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An investigation of the lateral spread of potentials in the octopus retina
Yision HIT.
Vol. 5, pp. 253-267.
Pcrgamon Press 1965.
AN INVESTIGATION POTENTIALS ALLEN
Printed inGreatBritain.
OF THE IN THE
LATERAL
OCTOPUS
C. NORTON,~ YOSHIRO FUKADA,~ Kom
SPREAD
OF
RETINA
MOTOKAWA
and KYOJI TASAKI
Department of Physiology, Tohoku University School of Medicine, Sendai, Japan (Received 15 June 1964) INTRODUCTION PREVIOUS
reports from this laboratory have described the spread of potentials around a locally illuminated region of the octopus retina (TASAKI et al., 1963a, 1963b). This spread of potentials was demonstrated by moving a spot of light around a fixed recording electrode. It was found that the surface response (a sustained negative potential recorded from the anterior surface of the octopus retina, see TASAKI ef al., 1963c) increases in amplitude as the light spot approaches the electrode from one side, reaching a maximum as the spot passes over the electrode and decreasing as the spot moves to the other side. The distribution, or position-amplitude relation, is symmetrical. The deep response (a sustained positive potential recorded from near the receptor cell bodies of the retina, see TASAKI et a/., 1963c) shows a different and more complex positionamplitude relation. As the light spot approaches the recording electrode from one side the usual positive responses increase in amplitude and reach a maximum as the spot passes over the recording electrode. As the spot passes to the other side of the electrode, however, negative responses suddenly appear. These negative potentials show first an increase and then a decrease in amplitude as the light spot is moved away from the recording electrode. The distribution is markedly asymmetrical, positive responses occurring as the light spot is presented on one side of the electrode and negative responses occurring from stimulation of the other side. Transition (biphasic negative-positive) responses can occasionally be recorded from the boundary between the illuminated and unilluminated portions of the retina. The origin and significance of the deep negative responses is discussed below (see Discussion). A previous study used this asymmetrical distribution of the deep response as a means for investigating various aspects of the electrophysiology of the octopus retina (TASAKI et al., 1963b). The following properties of the position-amplitude relation of the deep response wcrc shown : (1) The appcarancc of the negative responses depends upon the diameter of the stimulus spot. With stimulus spots below l/2-1/4 mm, negative responses do not occur and a symmetrical distribution of only positive responses is recorded. (2) The deep (positive) responses, but not the surface (negative) responses, can be blocked by cutting the retina mechanically. (3) Diffuse background illumination has little effect on the relative amplitude of the positive responses, but it will selectively decrease the amplitude of the negative responses. 1 This investigation .was carried out during the tenure of a postdoctoral fellowship from the National Institute of Mental Health, United States Public Health Service. Present address: Institute of Medical Research, Huntington Memorial Hospital.Pasadena,Califomia, U.S.A. 2 Present address: Technical Research Laboratories, Japan Broadcasting Corporation (N.H.K.), Tokyo, Japan. 253
254
ALLEN C. NORTON, YOSHIRO FUKADA,
KOITI MOTOKAWA AND
KYOJI TASAKI
In addition to these characteristics of the lateral spread of the deep responses, it was frequently observed that the asymmetrical distribution of potentials was different along different lines passing over the recording electrode and also that the direction (from the recording electrode) in which maximum amplitude negative responses were elicited was different in different parts of the retina. The present paper explores these directional and regional differences in the spread of deep potentials in the octopus retina; a unique central area of the retina is also reported. A preliminary report of these phenomena has been published previously (TASAICI ef al., 1963a). METHODS The experimental animals were twenty-five octopuses (one Octopus dojfeini, Wiilker, and the remainder 0. vu/gut-is). Before use they were dark adapted for at least 1 hr. Under a dim photographic safety-lamp, they were decapitated and the eyes were enucleated. The anterior parts of the eye were dissected away, lcaving only the retina and attached sclera. Excess vitreous fluid was removed by absorption onto filter paper. The retina was placed, sclera downward, on a small piece of black cloth which had been moistened with the vitreous fluid from the same eye. The retina and cloth were then placed on a plastic mount which contained an Ag-AgCl referenceelectrode. To flatten the preparation, small pieces of tissue were cut from the periphery of the retina in areas which would not lie flat, the cuts being made from periphery to periphery, not from periphery to center. The retina and mount were placed on a microscope stage which had a micrometer-driven slide carriage. With the electrode position fixed, the mount could be moved accurately to allow penetration in any region of the retina; the electrode was withdrawn whenever the retina was moved. For convenience of discussion in the following sections, the electrode, rather than the retina, is referred to as having been moved. Special care was taken to preserve the in vivo orientation of the retina. In the intact animal the pupil, a nearly rectangular slit, is always kept in a horizontal position (WELLS, 1962). This orientation was maintained by placing the retina (and mount) on the experimental table so that the mid-horizontal