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Is light receptor a dipole? Electrophysiological study in an arachnid
:Neuras~ienee Le~,ers, 5 (1977) 51--55 ©iElse~ier/North-Holland Scientific Publishers Ltd.
51
IS LIGHT RECEPTOR A DIPOLE? ELECTROPHYSIOLOGICAL STUDY IN AN ARACHNID
T. R A M A K R I S H N A * Department of Biophysfcs, National Institute of Mental Health and Neurosciences, Bangatore (India)
(Received June 14th, 1976) (Accepted March 16tb0 19'77 )
SUMMARY
Retinal action potential (RAP) was recorded at different depths in the median eye of scorpion, using a glass micropipette electrode (tip diameter 2--3 ~m). The amplitude of RAP was largest when t0he tip of the electrode t o u c h e d the distal process of the receptor cell (RC). The polarity of RAP became reversed when the tip was inside the cell body. It is suggested that RC acted as a dipole with the outer segment being the cu~ent sink and the cell body, the source. This situation is similar to the findings in the light receptors of insects, crustaceans, molluscs and also in vertebrate retina. Depth recordings through conventional electrophysiological methods have been made in such divergent preparations ranging from light receptors of Aplysia [5] to the olfactory bulb of rabbits [ 3]. What lends importance of wider implications to these studies is the interpretation in terms of a dipole field structure that is responsible for the reversal of polarity of potential when recorded from the surface to the depths in these preparations. In the light of such studies Tomita's [ 12] formulation of a thecretical model of the assumed dipole iayer within the vertebrate retina calls for more intensive efforts at identifying the occurrence or otherwise of such reversal of polarity in the receptor cells, when recorded from one end to the other, in va~ed preparations in an attempt to generalize such models. A simple median eye ira t h e scorpion, Heterometrus fulvipes, was chosen for the purpose in the present study because of its morphological suitability and experimental amenability. Bullock and Horridge [2] considered them as attractive for physiological analysis. i Stimulusav~zmbly. A tungsten filament bulb (12 V, 5.~ W) from a microscope lamp was the source. The bulb was fixed at one end of a light guide, which had *Present address: Fulbright-Hays Senibr Scholar, N.C. Department of Mental Health, Division of Research, P.O. Box 7532, Raleigh, N.C. 27611, U.S.A.
52
two condenser lenses. At a distance of 12 in. the source emitted a circular spot of light (diameter 0.4 ram). Light intensity was measured through a lux meter (Technolab Co., India). Duration of stimulus was controlled through a sectored disc, attached to a DC motor, powered by a storage battery through a linear potentiometer.
Recording deoices. Glass pipette electrode with the inside tip diameter pulled to 2--3 ~m through a vertical electrode puller and filled with scorpion Ringer [8] was used as the recording electrode (RE). Platinum wire inserted into the glass pipette was connected to the grid G I of a Grass preamplifier. A steel pin etched electrophysiologically and insulated except at the tip with the same diameter as the active electrode served as an indifferent electrode (IE). Potentials were displayed on a duat beam oscilloscope (Tektronix 502A) and were photographed with a Grass C4 camera. Stimulus was monitored
through a photocell. Preparation. Scorpion was restrained with the dorsal side up on a metal base by using plasticine. The surface of the eyes was scraped gently ~ith a microscalpel to ~emove the wax coat. While the RE was placed over the illuminated eye, the IE was placed over the adjacent unilluminated eye. As a further meamre against light-leakage, the latter was covered with black paper. Preparation as well a~ the stimulus assembly were e n c l o ~ d in a metal cage which was grounded to a common point as the other instmr~ents. Retinal action potential (RAP) was displayed on the oscilloscope every 5 rain. When the potential reached a stable level of amplitude, which it did usually after 40--60 min, the experiment was begun. All the experiments were carried ou~ in a dark room under a dim red light and at the temperature 26 +I°C. Locutiou of the recording site. The depth of penetration of RE was determined in two ways. In the first method the vernier on the micromanipulator was read to estimate the d~pth from which a particula~ recording was obtained. In the second method the RE was inserted to a depth at which a particular response was obtained. The electrode was then sprayed in situ with the fast drying opaque India ink and withdrawn. The length of the unpainted tip was measured with an ocular micrometer on a compound microscope. These methods were similar to those used by Karnpa et al. [6]. Histology. After adapting the scorpion for about 3 h in the dark, the cephalic parts of the prosoma with the eyes intact was removed under a dim red light and p~served in Bouin's fixative. Paraffin blocks were made of the median eyes and transvers(~ as well as longitudinal sections of 6--8/~m thickness were cut and stained w~ ~h eosin-hemotoxylin [ 1]. The median eyes have a biconvex cuticular lens. Underneath the lens ~s a vitreous body which is a specialized hypodermis. The retina lies beneath the constituent visual cells grouped into retinuli. The retina is separated from the vitreous body by a membrm~. The rhabdoms appear as club.shaped gr~yish-bIue rods. (Fox-~details of the structure of the eye see Fig. la.)
