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The electrical response of the ocellus of Phalangium opilio
RESEARCH NOTE
THE
ELECTRICAL RESPONSE OF PHALANGIUM
OF THE OCELLUS OPILIO
MARIELLA FRELATO and FABR~ZIOVEGLIA Institute of General Physiology. University of Torino. Italy
DAMDE Lovtsoto,
(Rewired
KVJ Words-eye:
27 July
Arachnid: photoreceptor
rl~lirw/ Classificarion-Arachnid
(Phahyiurn
1977; in recisrdfoun
16 Janrrar_v 1978)
response: temporal summation. Opilio).
The origin of the extracellular potentials recorded from Arachnid eyes has been discussed by various workers (Giulio, 1962; DeVoe, 1963; Magni and Strata. 1965). It has been proposed that the ERG in these animals may be ascribed to the receptor cells only, and this interpretation is supported by anatomical data. at least for the principal eyes (Magni and Strata. 1965). confirming the absence of synaptic endings in the eye. Among Arachnids, the Phalangida present some striking differences in their visual system, having only two eyes, each with a single lens, like that of simple eyes common to all Arachnids, but with each “retinula” formed by a “central cell” and three “peripheral cells” (Purcell. 1894). and with a characteristic fusion of the microvilli of the rhabdoms of different retinulae. This peculiar structure has recently stimulated more refined ultrastructural work (Curtis. 1970) that has confirmed in more detail the existing findings. Here, too, no evidence of the presence of synaptic structures in the eye was found. The present work was begun to investigate the main features of the physiological responses of this photoreceptor structure. Freshly collected animals were immobilized with adhesive tape on a perspex base, and the eye was pierced with a very thin needle to allow the electrode to reach the retina without breaking. The oceliar potential was recorded by means of a glass microelectrode filled with 2.5 M KCI; electrical impedances ranged from 5 to 20 MR. The reference electrode consisted of an Agar-Silver chloride electrode that was placed near the’ eye into a drop of physiological saline solution (DeVoe, 1972) that moistened a small area around the eyes. Potentials recorded by the microelectrode were sent to a capacity-neutralized cathode-follower (Instrumentation Laboratories Picometric Amptier IL 181) that was connected to the differential plug-in of a Tektronix 5103N oscilloscope. The use of microelectrodes allowed us to record activity from only one, or at most, a few units (as demonstrated by occasional recordings from unidentified nerve cells, when the electrode was advanced through the eye-cup, in which unit activity was observed). The eyes were dark-adapted for about 20 min before each Set of experiments.
Light stimulation consisted of Rashes of light obtained from a Glow Modulator Tube (Sylvania, R 1131-C) driven by an American Electronic Laboratories Laboratory Stimulator 104A. The light beam was focused on a small spot (2 mm diameter). The illuminance of the unattenuated light was 1200 Ix (unit intensity); neutral density filters (Kodak Wratten) were inserted when lower intensities were used. Figure 1 shows the typical ocellar potential recorded by an electrode at the level of the distal part of the retina (just below the lens). This potential was very similar in shape to the electroretinographic or ocellar potentials recorded from other Arachnids, particularly to those recorded in Lycosa (DeVoe, 1963) and in Tegenaria (Giulio, 1962). It consisted of a negative wave, basically monophasic. which varied in a graded way with light intensity. according to a logarithmic law, at least within the range of intensities tested (Fig. 2). Peak latency time decreaskd with increasing light intensities, as shown in Fig. 2. No sign of all-or-none activity was ever recorded at the level of the retina. Effects of stimulus duration were investigated. With short flashes (up to about 40 msec) the peak amplitude of the response increased with flash duration;
I
Fig. 1. Response to a 2.5 msec flash of unit intensity. Upper trace-photocell response. Lower trace-ocellar potential. Calibration, 0.5 mV, 50 msec.
1423
Research
I -2
-1.5
I
I
-I w
-05
Note
I
1
Fir 2. Peak amphtude (top ordinates. mV) and peak la&cy time (bottom ordinates. msec) of the ocellar potenteal as a function of the logarithm of light intensity (log I). Lines drawn by eye.