line of the retina (corresponding to the long axis of the pupil) was parallel to the front edge of the table, with the ventral portion of the retina toward the front of the table. From this standard orientation, local regions of the retina were referred to by clockfact notation: 12:OOo’clock upward (away from the experimenter), 3:00 to the right, etc. This is the notation used in the present paper. The position of the electrode and light spot were viewed directly through a dissecting microscope in front of the preparation. The recording clcctrodcs wcrc glass capillaries filled with 3 M KCI; the tip diameter was less than l/l. Electrodes with especially long, thin shanks were selected to minimize reflection of light from the electrode as the electrode or spot was moved across the retina. The signals were led through cathode follower preamplifiers and d.c. coupled amplifiers and displayed on a dual beam cathode ray oscilloscope. The light stimulus was provided by a tungsten projection bulb projected through a simple optical system (see Fig. 1 in TASAKI ef nl., 1963b). Circular masks in the light beam limited the projected stimulus spot to 2 or 3 mm diameter. To obtain displacement of the stimulus spot across the retina, the final focussing lens of the optical system was moved through a plane perpendicular to the optical axis by the use of a 2-dimension micromanipulator. The spot could be moved 20 mm over the retinal surface without significantly altering its focus, and displacements as small as l/4 mm could be made with reasonable accuracy. The stimulus duration (500 msec in all experiments) was controlled by a magnetic shutter driven by an electronic square pulse generator. The stimulus repetition was one flash/l0 set, or one flash/20 set in series which required more time for adjustments between flashes. RESULTS
When a small region of the octopus retina is illuminated, a predictable sequence of responses can be recorded as a microelectrode is inserted through the retina within the illuminated area (see Fig. 1, TASAKI et al., 1963~). High amplitude negative responses are recorded as the electrode touches the vitreal surface; these responses are of maximum amplitude near the surface of the retina and decrease in amplitude as the electrode is inserted deeper into the retina. At around 400 ,Ufrom the surface there is an equipotential region in which only small deflections are recorded at the “on” and “off)’ of the stimulus flash. Still deeper in the retina, around the receptor cell bodies, positive responses are recorded. The following experiments studied only the deep (normally positive) responses (in
Lateral Spread in the Octopus Retina
255
section 13the surface and deep responses are compared). The deep responses were recorded at a depth from which maximum amplitude positive responses were obtained when the light spot was centered on the recording electrode. A, Equivalency of the moving spot and moving electrode procedures Previous cxpcrimcnts on the spread of potentials in the octopus retina employed only the moving spot procedure (TASAKI et al., 1963b, 1963c). Since regional differences were observed (see below), it was necessary to determine whether the same spread of potential could be recorded when the electrode rather than the light spot was moved,
-
-v
v-V-7
FIG. 1. Moving electrode (above) and moving spot Wow) procedures. Recordings along the mid-hori~n~ axis on the right side of the retina (around 3:00 o’clock); traces represent the deep response at successive 1 mm displacements across the retina; circles indicate the diameter of the light spot (2 mm). Vertical bars {right), 2 mV; positivity downward in these and following records.
One series of records was taken with the electrode position fixed while the stimulus spot was moved. In the same area of the retina, a second series was taken with the position of the light spot fixed while the electrode was moved. After each displacement the electrode was reinserted in the retina to record maximum amplitude positive responses when the light spot was centered on the electrode, records being taken from a constant functional (rather than physical) depth. With careful procedure, comparable deep responses could be obtained with each penetration in a series. The results of a typical experiment are shown in Fig. 1; these records were taken from the right side of the retina around 3:oO o’clock. The upper line of black traces represents the moving electrode series, while the lower line of white traces represents the moving spot series. The circles indicate the area of the stimulus spot in relation to the distance the electrode (or spot) was moved; the traces represent the response at 1 mm intervals. With both procedures relatively high amplitude positive responses were recorded from within the illuminated area. Outside the illuminated area negative responses were recorded as the electrode was moved to the right (top row) and as the spot was moved R
256
ALLEN C. NORTON, YOSHIRO FUKADA, Korrr
MOTOKAWA
AND
KYOJI TASAKI
to the left (bottom row). Thus, although the actual direction of movement was opposite in the two cases, the orientation of the spot and electrode with respect to each other remained constant when negative responses appeared. It was concluded that, at least for smali areas of the retina, the moving electrode or moving spot procedures would yield equivalent results with respect to the lateral distribution of the deep potentials. Since the depth of the electrode in the retina has considerable influence on the form and amplitude of the deep response recorded (TASAKIet al., 1963c), the moving spot procedure was employed in most of the following experiments, thus assuring a constant vertical depth from which responses were recorded.
