Two Components from the Compound eye of the Crayfish^*

In recent years the electrical response from the crustacean compound eye was observed by Ruck & Jahn (1954) from Ligia and by Hanaoka and his co-workers (Hanaoka, 1950a, b;Hanaoka et al. 1957) from the crayfish. In the previous paper Naka & Kuwabara (1956) recorded the electroretinogram of the crayfish from the corneal surface of the eye and found that the e.r.g. consisted of two components which they referred to as H-I and H-II. H-I responded only to the ‘on’ of illumination, while the amplitude of H-II was maintained during the stimulus. The present paper deals with the two components separated by means of the microelectrode.

Materials used were the crayfish, Procambarus clarkii,^† which were collected in the field nearby and maintained in the laboratory. Before the experiments the eye was detached from the body. Two methods of preparation were employed. In one series of experiments the compound eye was bisected through the axis of the eye. In the other series a small hole was made at the centre of the corneal surface so as to insert the microelectrode vertically into the receptor layer. Both types of preparation were so fixed in a Ringer pool with vaseline that only the basal part of the eyestalk was in contact with the Ringer, an indifferent lead being taken from the Ringer pool. Harreveld’s physiological solution for crayfish was used throughout this experiment as the Ringer (Harreveld, 1936). A capillary electrode filled with 3M-KCI with a tip diameter of less than 0·5μ was positioned and inserted into the preparation by means of a micromanipulator. The vertical movement of the electrode was measured by a micrometer attached to the manipulator. The potential was picked up by a 954 type tube that served as an electrometer preamplifier (Elmore & Sands, 1949), which was followed by a two-stage d.c. amplifier. The other apparatus was as described elsewhere (Naka & Kuwabara, 1956). The experiments were carried out at room temperature ranging from 10° to 15° C. Under these experimental conditions a normal response (i.e. an e.r.g. consisting of two components) was recorded for about 1 hr., but this ‘normal time’ varied greatly from preparation to preparation suggesting that minute damage to the preparation during the operation might have had great effect on its condition.

The compound eye of the crayfish consists of three layers: the crystalline cone layer, the receptor layer, and the layer proximal to the basement membrane as shown in Fig. 1.^* The crystalline cone layer is a layer between the cornea and the receptor layer. The receptor layer can also be divided into two parts, the inner and outer parts. The outer part corresponds to the distal part of the retinula cell and the inner part to the basal part of the cell. The inner part also contains the rhabdome, which is surrounded by seven retinula cells. The region proximal to the basement membrane consists of the proximal process of the retinula cell (i.e. the nerve fibre) which proceeds toward the optic ganglion.

The electrode was inserted into four layers of the bisected eye : the crystalline cone layer, the outer and inner part of the receptor layer and the region proximal to the basement membrane. Three e.r.g.’s were recorded at each layer using light stimuli of three different durations.

The e.r.g. recorded by the electrode inserted into the crystalline cone layer was a negative wave consisting of a negative peak, followed by a slow plateau (Fig. 2).

According to Naka & Kuwabara (1956) the initial peak was due to H-I which responded only to the ‘on ‘of the light stimulus and the slow plateau was due to H-II which was maintained during stimulation. In Fig. 2 the slow potential became smaller in amplitude with a shorter duration of the flash of light, while the amplitude of the initial potential remained the same.

From the outer part of the receptor layer a diphasic e.r.g. with an initial positive spike-like potential and a following slow negative potential were recorded (Fig. 3). The amplitude and duration of the positive spike-like potential were independent of the duration of the flash of light, but the slow potential increased in both amplitude and duration when the duration of the light stimulus was increased. The positive potential had the same features as H-I with the exception of the polarity of the potential. It was concluded: (1) that the positive potential corre-sponded to H-I, whose polarity was reversed at this part of the receptor layer ; and (2) that the negative potential which responded to both ‘on’ and ‘off’ of the light stimulus corresponded to H-II.

The inner part of the receptor layer was distinct because of its white guanin deposit. The e.r.g. recorded from this part consisted of only a spike-like positive potential which had the same wave form as that of the positive potential in the diphasic e.r.g. from the outer part of the receptor layer (Fig. 4). The e.r.g. responded only to the ‘on ‘of the light stimulus and the duration and amplitude of the potential was independent of the duration of the light stimulus. It was therefore concluded that the e.r.g. from the inner part of the receptor layer was composed of only H-I, with H-II not being recorded from this part of the receptor layer.