3
3 9 2
for longer flashes, the peak amplitude remained constant and only a plateau could be detected (Fig. 3A). The experimental data fit we!l a theoretical curve (broken line. Fig. 3B) calculated on the basis of the
I
-------_ ___ ---1 ii-I
I”
0
,’
f
i/
1’1
exponential equation: P..P; = [(V/lQ,
- I][1 - exp(-t!:)]
+ I
uhere (L’;Lb)r = 3.25 and 5 = 9.7 .msec. The effect was observable within the whole range of intensities used and confirms “temporal summation” effects described by various authors (see Hood and Grover. 1974). Dnrk-adaptarion: After exposure to a IOsec adapting flash of unit intensity, sensitivity recovered as shown in Fig. 4, and after about 100 set the recovery was almost complete. as judged by the height of the peak response to a test flash of 5 msec, unit intensity. Critical fusion frequency was tested stimulating the dark-adapted eye with trains of flashes of unit intensity at various frequencies, with a light/dark ratio of 1. The critical frequency, at which the amplitude of the second and following responses became neglegible and fused in a steady negative polarization. was of 25 Hz. No tests were made with flashes of lower intensities. Finally. all these measurements were repeated after application on the retina of I’&, of Nicotine Hydrochloride, a substance that has been found by various authors (Autrum. 1958; Giulio, 1962) to block postsynaptic activity in the Arthropod visual system. Nicotine Hydrochloride was applied on the eye after cutting off the greatest part of the lens. Even if it was not possible to have a direct check that Nicotine Hydrochloride actually reached the synaptic level, we started the recordings about 20 min after the application of the substance-a time considered to be enough to allow penetration through the receptor cell layer. In these conditions, in accordance with data by Giulio (1962), no modification in the shape of the ocellar potential, nor in the intensity or time effects. was recorded (see Fig. 3C, where the experimental data fit the same equation as for normal physiological solution, with (V/V,), = 3.28 and r = 10.5 msec). In conclusion, our results seem to be consistent
10
M
100
Insa
Fig. 3. A. Responses to Rashes of increasmg duration (from 5 to 100 msec). Calibration. 0.5 mV. 10 msec. B. Increase in peak amplitude of response as a function of stimulus duration. E>e bathed in normal physiological solution. Ordinates: peak amphtude--C: referred to lb-peak amplitude in response to a 5 msec flash. Broken line: theoretical curve calculated as described in the text. C. Same as in B. but with the eye bathed in 19, Nicotine Hydrochloride.
with the view that extracellular recordings from Arachnid eyes are interpretable in terms of purely receptor electrical activity. The cornea-negative potential could thus be considered the extracellular recording of a depolarizing receptor potential in agreement with the results of DeVoe (1972). Moreover, we think the fact that the temporal summation effect described in Fig. 3 reaches a plateau for times of about 40 msec is worthy of note; this value is about the same as for the latency period of the response (i.e. the interval between the start of the stimulus and the beginning of the rising phase of the ocellar potential). Again. this value corresponds to the interval between t&o flashes in the 25 Hz critical fusion
1425
Research Note
3
ez Fig. 4. Recovery of the ocellar potential (response to a S msec Rash. unit intenstty) in the dark folloumg a IO set light adaptation of the same intensity. The peak amplitude of the S msec test flash without light adaptation was 3.5 mV. Curve drawn by eye
frequency described above. A possible interpretation of this fact could be that, at the end of the latency
period, while the response begins to develop. a block in the transduction process leading to no further in-
crease in peak amplitude could occur, but this problem requires some more detailed work (e.g. intracellular recordings) to be dealt with. REFERESCES
Aurrum H. (1958) Eiectrophysiologicdl analysts of the VISual systems in insects. Expi ccl/ Rrs. Suppl. 5. 426-439. Curtis D. (1970) Comparative aspects of the fine structure of the eyes of Phalangida (Arachnida) and certain correlations with habitat. J. Zool.. Lond. 160. 231-265. DeVoe R. D. (1963) Linear superimposition of retinal actron potentials to predict electrica flicker responses from the eye of the Wolf Spider. Lyeosa ~u~rr~o~ianu (Keyserlmgk J. gen. Physioi. 46, 75-96. DeVoe R. D. (1972) Dust sensitivities of cells in Wolf Spider eyes to ultraviolet and visible wavelengths of light. J. gm. Physiol. 59, 247-269. Giulio L. (1962) L’elettroretinogramma ocellare in Tegenariu (Araneaz .~gelumdm) I. Caratteristiche de1 potenziale di ~ilurn~n~~one. Boll. Sue. Ir. Biol. Sper. 38, 910-912. Hood D. C. and Grover B. G. (197-Q Temporal summation of light by a vertebrate visual receptor. Science t84. 1003-1005. Magni F. and Strata P. (1965) Electroretinographic responses from the eyes of the Wolf Spider Lvcosa Turenr& (Rossi) Archs itat Riot. 103. 694-704. _ Purcell F. (18%) Uber der bau der Phaiangidenaugen. Zritsch Wis. Zwl. 58. I-60.