FIG. 2. Two-dimensionexperimentaround 9:00 o’clockwith movingspot procedure. Horizontal series A-A’, left; and vertical series B-B’, right. Co-ordinates: Amplitude of the deep response, and distance light spot was moved betweensuccessiverecordings. Insets top and right indicate oscilloscoperecords at position indicated; lower traces (top and left) show
stimulus and time (10 c/s) marks, vertical bars, 1 mV. Dotted line shows diameter of the spot. Lower insert shows the retinal position in which thb electrode was inserted and the lines along which the light spot was moved.
B. Directionaldifferences in the distributionof the deep response The previous experiments considered the distribution of potentials along only one line passing over the active electrode. The following experiments investigate more fully the distribution of potentials around locally iiluminated areas of the octopus retina. Figure 2 illustrates a typical two-dimension experiment. The light spot was first moved along the mid-horizontal line from A to A’; the distribution of response recorded is shown on the horizontal coordinates. Next the light spot was moved vertically aiong a second line passing over the electrode from B to B’; the distribution of responses is shown on the vertical coordinates. Although the horizontal experiment showed the asymmetrical distribution of potentials described above, the vertical experiment showed a symmetrical distribution with positive responses appearing for short distances on either side of the illuminated area and negative responses at still more distant points. A symmetrical distribution of potentials along lines perpendicular to the line of maximum asymmetry was a consistent finding. It will be noted in Fig. 2 (and following figures) that there are slight deviations from strict
Lateral Spread in the Octopus Retina
257
symmetry in distributions
which are called symmetr~~l. Since the mechanical manipulations could only approximate the texture of the actual potential gradients, the present records can Experimental error is particularly show only approximately symmetrical distributions, obvious with respect to centering the light spot on the electrode and determining the edge of the light spot.
FIG. 3. Scanning experiment around 9:00 o’clock, moving spot procedure. Traces indicate the deep responses at successive 1 mm intervals within a 13 x 13mm array; electrode was in the center of the array. Stimulus and time mark (10 c/s) lower left, calibration (-2 mV) lower right, millimeter scale lower center.
More detailed scanning experiments were next undertaken. The light spot was moved from right to left and from top to bottom (similar to the scanning on a television screen) around the area in which the electrode was inserted. Responses were recorded at 1 mm intervals; one flash was presented every 10 sec. Figure 3 shows the array of responses obtained from such an experiment; the electrode was in the center of the array, in the area showing high amplitude positive responses. This same data is presented in schematic form in Fig. 4 which shows the response amplitude as proportional to the area of the circles; the
258
ALLEN C. NORTON, YOSHIRO FUKADA, KOITI MOTOKAWA
AND
KYOJI T’ASAKI
filled circles represent negative responses and the open circles positive responses. The inset at the lower left shows the position of the electrode and the area scanned in relation to the mid-horizontal and mid-vertical axes of the retina. Since it was shown above that, at least for small areas of the retina, the moving spot and moving electrode procedures provide equivalent results, the pattern of potentials shown in Fig. 4 may be considered as illustrating the spread of potentials around a locally illuminated region. Considering the actual position of the light spot with respect to the electrode, it is apparent that in this region of the retina (around 903 o’clock) positive potentiaIs spread toward the right and negative potentials to the left of the illuminated area.
0
+SmV
0 -ImV
/--
8 FIG. 4. Schematicrepresentationof data shown in Fig. 3. Response amplitude is shown as proportional to the area of the circles; positive responses open circles, negative responses filled circles. Position of the electrode and area scanned shown at left.
To confirm the previous finding that the asymmetrical distribution of deep responses is not found in the surface (negative) response, simultaneous records of the surface response were also taken. Figure 5 shows the surface responses corresponding to the deep responses shown in Fig. 4 (the two responses were recorded simultaneously); the same schematic representation is employed in both figures. Figure 5 shows that there is no asymmetry in the distribution of the surface responses around a locally illuminated area. This finding, it may be noted, provides additional support to the previous proposition that the surface and deep responses behave differently with respect to stimulus variables and probably reflect two origins of potential within the octopus retina (TASAKI et al., 1963b, 1963~).