53
General features of the profile of RAP. When recorded with the tip of the RE at the depth of 0.48 mm or above in the eye, RAP has two prominent phases: (i) a fast rising negative wave (upward deflection) and (ii) a relatively slow positive wave, and hence is diphasic. The latency measured from the onset of stimulus to the appearance of upward rising negative phase is 70 + 7 msec. The negative wave ('a' wave) reaches its peak in 61 + 7 msec. The positive wave ('b' wave) generally has a somewt~at lower magnitude than the 'a' wave ( Fig. lb). Profile changes in RAP beyond the depth of 0:4'8 ram. When the electrode is moved downwards beyond this depth, the negative phase gets reduced in amplitude. Following this transient negative phase is the positive phase. But interestingly, the latter is followed by another prominent negative wave (Fig. lc). When the RE is moved further downwards, up to about 0.7 mm, the transient negative phase becomes reduced further and disappears completely beyond this depth; so much so the profile of RAP at this depth and beyond looks like a complete reversal of the one obtained at the 'surface' i.e. up to 0.48 mm from above (Fig. ld). Thus three distinct phases could be recognized in the depth-profile of RAP. These three phases are" (i) a negative-positive phase, (ii) a transient negative~.L
/
/ J
/
/ /
j /
/
/
VB
f
~/J/ f.11I"F Ps
(b)
°51 o.71
;~0.N~d:t
(a)
\,CT
_._l
(d}
Fig. 1. a: structural details of the median eye, camera lucida drawing. L, lens; VB, vitreous
body; M, membrane separatir~gV B from the distalsegments of the receptor; PS, pigment sheath between which the distalsegments arE:interposed; RC, receptor cell;CT, connective tissue cells,b--d: oscilloscope records of RAP, shown to originate at different loci in the eye. The lower trace in these records is the photocell-response to the stimulus. Light intensity used is 100 lux. C~lib~ation, 200 msec; I inV.
54
J
I'
>.
E ¸ 2uJ £3 I-.-
Z3
0
D
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.,J
2
2
< !
0.4
I
0.5
!
0.6
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0.7
D E PT H ( r n m )
Fig. 2. Changes in amplitude and polarity of the RAP, plotted in relation to the receptor cell (RC). Amplitude above 0 represents the negative phase and below 0, the positive phase. 1 and 2 at the depth of 0.4 mm represent the negative-positive phases, respectively, of RAP, obtained at this depth. Similarly, 1, 2 and 3 at the depth of about 0.55 mm repT~esent the negative-positive-negative phases of RAP. I and 2 at the depth of 0.7 mm re~re~ent the positive-negative phases of RAP obtained at this depth. For each phase, the iow-~st and highest values obtained from six experiments are shown. '.
positive-negative phase and (Tii) a positive-negative phase and they could be ascribed to the following histological levels: the first phase arises from the distal processes of the receptor cell since the amplitude of this phase is largest beneath the membrane, separating the vitreous body from the receptor cells; the second phase, which can possibly be termed as transitory between the first and the third phases, arises from the mid-retina. Thus this transitory phase seems to have been obtained when the electrode tip approaches the cell body of the receptors. When the tip of the electrode is pushed further down, into the cell body, the third phase in RAP res~ting in the reversal of polarity can be seen (Fig. 2). It is reasonable to assume, however, that the principal contributor to the RAP seems to be the receptor distal segments as is indicated by the amplitude. Reversal of polarity of RAP and its significance. Such reversal of polarity of RAP in the depths of the eye is not unknown. Reports similar to the present study are available in crustaceans [6:9] insects [10] and molluscs [4,5,7,11]. However, the present study is the first report of its kind among arachnids. What invests these reports with significance is Jacldet's [ 5] interpretation of his study on the electrophysiolo$cal organization of the eye of Aplysia, based on a dipole hypothesis, The electroretinogram reco~ed from this eye showed a maximum negativity at the distal segments, neutr~ity near the cell body layer and positivity on the outside of the eye capsule. Jacklet concluded that the receptor cell acted as a dipole, with the outer segment being the
