adapted at 0% carbon dioxide for 20 min following which a unit intensity flash was given and the ERG recorded. The concentration of carbon dioxide was then increased to 5% as previously described and a 5-min period allowed before presentation of the test flash. The carbon dioxide was next increased to 10% and, after another 5-min period, the ERG recorded. This procedure was repeated with 5% step increases until the 60% level was reached. The chamber was then flushed with 100% oxygen for 2 min and the atmosphere returned to 0% carbon dioxide. The ERG was monitored every 2 min until recovery was complete, which could take as long as 30 min. The results for a particular fly were not accepted unless its ERG had recovered within this 30-min period. The 5-min lag between the gas increase and the stimulus was necessary to overcome the problems associated with mixing of each new atmosphere and to minimize the effects of any difference in carbon dioxide sensitivity between flies. The responses recorded using lag periods of up to 10 min were, in most cases, similar to those obtained using 5-min periods indicating a steady-state condition within the 5 to 10 min interval. However, after 10 min at a particular carbon dioxide concentration inconsistent changes were often seen in the response waveforms, particularly for those obtained below 15% and above 40% carbon dioxide. Responses typical of 18 flies exposed to different levels of carbon dioxide are seen in Fig. 2. The percentages to the right indicate the carbon dioxide concentration. At 0% the response was identical to that observed in air (Fig. 1). Between 0 and 25% carbon dioxide there was an increase in the amplitude of the epsilon wave and an increased TTP for the delta and epsilon waves. There was also a slight increase in beta wave amplitude. At 30% carbon dioxide the amplitude of the delta and epsilon waves started to decrease until, between 45 and SO%, they disappeared from the response. At 50% the alpha wave was reduced to a small transient on the ascending limb of the beta wave which, under these circumstances, had a faster time-course than the alpha wave. Oscillations with an average frequency of 150 Hz were present on the descending limb of the beta wave at 50% carbon dioxide. In addition, the 50% record shows a positive wave following the beta wave which is not comparable to the epsilon wave. The oscillatory activity extends onto this positive wave. Above the 50% carbon dioxide level, the response simplified to a monophasic negative wave (60%) apparently associated with the beta wave as suggested by the progression of waveform changes between 0 and 60% carbon dioxide. A return to 0% carbon dioxide led to recovery of the ERG indicating that the effects of carbon dioxide were reversible. (-
186
ELLIS R.
LOEW
50%
55%
25%
60%
0%
I
125MSEC.
FIG. 2. Effect of carbon dioxide on the ERG from dark-adapted eyes. The percentages to the right of each record indicate the carbon dioxide concentration (at atmospheric pressure) in the gas mixture. The remaining gases were 20% oxygen and nitrogen to make 100%. All stimuli were unit intensity. A striking feature of carbon dioxide narcosis in insects is the speed with which induction and recovery occur. Exposure of Sarcophaga to 80% carbon dioxide produces narcosis within 30 sec. Experiments were performed to determine the rate with which carbon dioxide affects the ERG. A fly was dark adapted for 15 min at 0% carbon dioxide and the response to a unit intensity flash recorded. The carbon dioxide concentration was then rapidly increased to 80% allowing 2 min between the flash and the increase for the eye to recover. At specific intervals, after the increase in carbon dioxide, a unit intensity stimulus was given and the response recorded. Fig. 3 shows typical records for some 30 flies. The time to the right of each record indicates the interval between the carbon dioxide increase and the test
CO*-INDUCED CHANGES IN THE ERG OF SARCOPHAGA
I
187
50 mwc.
FIG. 3. Effect of 80% carbon dioxide on the responses to test flashes given at various intervals after carbon dioxide-dark adapted eyes. Record 0 is the response to a flash before carbon dioxide, while the record 10 set indicates a flash was delivered 10 set after carbon dioxide, etc. In each case the carbon dioxide was preceded by a flash such as record 0 given 2 min before the start of the carbon dioxide treatment. The carbon dioxide was maintained at 80% from the start of the experiment until the second test flash (i.e. 10 set, 20 set, etc.).