259
Lateral Spread in the Octopus Retina
C. Regional differences in the distribution of the deep response
The experiments described in section B were all performed on the left side of the retina (around 9:00 o’clock). Similar directional differences in the distribution of the deep response were observed in other regions of the retina, however, the direction of the asymmetry was found to be different in different regions. Around 3:OO o’clock on the retina the vertical record was symmetrical and the horizontal record asymmetrical. However, unlike the recordings taken at 9:00 o’clock, those taken at 3:00 o’clock revealed negative responses
0
l
-lOmV
-ImV
FIG. 5. Scanning experiment showing distribution of the surface response around 9:OO o’clock. Records taken simultaneously with those shown in Figs. 3 and 4; schematic representation is the same as in Fig. 4.
when the light spot was to the left of the electrode and positive responses when it was to the right (see lower row in Fig. l), the opposite of that seen in Fig. 3. Figure 6 shows the results of a two-dimension experiment performed at 12:00 o’clock. At this position, the horizontal record is symmetrical, but the vertical record shows asymmetry with negative responses occurring toward the bottom. Experiments at 6:00 o’clock showed a similar symmetry of the horizontal records and asymmetry of the vertical records, but in the latter case the negative responses appeared when the light spot was above the electrode. These findings suggest that there is either a horizontal-vertical or a radial organization within the retina. In reference to the first alternative, it may be noted that SUTHERLAND (1960) has proposed a theory of pattern discrimination in the octopus which involves coding of pattern information along the horizontal and vertical extents of the figures. However, since the above experiments were performed only at 3 :OO,6:00,9:00 and 12:OOo’clock, the second alternative cannot be excluded.
+mV
FIG. 6. Two-dimension
@
-mv
experiment around 1290 o’clock. Otherwise same as Fig. 2. Vertical bars, 2 mV.
$ u
4 IOmV
- Imv
Fro. 7. Scanning experiment performed around 4:30 o’clock. Otherwise same as Fig. 4.
261
Lateral Spread in the Octopus Retina
The two alternatives were tested by performing similar experiments at intermediate positions. Figure 7 shows the results of a scanning experiment performed at 4:30; the procedure and schematic representation are the same as above (Figs. 4 and 5). It is seen here that although some asymmetry occurs along the horizontal and vertical lines, the maximum asymmetry is along a diagonal directed toward thecenter of the retina. It was thus concluded that the directional differences of the deep response always occur with respect to the center of the retina. In view of the equivalence of the moving electrode and moving spot procedures, it seems clear that: from any locally illuminated region of the retina there is a spread of positive potentials toward the center of the retina and a spread of negative potentials toward the periphery,
mV -I
l
A
. . . . .
A’
0 +I +2 +3
:~~~
-I 0 +I +2 +3 -I 0 +I +2 +3 -I 0 +I +2 +3 -mV
FIG. 8. At physiological center, 44rection experiment. Inset (Iower right) shows position of electrode and lines along which tight spot was moved. Abscissae: amplitude of the deep response (mv); coordinates: distance light spot was moved (mm). Dotted line (top) shows diameter of stimulus spot.
262
ALLEN C. NORTON, YOSHIRO FUKADA, KOITI MOTOKAWA AND KYOJI TASAKI
D. The physiological center
ofthe octopus retina
ft follows from the above findings that there should be some central region of the retina toward which the maximum asymmet~ is directed. It would be expected that recordings taken from this region would show a symmetrical distribution of potentials along any line passing over the recording electrode. The term physiological center has been adopted to refer to this central region, the region being defined by its physiological properties. The following method was used to locate this central region. With the electrode inserted near the center of the retina to record maximum amplitude positive responses, a two-dimension moving spot experiment was performed. The appearance of asymmetrical negative responses was noted and the electrode was moved in the direction from which the negative responses
w +5mV
-Imv
~ FIG. 9.
allying
cxpcziment ~rform~
at the physiologica ccntcr. Otherwise same as Figs. 4, 5 and 7.
had been recorded. The two-dimension experiment was repeated, and further adjustment of the electrode was made as indicated. In several experiments it was indeed possible to locate a region from which symmetrical distributions were obtained as the light spot was moved along any line passing over the electrode. Since the edges of the retina had been trimmed irregularly in order to flatten the preparation, it could not be determined whether the physiological center coincided with the geometrical center of the whole retina. Examples of the distribution of potentials as the light spot was moved around such a central region are
Lateral Spread in the Octopus Retina
263
shown in Fig. 8 (graphically along four lines passing over the electrode) and Fig. 9 (a scanning experiment shown schematically). In both examples it is to be noted first that there is a symmetrical distribution of intermediate amplitude negative potentials around the central positively responding area, and second that the positively responding region occupies a far more narrow area than is the case in other parts of the retina (compare Fig. 8 with Figs. 2 and 6, or Fig, 9 ~virh Figs. 4 and 7). It was demonstrated for other regions of the retina that the moving spot and moving electrode procedures were equivalent (Section A, above). Since the dir~tionality of potentials observed in other parts of the retina is not seen at the point which the moving spot
FIG. IO. Two-dimension, moving clcctrodo cxpcrimcnt at the physiological ccntcr, Tracts indicate amplitude and wave form of the deep responses at successive one mm intervals along the mid-horizontal and mid-vertical lines of the retina. Verticalbar, 2 mV.
procedure indicates to be the physiological center, it is to be expected that similar findings would be shown by the moving electrode procedure. This expectation was tested directly. The light spot was fixed at the point indicated as the physiological center and the electrode was moved along orthogonal lines passing over this point. The results of this procedure are shown in Fig. 10. As was demonstrated with the moving spot procedure, there is a symmetrical distribution of intermediate amplitude negative responses around the illuminated area, and the positively responding area is relatively narrow (compare with Fig. 1) with the positive responses limited to the iIluminated area. The fun~tiona1 si~ificance of this central part of the retina is considered below.