55
cun'ent sink and the cell body the source. It is suggested that a similar situation possibly prevails in the median eye of the scorpion. ACKNOWLEDGEMENTS
The equipment used in this study was a gift,from U S A F O S R to Professor T.H. Bullock and Professor K.P. Rao, and the work was carried out at the Department of Zoology, Bangalore University..Ithank D~. R.V. Krishnarnoorthy for advice and Dr. A.R. Kasturi Bai for facilities. REFERENCES 1 Ages, H.R., Histology of the compound eye of Heliothis virescens (Lepidoptera: Nocmidae), Ann. Entomol. Soc. Amer., 65 (1972) 767--768. 2 Bullock, T.H. and ]~orzidge, G.A., Structure and Function in the Nervous Systems of Invertebrates,Vol. II. Freeman and Coo, San Francisco, Calif.,1965, 1107 pp. 3 Freeman, W.J.~ Depth recording of averaged evoked potentialof olfactory bulb, J. Neuzophysiol., 35 (1972) 780--796. 4 Hagins, W., Zonana, H. and Adams, R., Local membrane currents in the outer seg° ment~ of squid photoreceptor~, Nature (Lond.), 194 (1962) 844. 5 Jacklet, J.W., Electrophysiological organization of the eye of Aplysia, J. gen. Physiol.~ 53 (1969) 21--42. 6 Kampa, E.M., Abbott, B.C. and Boden, B.P., Some aspects of vision in the lobster, Homarus vulgari#, in relation to the structure of its eye, J. mar. biol. Ass. U.K., 43 (1963) 683--699. 7 Karita,K., Ito, S. and Tasaki, J., Fast and slow components in the electroretinograrn of the gastropod, Tohoku J. exp. Med., 109 (1973) 77--84. 8 Naidu, B.P., Perfusion fluid for the scorpion. Heterometrus fulvipes, Nature (Londo), 213 (1967) 410. 9 Naka, J. and Kuwabara, M., T w o components from ~he compound eye of the crayfish, J. exp. Biol., 36 (1959) 51--61. 10 Swihart, S.L., Variability and the nature of the insect electroretinogram. J. Insect Physiol., 18 (1972) 1221--1240. 11 Tasaki, K., Oikawa, T. and Norton, A., The dual nature of the octopus electroretinogram. Vision Res., 3 (1963) 61. 12 Tomita, R., The electrozetinogram as analyzed by microelectrode studies. In Handbook of Sensory Physiology, VII/2, Springer, New York, 1972, pp. 635--665.
51
IS LIGHT RECEPTOR A DIPOLE? ELECTROPHYSIOLOGICAL STUDY IN AN ARACHNID
T. R A M A K R I S H N A * Department of Biophysfcs, National Institute of Mental Health and Neurosciences, Bangatore (India)
(Received June 14th, 1976) (Accepted March 16tb0 19'77 )
SUMMARY
Retinal action potential (RAP) was recorded at different depths in the median eye of scorpion, using a glass micropipette electrode (tip diameter 2--3 ~m). The amplitude of RAP was largest when t0he tip of the electrode t o u c h e d the distal process of the receptor cell (RC). The polarity of RAP became reversed when the tip was inside the cell body. It is suggested that RC acted as a dipole with the outer segment being the cu~ent sink and the cell body, the source. This situation is similar to the findings in the light receptors of insects, crustaceans, molluscs and also in vertebrate retina. Depth recordings through conventional electrophysiological methods have been made in such divergent preparations ranging from light receptors of Aplysia [5] to the olfactory bulb of rabbits [ 3]. What lends importance of wider implications to these studies is the interpretation in terms of a dipole field structure that is responsible for the reversal of polarity of potential when recorded from the surface to the depths in these preparations. In the light of such studies Tomita's [ 12] formulation of a thecretical model of the assumed dipole iayer within the vertebrate retina calls for more intensive efforts at identifying the occurrence or otherwise of such reversal of polarity in the receptor cells, when recorded from one end to the other, in va~ed preparations in an attempt to generalize such models. A simple median eye ira t h e scorpion, Heterometrus fulvipes, was chosen for the purpose in the present study because of its morphological suitability and experimental amenability. Bullock and Horridge [2] considered them as attractive for physiological analysis. i Stimulusav~zmbly. A tungsten filament bulb (12 V, 5.~ W) from a microscope lamp was the source. The bulb was fixed at one end of a light guide, which had *Present address: Fulbright-Hays Senibr Scholar, N.C. Department of Mental Health, Division of Research, P.O. Box 7532, Raleigh, N.C. 27611, U.S.A.