THE
ELECTRICAL RESPONSE OF PHALANGIUM
OF THE OCELLUS OPILIO
MARIELLA FRELATO and FABR~ZIOVEGLIA Institute of General Physiology. University of Torino. Italy
DAMDE Lovtsoto,
(Rewired
KVJ Words-eye:
27 July
Arachnid: photoreceptor
rl~lirw/ Classificarion-Arachnid
(Phahyiurn
1977; in recisrdfoun
16 Janrrar_v 1978)
response: temporal summation. Opilio).
The origin of the extracellular potentials recorded from Arachnid eyes has been discussed by various workers (Giulio, 1962; DeVoe, 1963; Magni and Strata. 1965). It has been proposed that the ERG in these animals may be ascribed to the receptor cells only, and this interpretation is supported by anatomical data. at least for the principal eyes (Magni and Strata. 1965). confirming the absence of synaptic endings in the eye. Among Arachnids, the Phalangida present some striking differences in their visual system, having only two eyes, each with a single lens, like that of simple eyes common to all Arachnids, but with each “retinula” formed by a “central cell” and three “peripheral cells” (Purcell. 1894). and with a characteristic fusion of the microvilli of the rhabdoms of different retinulae. This peculiar structure has recently stimulated more refined ultrastructural work (Curtis. 1970) that has confirmed in more detail the existing findings. Here, too, no evidence of the presence of synaptic structures in the eye was found. The present work was begun to investigate the main features of the physiological responses of this photoreceptor structure. Freshly collected animals were immobilized with adhesive tape on a perspex base, and the eye was pierced with a very thin needle to allow the electrode to reach the retina without breaking. The oceliar potential was recorded by means of a glass microelectrode filled with 2.5 M KCI; electrical impedances ranged from 5 to 20 MR. The reference electrode consisted of an Agar-Silver chloride electrode that was placed near the’ eye into a drop of physiological saline solution (DeVoe, 1972) that moistened a small area around the eyes. Potentials recorded by the microelectrode were sent to a capacity-neutralized cathode-follower (Instrumentation Laboratories Picometric Amptier IL 181) that was connected to the differential plug-in of a Tektronix 5103N oscilloscope. The use of microelectrodes allowed us to record activity from only one, or at most, a few units (as demonstrated by occasional recordings from unidentified nerve cells, when the electrode was advanced through the eye-cup, in which unit activity was observed). The eyes were dark-adapted for about 20 min before each Set of experiments.
Light stimulation consisted of Rashes of light obtained from a Glow Modulator Tube (Sylvania, R 1131-C) driven by an American Electronic Laboratories Laboratory Stimulator 104A. The light beam was focused on a small spot (2 mm diameter). The illuminance of the unattenuated light was 1200 Ix (unit intensity); neutral density filters (Kodak Wratten) were inserted when lower intensities were used. Figure 1 shows the typical ocellar potential recorded by an electrode at the level of the distal part of the retina (just below the lens). This potential was very similar in shape to the electroretinographic or ocellar potentials recorded from other Arachnids, particularly to those recorded in Lycosa (DeVoe, 1963) and in Tegenaria (Giulio, 1962). It consisted of a negative wave, basically monophasic. which varied in a graded way with light intensity. according to a logarithmic law, at least within the range of intensities tested (Fig. 2). Peak latency time decreaskd with increasing light intensities, as shown in Fig. 2. No sign of all-or-none activity was ever recorded at the level of the retina. Effects of stimulus duration were investigated. With short flashes (up to about 40 msec) the peak amplitude of the response increased with flash duration;
I
Fig. 1. Response to a 2.5 msec flash of unit intensity. Upper trace-photocell response. Lower trace-ocellar potential. Calibration, 0.5 mV, 50 msec.
1423
Research
I -2
-1.5
I
I
-I w
-05
Note
I
1
Fir 2. Peak amphtude (top ordinates. mV) and peak la&cy time (bottom ordinates. msec) of the ocellar potenteal as a function of the logarithm of light intensity (log I). Lines drawn by eye.