The e.r.g. from the region proximal to the basement membrane was a slow positive potential which increased in both amplitude and duration when the light stimulus was increased in duration (Fig. 5).

The wave form of the potential was symmetrical with the slow negative phase of the diphasic e.r.g. from the outer part of the receptor layer, suggesting that the potential corresponded to H-II with an inverted polarity.

The central portion of the cornea of an excised eye was cut off with scissors to make a small hole through which the electrode was inserted vertically into the eye. The preparation was fixed in the Ringer pool and the axis of the electrode was made to coincide as closely as possible with that of the eye.

When the electrode touched the surface of the preparation, the beam of the oscilloscope was adjusted to the zero level.

The e.r.g. recorded at the position of the electrode, where it touched the surface of the material, was identical with the e.r.g. from the crystalline cone layer and also with the e.r.g. recorded from the whole eye (Fig. 6A).

As the electrode was then moved toward the receptor layer the movement of the electrode did not at first produce any change in the form of the e.r.g., though the response increased in amplitude. When the electrode was inserted deeper there appeared an initial positive potential in the e.r.g. which had the same wave form as that from the outer part of the receptor layer, with the initial positive potential corresponding to H-I and the slow negative potential to H-II. The diphasic e.r.g. was recorded for about 50-100 μ in depth. If the electrode was inserted deeper the e.r.g. was suddenly transformed into a positive spike-like potential which was recorded for 50-150 μ in depth (Fig. 6c). Apparently this potential corresponded to H-I and the electrode seemed to be in the inner part of the receptor layer. Even at this depth a slow potential which seemed to be a remnant of H-II was recorded, though of very small amplitude. If the electrode was near the outer part of the receptor layer the slow potential was negative in sign, and if the electrode was near the basement membrane the potential was positive in sign.

The further insertion of the electrode produced a sudden change in the steady potential, and the resting potential became 10-50 mV negative as if the electrode had gone through a membrane. This membrane seemed to be the basement membrane, because there was no other membranous structure which might account for this sudden change in the resting potential. With the change in the resting potential there appeared a positive sustained potential which had approximately the same wave form as the e.r.g. from the region proximal to the basement membrane (Fig. 6D).

From the basement membrane to the region corresponding to the optic ganglion the resting potential changed irregularly, ranging from—50 to—10 mV., and the e.r.g. recorded from this region was composed mainly of the slow potential, H-II, which became smaller in amplitude as the electrode was inserted deeper.

The distance of the electrode from the basement membrane was obtained by assuming that the change in the resting potential occurred when the electrode went through the basement membrane. According to this recalculation, the e.r.g. composed of only H-I was recorded when the electrode was 0-150 μ from the basement membrane, while the diphasic e.r.g. composed of both H-I and H-II was recorded when the electrode was 150-200 μ from the basement membrane.

The e.r.g. from the inner surface of the basement membrane usually had the wave form shown in Figs. 5 or 6D, which seemed to consist of H-II or both H-I and H-II. Direct evidence for this assumption is shown in Fig. 7, which shows three e.r.g.’s recorded with stimuli of different durations, with the electrode in the same position as in Fig. 6D.

The amplitude and duration of the spike-like potential, H-I, was independent of the duration of the light stimulus, while that of the slow potential increased when the light stimulus was increased in duration.

The electrode was inserted vertically into the receptor layer through a hole made at the centre of the corneal surface until a diphasic e.r.g. was recorded. When a diphasic e.r.g. was recorded the preparation was dark-adapted for an hour without changing the position of the electrode. Judging from the wave form of the e.r.g. the electrode seemed in the outer layer of the receptor layer. The response to the first flash is shown in Fig. 8 A and is composed of H-I only. The second flash caused the appearance of the negative component, H-II, which indicated that the preparation was brought into a condition of light-adaptation (Fig. 8B).

When the preparation was further light-adapted the negative component, H-II, became larger in amplitude and the e.r.g. became almost negative in sign (Fig. 7c). The decrease in the amplitude of H-I seemed to be caused by light-adaptation and also by interaction with H-II whose latency decreased as a result of light-adaptation.