flash except for record 0 which is the response to the initial test flash. It is seen that very soon after the carbon dioxide increase (10 set) the epsilon wave apparently disappeared. This was followed by the loss of the gamma and delta waves at 30 set and the alpha wave at 60 sec. After 120 set at 80% carbon dioxide only a monophasic negative wave remained. 6
188
ELLIS R. Lo~w
After 120 set at 80% carbon dioxide, flushing the chamber with 100% oxygen atmosphere (i.e. 80% nitrogen and 20% from narcosis are seen in Fig. 4. In this
the atmosphere was returned to 0% by for 30 set and re-introducing the control oxygen). The responses during recovery case the time of each record is measured
IO
60
FIG. 4. Recovery of the ERG after exposure to 80% carbon dioxide for 120 secdark adapted eyes. Record 0 is the response while the fly was still exposed to 80% carbon dioxide. The other records were taken at various intervals after the removal of the carbon dioxide. After the 0 set flash, the chamber was flushed with 100% oxygen for 30 set, following which an atmosphere of 20% oxygen and 80% nitrogen was admitted. from the return to the control atmosphere
except for record 0 which is the response recorded just prior to the oxygen flush. It is seen that recovery proceeded in a manner almost the reverse of induction. The first wave to recover was the alpha followed, in order, by the delta, gamma, and epsilon waves. Recovery was usually complete by 180 set after carbon dioxide removal. It is interesting that the time necessary for recovery depends upon the duration of narcosis. In cases where the fly was kept at 80% carbon dioxide for 30 min, recovery could take as long as 45 min.
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189
Light-adapted eyes (1) Con&o1 at-e
(0% carbondioxide). Flies were dark adapted for 15 mm, their ERG recorded, and then light adapted at maximum background intensity (10 mW . cm-“) for 20 min before testing. Typical responses to stimuli of various intensities superimposed on the background are seen in Fig. 5. The unit intensity
i
E
m
L50 msec.
FIG.
5.
Control conditions-the ERG of the light-adapted eye with decreasing stimulus intensity (nomenclature as in Fig. 1).
response (0) consisted of the same five waves recorded from dark-adapted eyes. However, the slopes of the waves were much steeper in the light-adapted state which reduced the duration of the ERG to about 100 msec. A fast negative transient preceded the alpha wave at unit intensity, but disappeared with a slight intensity decrease (- 1). Light adaptation reduced the amplitude of all the waves, with the epsilon wave showing the greatest reduction. In fact, in about 40 per cent of the light-adapted eyes studied, an epsilon wave could not be detected in the unit intensity response. The sequence of changes in waveform with decreasing stimulus intensity for light-adapted eyes was similar to that seen for dark-adapted eyes (Fig. 1). There was a decrease in amplitude of the negative beta and delta waves, and the positive
190
ELLIS R. LOJSW
epsilon wave; and an increase of the positive alpha and gamma waves. As with the dark-adapted eye, only a positive-dominant wave remained at low stimulus intensities (Fig. 4). (2) Eflects of carbon da’oxide. Responses typical of 22 light-adapted flies at various intervals after rapidly increasing the carbon dioxide concentration to 80% are seen in Fig. 6. The first effect was a slight increase in epsilon wave amplitude 0
30
ICC.
60
sec.
75
sec.
FIG. 6. Effect of 80% carbon dioxide on the responses to test flashes given at various intervals after carbon dioxide-light adapted eyes. Experimental procedures were like those for Fig. 3.
reaching a maximum at 30 sec. During the next 45 set there was an increase in amplitude, TTP, and time-course of the delta wave which was never observed in similar experiments using dark-adapted flies (Fig. 3). During this same period (O-75 set) there was little change in the alpha and beta waves. After 75 set the amplitude of the delta wave decreased and the alpha wave was lost leaving a monophasic negative wave similar to that isolated from dark-adapted eyes (Fig. 3).
191
COI-PIDUCBDCHANCEBINTHBBRGOFSRRCOPHRGA
Recovery of the response after removal of the carbon dioxide followed a course that was the reverse of that seen during induction. As seen in Fig. 7, the first wave to recover was the alpha, followed by the gamma, delta, and epsilon waves. Again, the recovered response (60 set) was identical to that recorded before carbon dioxide exposure.
40rrc.