3
3 9 2
for longer flashes, the peak amplitude remained constant and only a plateau could be detected (Fig. 3A). The experimental data fit we!l a theoretical curve (broken line. Fig. 3B) calculated on the basis of the
I
-------_ ___ ---1 ii-I
I”
0
,’
f
i/
1’1
exponential equation: P..P; = [(V/lQ,
- I][1 - exp(-t!:)]
+ I
uhere (L’;Lb)r = 3.25 and 5 = 9.7 .msec. The effect was observable within the whole range of intensities used and confirms “temporal summation” effects described by various authors (see Hood and Grover. 1974). Dnrk-adaptarion: After exposure to a IOsec adapting flash of unit intensity, sensitivity recovered as shown in Fig. 4, and after about 100 set the recovery was almost complete. as judged by the height of the peak response to a test flash of 5 msec, unit intensity. Critical fusion frequency was tested stimulating the dark-adapted eye with trains of flashes of unit intensity at various frequencies, with a light/dark ratio of 1. The critical frequency, at which the amplitude of the second and following responses became neglegible and fused in a steady negative polarization. was of 25 Hz. No tests were made with flashes of lower intensities. Finally. all these measurements were repeated after application on the retina of I’&, of Nicotine Hydrochloride, a substance that has been found by various authors (Autrum. 1958; Giulio, 1962) to block postsynaptic activity in the Arthropod visual system. Nicotine Hydrochloride was applied on the eye after cutting off the greatest part of the lens. Even if it was not possible to have a direct check that Nicotine Hydrochloride actually reached the synaptic level, we started the recordings about 20 min after the application of the substance-a time considered to be enough to allow penetration through the receptor cell layer. In these conditions, in accordance with data by Giulio (1962), no modification in the shape of the ocellar potential, nor in the intensity or time effects. was recorded (see Fig. 3C, where the experimental data fit the same equation as for normal physiological solution, with (V/V,), = 3.28 and r = 10.5 msec). In conclusion, our results seem to be consistent
10
M
100
Insa
Fig. 3. A. Responses to Rashes of increasmg duration (from 5 to 100 msec). Calibration. 0.5 mV. 10 msec. B. Increase in peak amplitude of response as a function of stimulus duration. E>e bathed in normal physiological solution. Ordinates: peak amphtude--C: referred to lb-peak amplitude in response to a 5 msec flash. Broken line: theoretical curve calculated as described in the text. C. Same as in B. but with the eye bathed in 19, Nicotine Hydrochloride.
with the view that extracellular recordings from Arachnid eyes are interpretable in terms of purely receptor electrical activity. The cornea-negative potential could thus be considered the extracellular recording of a depolarizing receptor potential in agreement with the results of DeVoe (1972). Moreover, we think the fact that the temporal summation effect described in Fig. 3 reaches a plateau for times of about 40 msec is worthy of note; this value is about the same as for the latency period of the response (i.e. the interval between the start of the stimulus and the beginning of the rising phase of the ocellar potential). Again. this value corresponds to the interval between t&o flashes in the 25 Hz critical fusion
1425
Research Note
3
ez Fig. 4. Recovery of the ocellar potential (response to a S msec Rash. unit intenstty) in the dark folloumg a IO set light adaptation of the same intensity. The peak amplitude of the S msec test flash without light adaptation was 3.5 mV. Curve drawn by eye
frequency described above. A possible interpretation of this fact could be that, at the end of the latency
period, while the response begins to develop. a block in the transduction process leading to no further in-
crease in peak amplitude could occur, but this problem requires some more detailed work (e.g. intracellular recordings) to be dealt with. REFERESCES
Aurrum H. (1958) Eiectrophysiologicdl analysts of the VISual systems in insects. Expi ccl/ Rrs. Suppl. 5. 426-439. Curtis D. (1970) Comparative aspects of the fine structure of the eyes of Phalangida (Arachnida) and certain correlations with habitat. J. Zool.. Lond. 160. 231-265. DeVoe R. D. (1963) Linear superimposition of retinal actron potentials to predict electrica flicker responses from the eye of the Wolf Spider. Lyeosa ~u~rr~o~ianu (Keyserlmgk J. gen. Physioi. 46, 75-96. DeVoe R. D. (1972) Dust sensitivities of cells in Wolf Spider eyes to ultraviolet and visible wavelengths of light. J. gm. Physiol. 59, 247-269. Giulio L. (1962) L’elettroretinogramma ocellare in Tegenariu (Araneaz .~gelumdm) I. Caratteristiche de1 potenziale di ~ilurn~n~~one. Boll. Sue. Ir. Biol. Sper. 38, 910-912. Hood D. C. and Grover B. G. (197-Q Temporal summation of light by a vertebrate visual receptor. Science t84. 1003-1005. Magni F. and Strata P. (1965) Electroretinographic responses from the eyes of the Wolf Spider Lvcosa Turenr& (Rossi) Archs itat Riot. 103. 694-704. _ Purcell F. (18%) Uber der bau der Phaiangidenaugen. Zritsch Wis. Zwl. 58. I-60.