264
ALLEN
C. NORTON, YOSHIRO FIIKAUA,
Ken-I MOTOKAWA
AND KYOJI ‘TASAKI
DISCUSSION The present study reveals a highly organized directionality in the spread of the deep responses in the octopus retina. Around any locally illuminated area in the retina there is a spread of positive potentials toward a unique central part of the retina. Negative responses appear in the opposite direction, radially toward the near periphery. Thus an asymmetrica distribution of positive and negative potentials is observed along lines passing between an illuminated area and the unique central area. Given this basic organization of the spread of potentials it follows that there is a symmetrical distribution of potentials along lines per~ndicular to the line of maximum asymmetry. The unique central part of the retina has been termed the~~y~~o~Qg~~fff cenfer; this area is defined by its physiological properties: that there is a symmetrical distribution of potentials along any tine passing over it. It seems highly unlikely that the organization reported above is due to artifacts in the experimental procedure. The phenomena reported were consistent, predictable and easily replicated. The possibility that the deep negative responses, for example, arose from the scattering of stray light from the recording electrode can be rejected by the finding that the ncgativc rcsponscs appear in relation to the radial organization of the retina (described above), while the electrode was always inserted from the right of the preparation (3:OO o’clock). The orthogonal arrangement of the experimental apparatus, in contrast to the radial organization found in the spread of the deep potentials, excludes the possibility of many other procedural errors. The possibility that radial gradients arose from the placement of the indifferent electrode may also be rejected; the same results were obtained whether the indifferent electrode was placed in the center or near the periphery of the preparation. Furthermore the black cloth soaked with vitreous fluid between the sclera and the indifferent electrode served to distribute the potential of the reference electrode over the entire surface of the sclera. Attempts to measure the conduction velocity of the spread of potentials were unsu~essful. Because of the small distance in which responses of the same polarity could be recorded and the relativeiy slow rise time of the responses, conduction velocities as slow as a few meters per second could not be proved. It should be noted, however, that the conduction velocity of the slow potentials of the carp retina was found to be as low as 100 mmjsec (MOTOKOWA et al., 1959b). [Our thanks are expressed to Prof. J. Z. YOUNG (personal communication) for pointing out the significance of the latter two factors.] Although there was considerable variabjIjty in amplitude of response between preparations, little variability of response amplitude was observed in different parts of the same retina. The origin of the surface (negative) and deep (normally positive) potentials of the octopus retina has been considered previously (TASAKI et al., 1963b, 1963~). It was tentatively suggested that the surface responses arose from the pigment cells and the deep responses from the receptor cells; the likelihood that the recorded responses actually represented an interaction of these two potentials was also noted. The origin of the negative potentials in the deep portions of the retina is unclear. The previous findings (TASAKI et al., 1963b), first that the deep negative responses do not occur when the stimulus spot is very small and second that the deep responses are entirely negative when the stimulus spot is very large, suggest a complex spatial interaction in the generation of the deep responses, both positive and negative. At present, experimental findings are not available to account for the origin of the deep responses, their dependence upon the area of the stimulus spot, and the basis of their radial organization (as reported in the present paper). These three factors are obviously interrelated, but a clearer understanding must await further experimental study. The mechanism by which responses appear to spread through the octopus retina is also
Lxtcral Sprcact in the Oclopus Rclina
265