FIG. 7. Recovery of the ERG after exposure to 80% carbon dioxide for 120 seclight adapted eyes. Experimental procedures were like those for Fig. 4. INTERPRETATION
OF RESULTS
(1) The number of components
The records obtained with both light and dark adapted eyes under control conditions (Figs. 1,s) indicate at least two components-a positive and a negative. In no other way can the positive alpha and epsilon waves, and the negative beta
192
ELLISR.LOEW
and delta waves be explained. Assuming only these two components, the beta and delta waves would be the product of the negative component, while the epsilon wave would be the tail end of the positive component producing the alpha wave. Differences in latency and waveform of the two components would account for the gamma wave. The positive component would have the lowest threshold since only a positive wave remains at low stimulus intensities (Figs. 1, 5). The similarities between the responses from dark- and light-adapted eyes suggest that light adaptation does not alter the waveform of the components, but only alters their amplitude and latency. While such a two-component scheme might be manipulated so as to explain the responses recorded under control conditions, it fails to explain those recorded at elevated carbon dioxide concentrations. In Fig. 2 it is clear that alterations in both the amplitude and TTP of the delta wave occur without any observable effect on the beta wave. This is particularly evident from the responses at 30, 35, and 40% carbon dioxide. This independence of the beta and delta waves is also seen in the records of Fig. 6 from light-adapted eyes. The marked changes in the delta wave between 30 and 90 set after the carbon dioxide increase have little effect on the beta wave. The records from the recovery period also demonstrate this independence (Figs. 4, 7). The most reasonable explanation for this behaviour is that a separate component produces each negative wave of the ERG. It can also be argued, though less convincingly, that there are two positive components. This separation of the positivity into two components is based on the behaviour of the epsilon wave. It is seen in Fig. 1 that the epsilon wave disappeared with a tenfold decrease from unit stimulus intensity. To explain this loss using a single positive component scheme would require either that the duration of the component producing the delta wave increases, or that the duration of the positive component decreases. Both of these changes would reduce the summed potential in the temporal region of the epsilon wave. However, such changes would be expected to affect the slopes of the delta wave. The loss of the epsilon wave does not, however, alter the slopes of the delta wave or, for that matter, the slopes of any of the remaining waves. Thus, the loss of the epsilon wave must result from an amplitude decrease of a second positive component in addition to that producing the alpha wave. The only other explanation consistent with a single positive component is one that assumes changes in the waveform and amplitude of the two negative components in synchrony with changes in the positive component resulting in a loss of the epsilon wave. The sum of such synchronous changes would then fortuitously yield the response seen at density 1 in Fig. 1 which is almost identical to the unit intensity response except for the absence of the epsilon wave. Such exact and specific compensation, though possible, seems improbable in view of the regular changes in response waveform with further decreases in stimulus intensity (Fig. 1). The responses with carbon dioxide also support two positive components. It is seen in Fig. 2 that alterations in amplitude and TTP of the epsilon wave are not attended by changes in either the alpha or gamma waves. This is particularly
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193
noticeable between 5 and 25% carbon dioxide. While it is possible to explain these changes in terms of complicated alterations of two negative and one positive component, the proposal of a second positive component explains the data in the simplest way. In summary, the data are best explained by a component scheme of two positive and two negative components. (2) Waveform and origin of thefour proposed components The waveform, relative amplitude, and temporal relationships of the four proposed components are seen in Fig. 8, as is their algebraic summation. I believe
FIG. 8. The four components proposed for the ERG of SarcqAaga based on the data presented here. The dashed line is zero potential with an upward deflection indicating positivity of the cornea with respect to the indifferent electrode. The waveform in the lower half of the figure is the algebraic summation of the four proposed components. that
three of these components correspond to those proposed by FOUCI-IARD and CARRICABURU (1972) and have, therefore, adopted their terminology for the components NR, P, and NS. The fourth component, from this study, has been labelled PL because it is positive and appears late in the response.