52
two condenser lenses. At a distance of 12 in. the source emitted a circular spot of light (diameter 0.4 ram). Light intensity was measured through a lux meter (Technolab Co., India). Duration of stimulus was controlled through a sectored disc, attached to a DC motor, powered by a storage battery through a linear potentiometer.
Recording deoices. Glass pipette electrode with the inside tip diameter pulled to 2--3 ~m through a vertical electrode puller and filled with scorpion Ringer [8] was used as the recording electrode (RE). Platinum wire inserted into the glass pipette was connected to the grid G I of a Grass preamplifier. A steel pin etched electrophysiologically and insulated except at the tip with the same diameter as the active electrode served as an indifferent electrode (IE). Potentials were displayed on a duat beam oscilloscope (Tektronix 502A) and were photographed with a Grass C4 camera. Stimulus was monitored
through a photocell. Preparation. Scorpion was restrained with the dorsal side up on a metal base by using plasticine. The surface of the eyes was scraped gently ~ith a microscalpel to ~emove the wax coat. While the RE was placed over the illuminated eye, the IE was placed over the adjacent unilluminated eye. As a further meamre against light-leakage, the latter was covered with black paper. Preparation as well a~ the stimulus assembly were e n c l o ~ d in a metal cage which was grounded to a common point as the other instmr~ents. Retinal action potential (RAP) was displayed on the oscilloscope every 5 rain. When the potential reached a stable level of amplitude, which it did usually after 40--60 min, the experiment was begun. All the experiments were carried ou~ in a dark room under a dim red light and at the temperature 26 +I°C. Locutiou of the recording site. The depth of penetration of RE was determined in two ways. In the first method the vernier on the micromanipulator was read to estimate the d~pth from which a particula~ recording was obtained. In the second method the RE was inserted to a depth at which a particular response was obtained. The electrode was then sprayed in situ with the fast drying opaque India ink and withdrawn. The length of the unpainted tip was measured with an ocular micrometer on a compound microscope. These methods were similar to those used by Karnpa et al. [6]. Histology. After adapting the scorpion for about 3 h in the dark, the cephalic parts of the prosoma with the eyes intact was removed under a dim red light and p~served in Bouin's fixative. Paraffin blocks were made of the median eyes and transvers(~ as well as longitudinal sections of 6--8/~m thickness were cut and stained w~ ~h eosin-hemotoxylin [ 1]. The median eyes have a biconvex cuticular lens. Underneath the lens ~s a vitreous body which is a specialized hypodermis. The retina lies beneath the constituent visual cells grouped into retinuli. The retina is separated from the vitreous body by a membrm~. The rhabdoms appear as club.shaped gr~yish-bIue rods. (Fox-~details of the structure of the eye see Fig. la.)
53
General features of the profile of RAP. When recorded with the tip of the RE at the depth of 0.48 mm or above in the eye, RAP has two prominent phases: (i) a fast rising negative wave (upward deflection) and (ii) a relatively slow positive wave, and hence is diphasic. The latency measured from the onset of stimulus to the appearance of upward rising negative phase is 70 + 7 msec. The negative wave ('a' wave) reaches its peak in 61 + 7 msec. The positive wave ('b' wave) generally has a somewt~at lower magnitude than the 'a' wave ( Fig. lb). Profile changes in RAP beyond the depth of 0:4'8 ram. When the electrode is moved downwards beyond this depth, the negative phase gets reduced in amplitude. Following this transient negative phase is the positive phase. But interestingly, the latter is followed by another prominent negative wave (Fig. lc). When the RE is moved further downwards, up to about 0.7 mm, the transient negative phase becomes reduced further and disappears completely beyond this depth; so much so the profile of RAP at this depth and beyond looks like a complete reversal of the one obtained at the 'surface' i.e. up to 0.48 mm from above (Fig. ld). Thus three distinct phases could be recognized in the depth-profile of RAP. These three phases are" (i) a negative-positive phase, (ii) a transient negative~.L
/
/ J
/
/ /
j /
/
/
VB
f
~/J/ f.11I"F Ps
(b)
°51 o.71
;~0.N~d:t
(a)
\,CT
_._l
(d}
Fig. 1. a: structural details of the median eye, camera lucida drawing. L, lens; VB, vitreous
body; M, membrane separatir~gV B from the distalsegments of the receptor; PS, pigment sheath between which the distalsegments arE:interposed; RC, receptor cell;CT, connective tissue cells,b--d: oscilloscope records of RAP, shown to originate at different loci in the eye. The lower trace in these records is the photocell-response to the stimulus. Light intensity used is 100 lux. C~lib~ation, 200 msec; I inV.
54
J
I'
>.
E ¸ 2uJ £3 I-.-
Z3
0
D
I2
.,J
2
2
< !