unclear. For the slow surface responses in the fish retina, MOTOKAWAet al. (1959a) have demonstrated that there is a negatively responding region which surrounds the local (positive) potentials and have suggested this as involved in visual contrast (MOTOKAWA eral., 1960). The negative potentials in the fish retina, however, are symmetrically distributed around the positively responding (illuminaled) area, in contrast to the asymmetrical distribution found in the octopus. The neural plexus of the vertebrate retina, which is generally held rcsponsiblc for lateral conduction, dots not exist in the cephalopod retina. One possible analog to such connections might be the axon collaterals described by YOUNG(1960, l962a). Another means by which an excited cell might influence the surrounding cells has been revealed by recent studies on the ultrastructure of the octopus retina. Electron-micrographs have shown points at which the unit membranes of two receptor cells are in direct contact. These points of contact appear similar to the apposition of cells at the synapse, but the area of contact is much greater than that of a tight junction (ROBERTSON,1963). The contact occurs between the rhabdome-free portion of the distal segment and the basement membrane. (Further studies on the ultrastructure of the octopus retina are now in progress.) At present, there are not sufficient data on the overall organization of either the axon collaterals or the cell-to-cell contacts to determine whether either could be responsible for the spread of potentials observed in the octopus retina. The significance of the radial spread of potentials to pattern vision in the octopus is uncertain. Behavioral studies on pattern discrimination have shown that the octopusis capable of a high level of pattern vision (SUTHERLAND,1958,1960,1961). It has been shown that not all pairs of patterns can be discriminated with equal ease; some discriminations are learned rapidly, others slowly or not at all. Attempts to analyze those discriminations which are learned rapidly and those which are not have led to the proposal of several theories of pattern vision in the octopus. At present, Sutherland’s theory (SUTHERLAND,1960) appears to account for most of the data on pattern discrimination in the octopus. This theory proposes a coding of pattern information with respect to the horizontal and vertical extents of the pattern. In light of the findings of the present paper it appears unlikely that such a coding occurs within the retina. Indeed SUTHERLAND(1960) himself suggests the optic lobes as a possible locus for such coding; the anatomical complexity of the optic lobes (YOUNG, 1962b) seems compatible with this suggestion. Also of interest with respect to visual function was the finding of a physiologically unique central area of the retina, the physiological center. YOUNG (1960) stressed the structural regularities throughout the cephalopod retina; in a later paper he (YOUNG, 1962a) noted some structural differences between the center and periphery of the retina but concluded that the existence of a differentiated central area remained unsettled. Still later he (YOUNG, 1963) described the differences between the center and periphery of the retina as marked and also noted differences in pigment migration and contraction of the rhabdomes in response to light stimulation between the center and periphery. Thus, although there is the possibility of some homologous relationship between the center of the cephalopod retina and the vertebrate fovea, the structural differences between the center and periphery of the cephalopod retina are subtle. Functional differences, on the other hand, are extreme. The physiological center forms a focus around which the spread of the deep responses of the entire retina is organized. The finding, that at the physiological center the positive responses are sharply limited to the illuminated region, suggests improved visual acuity in this area of the retina. It is of interest to point out these findings as an example of functional specialization preceding structural specialization.
266
ALLEN C. NORTON, YOSHIRO FUKADA, KOITI MOTOKAWA AND KYOJI TASAKI
SUMMARY
A detailed investigation of the lateral spread of the deep potentials of the octopus retina revealed the following: 1. The moving spot and moving electrode procedures yield equivalent results with respect to the distribution of potentials. 2. The deep response, but not the surface response, shows an asymmetrical distribution of potentials around a locally illuminated region. 3. The distribution of the deep response shows maximum asymmetry along lines directed toward the center of the retina. 4. There is a unique contra1 portion of the retina, the physiological center; symmetrical distributions of potentials are recorded along all lines passing over this area. It was concluded that there is a radial organization in the spread of potentials in the octopus retina, positive responses spreading toward the center and negative responses toward the near periphery. ~ck~~o~v/rlgo~tc~~/-lt is a plcasurc to thank the director and staR of the Onagawa Fishcrics Laboratory (Faculty of Agriculture, Tohoku University) for their aid in collecting and maintaining the octopuses used in this study.