The e.r.g. from the crustacean compound eye has been recorded by some authors, but no very extensive study has been made of the subject. The e.r.g. from the compound eye of Cambarus has been studied by Hanaoka and his co-workers. According to Hanaoka (1950) the e.r.g. reversed its polarity at the receptor layer, a diphasic e.r.g. being sometimes recorded from the receptor layer. As to the components in the Cambarus e.r.g., Hanaoka & Yasumi (1956) briefly reported that the rhabdome was the site of the slow potential which induced a ‘response potential ‘. Ruck & Jahn (1954) observed polarity reversal of the Ligia e.r.g. with leads from the central retina and optic ganglion, and this reversal occurred even in the absence of the ganglion.

In the previous report Naka & Kuwabara (1956) have analysed the e.r.g. from the Cambarus compound eye into two components according to the difference in the wave form under light- and dark-adaptation. The two components, which were referred to as H-I and H-II, could only be isolated by means of adaptation because the e.r.g. was recorded from the corneal surface. In the present investigation the two components were separated by the use of the microelectrode.

Some characteristic features of the two components which had been reported in the previous paper, and have been confirmed in the present report, are as follows : H-I responded only to the ‘on’ of illumination while H-II was maintained during the light stimulus. The amplitude of H-I was not affected by the duration of the light stimulus when the stimulus was longer than 10 msec., whereas H-II increased in amplitude when the duration of illumination was increased. The e.r.g. from the completely dark-adapted eye was composed of H-I alone, while light-adaptation was followed by the appearance of H-II, thus making the e.r.g. the sum of the two components. The change in the wave form of the e.r.g. resulted from the difference in adaptation as is clearly shown in Fig. 8. This phenomenon can be interpreted as a kind of facilitation of H-II.

In this investigation the diphasic e.r.g. was recorded when the electrode was in the outer part of the receptor layer or when the electrode was 150-200 ju. from the basement membrane, and the e.r.g. composed of H-I alone was recorded when the electrode was in the inner part of the receptor layer or when the electrode was 0-150 μ from the basement membrane. In the Procambarus compound eye the length of the retinula cell and that of the rhabdome are about 200 μ and 150μ respectively, the two structures thus differing in length by 50 μ. The diphasic e.r.g. seemed to be produced as the result of the difference in length of the structures which were responsible to the generation of the two components, H-I and H-II. These considerations lead to the conclusion that the two components originated from different structures in the receptor layer, probably from the retinula cell and from the rhabdome.

The e.r.g. from the insect compound eye was found by Kuwabara & Naka (1957b, 1958) and Naka & Kuwabara (1959) to be composed of two monophasic components with opposite polarity. The negative potential from the receptor layer of the insect compound eye was maintained during the illumination and it reversed its polarity at the basement membrane. Under favourable conditions the negative potential was recorded even 1 or 2 days after preparation, while the positive potential was recorded only for 1 or 2 hr. (Naka & Kuwabara, 1959). In ProcambarusNaka & Kuwabara (1956) reported that the slow potential, H-II, was reproduced for as long as 100 hr., whereas H-I disappeared within a much shorter time.

These similarities between the two potentials, the negative component from the receptor layer of the insect compound eye and H-II, suggest that the two potentials may play the same role in the visual mechanism of the compound eye.

Although the positive components from two kinds of compound eye were both variable, the two potentials differ in wave form. But it seems to be not unlikely that H-I is a primitive form of the positive component in the insect eye because after degeneration the positive component became somewhat like H-I. (Cf. Naka & Kuwabara 1959, fig. 4.)

The polarity reversal of the negative component from the receptor layer at the basement membrane was also observed in Lucilia by Naka & Kuwabara (1959). According to Naka & Kuwabara (1959) the reversal of the negative component at the basement membrane was an active process, which meant that the reversed positive potential was induced by the negative potential in the receptor layer. In further support of this assumption Naka (1959) reported on the insect compound eye that the positive potential from the region proximal to the basement membrane was preferentially abolished by the application of KC1 solution, while the negative potential from the receptor layer remained unaffected. These results on the insect compound eye suggest that the polarity reversal of H-II at the basement membrane was also an active process and that the slow positive wave from the region proximal to the basement membrane was induced by the slow negative wave, H-II, in the receptor layer.