0.4
I
0.5
!
0.6
i,
l, |
0.7
D E PT H ( r n m )
Fig. 2. Changes in amplitude and polarity of the RAP, plotted in relation to the receptor cell (RC). Amplitude above 0 represents the negative phase and below 0, the positive phase. 1 and 2 at the depth of 0.4 mm represent the negative-positive phases, respectively, of RAP, obtained at this depth. Similarly, 1, 2 and 3 at the depth of about 0.55 mm repT~esent the negative-positive-negative phases of RAP. I and 2 at the depth of 0.7 mm re~re~ent the positive-negative phases of RAP obtained at this depth. For each phase, the iow-~st and highest values obtained from six experiments are shown. '.
positive-negative phase and (Tii) a positive-negative phase and they could be ascribed to the following histological levels: the first phase arises from the distal processes of the receptor cell since the amplitude of this phase is largest beneath the membrane, separating the vitreous body from the receptor cells; the second phase, which can possibly be termed as transitory between the first and the third phases, arises from the mid-retina. Thus this transitory phase seems to have been obtained when the electrode tip approaches the cell body of the receptors. When the tip of the electrode is pushed further down, into the cell body, the third phase in RAP res~ting in the reversal of polarity can be seen (Fig. 2). It is reasonable to assume, however, that the principal contributor to the RAP seems to be the receptor distal segments as is indicated by the amplitude. Reversal of polarity of RAP and its significance. Such reversal of polarity of RAP in the depths of the eye is not unknown. Reports similar to the present study are available in crustaceans [6:9] insects [10] and molluscs [4,5,7,11]. However, the present study is the first report of its kind among arachnids. What invests these reports with significance is Jacldet's [ 5] interpretation of his study on the electrophysiolo$cal organization of the eye of Aplysia, based on a dipole hypothesis, The electroretinogram reco~ed from this eye showed a maximum negativity at the distal segments, neutr~ity near the cell body layer and positivity on the outside of the eye capsule. Jacklet concluded that the receptor cell acted as a dipole, with the outer segment being the
55
cun'ent sink and the cell body the source. It is suggested that a similar situation possibly prevails in the median eye of the scorpion. ACKNOWLEDGEMENTS
The equipment used in this study was a gift,from U S A F O S R to Professor T.H. Bullock and Professor K.P. Rao, and the work was carried out at the Department of Zoology, Bangalore University..Ithank D~. R.V. Krishnarnoorthy for advice and Dr. A.R. Kasturi Bai for facilities. REFERENCES 1 Ages, H.R., Histology of the compound eye of Heliothis virescens (Lepidoptera: Nocmidae), Ann. Entomol. Soc. Amer., 65 (1972) 767--768. 2 Bullock, T.H. and ]~orzidge, G.A., Structure and Function in the Nervous Systems of Invertebrates,Vol. II. Freeman and Coo, San Francisco, Calif.,1965, 1107 pp. 3 Freeman, W.J.~ Depth recording of averaged evoked potentialof olfactory bulb, J. Neuzophysiol., 35 (1972) 780--796. 4 Hagins, W., Zonana, H. and Adams, R., Local membrane currents in the outer seg° ment~ of squid photoreceptor~, Nature (Lond.), 194 (1962) 844. 5 Jacklet, J.W., Electrophysiological organization of the eye of Aplysia, J. gen. Physiol.~ 53 (1969) 21--42. 6 Kampa, E.M., Abbott, B.C. and Boden, B.P., Some aspects of vision in the lobster, Homarus vulgari#, in relation to the structure of its eye, J. mar. biol. Ass. U.K., 43 (1963) 683--699. 7 Karita,K., Ito, S. and Tasaki, J., Fast and slow components in the electroretinograrn of the gastropod, Tohoku J. exp. Med., 109 (1973) 77--84. 8 Naidu, B.P., Perfusion fluid for the scorpion. Heterometrus fulvipes, Nature (Londo), 213 (1967) 410. 9 Naka, J. and Kuwabara, M., T w o components from ~he compound eye of the crayfish, J. exp. Biol., 36 (1959) 51--61. 10 Swihart, S.L., Variability and the nature of the insect electroretinogram. J. Insect Physiol., 18 (1972) 1221--1240. 11 Tasaki, K., Oikawa, T. and Norton, A., The dual nature of the octopus electroretinogram. Vision Res., 3 (1963) 61. 12 Tomita, R., The electrozetinogram as analyzed by microelectrode studies. In Handbook of Sensory Physiology, VII/2, Springer, New York, 1972, pp. 635--665.