REFERENCES MOTOKAWA,K., OIKAWA, T. and OGAWA, T. (1959a). Slow potentials induced from the illuminated part into the surrounding area of the retina. Japan J. Physiol. 9, 218-227. MOTOKAWA, K., OIKAWA, T., TASAKI, K. and OGAWA, T. (1959b). The spatial distribution of electric responses to focal illumination of the carp’s retina. Tohoku J. exp. Med. 70, 151-164. MOTOKAWA, K., YAMASHITA,E. and OGAWA, T. (1960). The physiological basis of simultaneous contrast in the retina. Neurophysiologie und Psychophysik des Visuellen Systems, pp. 31-43 (Ed. JUNG, R. and KORNHUBER, H.), Springer, Berlin, Giittingen, Heidelberg. ROBERTSON,J. D. (1963). The occurrence of a subunit pattern in the unit membranes of club endings in Mauthner cell synapses in goldfish brains. J. ceN. Biol. 19.201-221. SUTHERLAND,N. S. (1958). Visual discriminations of shape by Octopus: squares and triangles. Quart. J. exp. Psychol. 10,40-47. SUTHERLAND,N. S. (1960). Theories of shape discrimination in Octopus. Nature, Land. 186, 840-844. SUTHERLAND.N. S. (1961). Discrimination of horizontal and vertical extents by Octopus. J. camp. physial. Psychol. 54,434. TASAKI, K., NORTON, A. C. and FUKADA, Y. (1963a). Regional and directional differences in the lateral spread of retinal potentials in the octopus. Nature, Load. 198, 12064208. TASAKI, K., NORTON, A. C., FUKADA, Y. and MOTOKAWA,K. (1963b). Further studies on the dual nature of the octopus ERG. Tohoku J. exp. Med. 80.7548. TASAKI, K., OIKAWA, T. and NORTON, A. C. (1963~). The dual nature of the octopus electroretinogram. Vision Res. 3, 61-73. WELLS, M. J. (1962). Brain and behaviour in Cephalopods. Heinemann, London. YOUNG, J. Z. (1960). Regularities in the retina and optic lobes of Octopus in relation to form discrimination. Nature, Land. 186,836-839. YOUNG, J. Z. (1962a). The retina of cephalopods and its degeneration after optic nerve section. Phil. Trans. Roy. Sot. Lond. B 245, 1-18. YOUNG, J. Z. (1962b). The optic lobes of Octopus vulgaris. Phil. Trans. Roy. Sot. Lond. B 245, 19-58. YOUNG, J. Z. (1963). Light- and dark-adaptation in the eyes of some cephalopods. Proc. Zool. Sm. Land. 140,255-272.
Abstract-A radial organization was found in the spread of the normally positive responses rccordcd from deep (near the sclcra) portions of the octopus retina. Around a locally illuminated region, positive rcsponscs arc rccordcd toward the ccntcr of the retina and negative responses toward the near periphery. A unique central area of the retina was also found.
Lateral Spread in the Octopus Retina R&snn&-On trouve une organisation radiale dans l’extension des reponses normalement positives enregistr&s dans des regions profondes (p&s de la sclerotique) de la n5tine de l’octopus. Autour dune region localement eclairee, on enregistre des resonses positives vers le centre de la &tine et des reponses negatives vers la p&ipherie. On trouve aussi une unique aire cent&e dans la r&tine. Zusammenfassung-In der Verteilung der normalerweise positiven Antworten, die in tiefen Schichten (nahe der Sklera) der Octopus Netzhaut registriert wurden, wurde eine radiale Organisation gefunden. In der Umgebung einer lokal beleuchteten Netzhautstelle wurden positive Antworten gegen das Netzhautzentrum hin und negative Antworten gegcn die Peripherie hin gemessen. Es ergab sich such eine einzelne zentrale Netzhautstelle.
267
Vol. 5, pp. 253-267.
Pcrgamon Press 1965.
AN INVESTIGATION POTENTIALS ALLEN
Printed inGreatBritain.
OF THE IN THE
LATERAL
OCTOPUS
C. NORTON,~ YOSHIRO FUKADA,~ Kom
SPREAD
OF
RETINA
MOTOKAWA
and KYOJI TASAKI
Department of Physiology, Tohoku University School of Medicine, Sendai, Japan (Received 15 June 1964) INTRODUCTION PREVIOUS
reports from this laboratory have described the spread of potentials around a locally illuminated region of the octopus retina (TASAKI et al., 1963a, 1963b). This spread of potentials was demonstrated by moving a spot of light around a fixed recording electrode. It was found that the surface response (a sustained negative potential recorded from the anterior surface of the octopus retina, see TASAKI ef al., 1963c) increases in amplitude as the light spot approaches the electrode from one side, reaching a maximum as the spot passes over the electrode and decreasing as the spot moves to the other side. The distribution, or position-amplitude relation, is symmetrical. The deep response (a sustained positive potential recorded from near the receptor cell bodies of the retina, see TASAKI et a/., 1963c) shows a different and more complex positionamplitude relation. As the light spot approaches the recording electrode from one side the usual positive responses increase in amplitude and reach a maximum as the spot passes over the recording electrode. As the spot passes to the other side of the electrode, however, negative responses suddenly appear. These negative potentials show first an increase and then a decrease in amplitude as the light spot is moved away from the recording electrode. The distribution is markedly asymmetrical, positive responses occurring as the light spot is presented on one side of the electrode and negative responses occurring from stimulation of the other side. Transition (biphasic negative-positive) responses can occasionally be recorded from the boundary between the illuminated and unilluminated portions of the retina. The origin and significance of the deep negative responses is discussed below (see Discussion). A previous study used this asymmetrical distribution of the deep response as a means for investigating various aspects of the electrophysiology of the octopus retina (TASAKI et al., 1963b). The following properties of the position-amplitude relation of the deep response wcrc shown : (1) The appcarancc of the negative responses depends upon the diameter of the stimulus spot. With stimulus spots below l/2-1/4 mm, negative responses do not occur and a symmetrical distribution of only positive responses is recorded. (2) The deep (positive) responses, but not the surface (negative) responses, can be blocked by cutting the retina mechanically. (3) Diffuse background illumination has little effect on the relative amplitude of the positive responses, but it will selectively decrease the amplitude of the negative responses. 