194
ELLISR. LOEW
Ideally, in order to analyse the waveform of each component of the ERG, it would be necessary to isolate each one from the others and to observe it in an uncomplicated form. Of the four proposed components, only the NR has been isolated in this manner. The waveform of this component was taken to be that of the negative wave remaining after increasing the carbon dioxide concentration (Fig. 2, 60%). In keeping with the known effects of carbon dioxide on the insect ERG, it is suggested that this negative potential arises from the receptor cells and is the receptor cell component (NR). The waveform of NR is similar to the receptor cell component isolated by selective recording techniques (AUTRUMand GALLWITZ, 1951; MOTE, 1968, 1970; HEISENBERG,1972), by physical isolation of the receptor cell layer (AUTRUM,1958; WOLBAR~HTet al., 1966; EICHENBAUMand GOLDSMITH, 1968; LOEW, 1973b), and by drugs (AUTRUMand HOFFMANN,1957; CARRICABURU, 1971). It is seen that the peak of the NR component corresponds to that of the beta wave in the control responses (Fig. 2). Knowing the waveform of the NR component, the waveforms and relative amplitudes of the remaining three components were derived by a process of successive subtraction. For this analysis each response was normalized by assigning to the unit intensity delta wave amplitude the value of 100 per cent and referring all other amplitudes to this level. It has previously been shown that responses normalized in this manner are congruent at all stimulus intensities (LOEW, 1973b). The rising phase of the P component must have a steep slope in order to account for the appearance of the alpha wave before the beta wave (Fig. 8). This slope and the relative amplitude of P were determined by subtracting the NR component from the unit intensity response and finding the magnitude of the positivity needed to account for the amplitude difference between the NR component and the beta wave (Figs. 1, 8). Two methods were used to derive the remainder of the P component (i.e. that part within the envelope of the NS component). The first involved extrapolation from the shape of the positive wave isolated by decreasing the stimulus intensity (Fig. l), assuming that only the amplitude and TTP of the P component changed with stimulus intensity. The second method was to subtract the 60% response from the 50% one of Fig. 2. Both of these operations yielded similar results which, when combined with the initial subtraction of the NR component, produced a positive wave similar in waveform to the potential recorded across the lamina ganglionaris in Sarcophaga (MOTE, 1968) and Drosophila ( HEISENBERG,1972). The analysis of the second negative component (NS) was made difficult by the interference of both the NR and P components which interact temporally with the NS (Fig. 8). Accordingly, the procedure was first to subtract the known components (i.e. NR and P) from the unit intensity response and thus to obtain the peak and a portion of the descending limb of NS. The first part of this component, from its origin at the baseline to within 75 per cent of the peak height, is purely speculative and could start somewhat later than is shown in Fig. 8. The terminal portion of the descending limb obtained from the above subtractions is obviously affected by the epsilon wave. This effect was abolished by applying the above subtractions to
COB-INDUCED
CHANGES
IN THE
ERG OF SL4RCOPHAGA
19.5
responses recorded after carbon dioxide exposure (Figs. 6,7) and to those recorded at low stimulus intensity (Fig. l), both of which are similar to the unit intensity response except for the presence of the epsilon wave. Using these procedures, the terminal portion of NS was established to fall to the baseline as shown in Fig. 8. The same procedure was used to derive the PL component. In this case responses comparable in all respects except for the presence of the epsilon wave were subtracted from each other. Thus, by subtracting record - 1 from record 0 of Fig. 1, after appropriate normalization, the waveform and relative amplitude of PL were ascertained. The initial portion of PL is uncertain and may be different from that seen in Fig. 8. That the P, NS, and PL components are of ganglionic origin can be assumed from their sensitivity to carbon dioxide, but by using carbon dioxide alone it is not possible to locate the site within the ganglionic layers from which each component arises. On the basis of developmental and surgical ablation studies on the compound eye of Sarcophaga, the P, NS, and PL components have been localized to the lamina ganglionaris, medulla, and lobula, respectively (LOEW, 1973a, b). Assuming these assignments to be correct, it appears that the order in which the components are affected by carbon dioxide is correlated with the position of its ganglionic layer of origin. That is, the effect of carbon dioxide seems to progress from the proximal ganglionic layers (e.g. lobular) distally, towards the receptor cell layer. This sequential effect of carbon dioxide on the ganglionic components is evident from the records of Figs. 2,3, and 6, in which first the PL, then NS, then the P component is affected and eventually lost from the response leaving the NR. The reversed order is observed during recovery of the components (Figs. 4, 7). It thus appears that the neuronal layers show a differential sensitivity to carbon dioxide. If this differential sensitivity is real it means that different neurons possess very different carbon dioxide sensitivities and that neurons with the same sensitivity are grouped within the same nervous layer. While this is possible, it seems unlikely since the neurons in the different layers are similar electrically and morphologically (TRUJILLO-CEN~Z, 1972). It is possible, however, that the differential sensitivity is only apparent and that all the neurons within the visual system have roughly the same carbon dioxide sensitivity. This follows if one assumes that within the ganglia a radial concentration gradient in equilibrium with the atmosphere is established such that the carbon dioxide concentration is greater proximally. This gradient could arise from physical barriers to diffusion of carbon dioxide such as that imposed by the basement membrane (KOOPOWITZ et al., 1974), or from differences in pH or buffering capacity within and around the neuronal layers. Such a gradient is consistent with the steady-state effects observed in Fig. 2 as well as the dynamic changes seen in Figs. 3 and 6. Unfortunately, no evidence for such a gradient is available. CONCLUSIONS From data obtained before, during, and after exposure to carbon dioxide, it is concluded that the ERG of Sarcophaga is resolvable into four components, one
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ELLIS R. LOEW
(NR) of receptor origin and three (P, NS, PL) arising from the ganglionic layers. In addition, all or part of the waveform of each component was derived by successive subtraction utilizing both control and experimental responses. The component scheme so obtained (Fig. 8) agrees with that previously proposed for Sarcophaga on the basis of records obtained during differentiation of the compound eye (LOEW, 1973a). The scheme is also consistent with the potentials recorded across individual visual layers of Sarcophqa (MOTE, 1968, 1970), and with generalized component schemes proposed for other dipterans (FOUCHARD and CARRICABURU, 1972). The amount of information which can be derived using carbon dioxide will generally depend on the number of carbon dioxide-sensitive components present. If, for example, the ERG under study is dominated by the receptor potential or consists of more than one receptor cell component, carbon dioxide will be of limited value. Such was the case in a recent analysis of the ERG of the beeswax moth, Galleria trzdonella, which was found to have three components only one of which was carbon dioxide-sensitive (KOOPOWITZ et al., 1974). However, even in this study carbon dioxide provided data on the site of origin of the components. It remains for carbon dioxide to be adopted as a routine tool in the resolution of the insect ERG. Acknowle&ments-I should like to thank Professor F. CRIBCITELLI for hia aid and support during the course of this investigation, and Professor H. J. A. DARTNALLand Dr. J. K. BOWMAKER for critically reading the manuscript. REFERENCES AUTRUMH. (1958) Electrophysiological analysis of the visual systems in insects. Exp. Cell Res. (Sup~l.) 5,426-439. AUTRUMH. and GALLWITZU. (1951) Zur Analysis der Belichtungspotentiale des Insektenauges. Z. vergl. Physiol. 33,407-435. AUTRUMH. and HOFFMANNE. (1957) Die Wirkung von P&rotor& und Nikotin auf das Retinogramm von Insekten. 2. Natutforsch 12b, 752-757. CARRICABURU P. (1971) Action du parathion sur 1’6lectror&inogramme de la mouche Musca domestica. C. R. Ad. Sci., Paris 273D, 2576-2578. DORMANS. C., HALE W. C., and HOPKINSV. M. (1938) Laboratory rearing of flesh&s. J. econ. Ent. 31, 44-51. EICHENBAUMD. and GOLDSMITHT. H. (1968) Properties of intact photoreceptor cells lacking synapses. J. exp. Zool.l69,15-32. FOUCH~~RD R. and CARRICABURU P. (1970) La response electroretinographique oscillante chez cinq especb de mouche. Vision Res. 10,655-667. FOUCHARDR. and CARRICABURU P. (1972) Analyse de l’Clectror&inogramme de l’insecte. Vision Res. 12, 1-15. GOLDSMITHT. H. (1960) The nature of the retinal action potential, and the spectral sensitivities of ultraviolet and green receptor systems of the compound eye of the worker honeybee. g. gen. Ph siol. 43,775-800. I-&&ENSTBINB. (1957) i; ber Belichtungspotentiale in den Augen der Fliegen Sarcophaga und Eristalis. J. Insect Physiol. 1, 124-130. HEISBNBERG M. (1971) Separation of receptor and lamina potentials in the electroretinogram of normal and mutant Drosophila. J. exp. Biol. 55, 85-100. KOOPOWIT~H., STONEG., and MARTINEZ D. (1974) Components of the ERG of the moth, Galleria mellonella. J. Insect Physiol. 20, 9-20.
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