1 This investigation .was carried out during the tenure of a postdoctoral fellowship from the National Institute of Mental Health, United States Public Health Service. Present address: Institute of Medical Research, Huntington Memorial Hospital.Pasadena,Califomia, U.S.A. 2 Present address: Technical Research Laboratories, Japan Broadcasting Corporation (N.H.K.), Tokyo, Japan. 253
254
ALLEN C. NORTON, YOSHIRO FUKADA,
KOITI MOTOKAWA AND
KYOJI TASAKI
In addition to these characteristics of the lateral spread of the deep responses, it was frequently observed that the asymmetrical distribution of potentials was different along different lines passing over the recording electrode and also that the direction (from the recording electrode) in which maximum amplitude negative responses were elicited was different in different parts of the retina. The present paper explores these directional and regional differences in the spread of deep potentials in the octopus retina; a unique central area of the retina is also reported. A preliminary report of these phenomena has been published previously (TASAICI ef al., 1963a). METHODS The experimental animals were twenty-five octopuses (one Octopus dojfeini, Wiilker, and the remainder 0. vu/gut-is). Before use they were dark adapted for at least 1 hr. Under a dim photographic safety-lamp, they were decapitated and the eyes were enucleated. The anterior parts of the eye were dissected away, lcaving only the retina and attached sclera. Excess vitreous fluid was removed by absorption onto filter paper. The retina was placed, sclera downward, on a small piece of black cloth which had been moistened with the vitreous fluid from the same eye. The retina and cloth were then placed on a plastic mount which contained an Ag-AgCl referenceelectrode. To flatten the preparation, small pieces of tissue were cut from the periphery of the retina in areas which would not lie flat, the cuts being made from periphery to periphery, not from periphery to center. The retina and mount were placed on a microscope stage which had a micrometer-driven slide carriage. With the electrode position fixed, the mount could be moved accurately to allow penetration in any region of the retina; the electrode was withdrawn whenever the retina was moved. For convenience of discussion in the following sections, the electrode, rather than the retina, is referred to as having been moved. Special care was taken to preserve the in vivo orientation of the retina. In the intact animal the pupil, a nearly rectangular slit, is always kept in a horizontal position (WELLS, 1962). This orientation was maintained by placing the retina (and mount) on the experimental table so that the mid-horizontal line of the retina (corresponding to the long axis of the pupil) was parallel to the front edge of the table, with the ventral portion of the retina toward the front of the table. From this standard orientation, local regions of the retina were referred to by clockfact notation: 12:OOo’clock upward (away from the experimenter), 3:00 to the right, etc. This is the notation used in the present paper. The position of the electrode and light spot were viewed directly through a dissecting microscope in front of the preparation. The recording clcctrodcs wcrc glass capillaries filled with 3 M KCI; the tip diameter was less than l/l. Electrodes with especially long, thin shanks were selected to minimize reflection of light from the electrode as the electrode or spot was moved across the retina. The signals were led through cathode follower preamplifiers and d.c. coupled amplifiers and displayed on a dual beam cathode ray oscilloscope. The light stimulus was provided by a tungsten projection bulb projected through a simple optical system (see Fig. 1 in TASAKI ef nl., 1963b). Circular masks in the light beam limited the projected stimulus spot to 2 or 3 mm diameter. To obtain displacement of the stimulus spot across the retina, the final focussing lens of the optical system was moved through a plane perpendicular to the optical axis by the use of a 2-dimension micromanipulator. The spot could be moved 20 mm over the retinal surface without significantly altering its focus, and displacements as small as l/4 mm could be made with reasonable accuracy. The stimulus duration (500 msec in all experiments) was controlled by a magnetic shutter driven by an electronic square pulse generator. The stimulus repetition was one flash/l0 set, or one flash/20 set in series which required more time for adjustments between flashes. RESULTS
When a small region of the octopus retina is illuminated, a predictable sequence of responses can be recorded as a microelectrode is inserted through the retina within the illuminated area (see Fig. 1, TASAKI et al., 1963~). High amplitude negative responses are recorded as the electrode touches the vitreal surface; these responses are of maximum amplitude near the surface of the retina and decrease in amplitude as the electrode is inserted deeper into the retina. At around 400 ,Ufrom the surface there is an equipotential region in which only small deflections are recorded at the “on” and “off)’ of the stimulus flash. Still deeper in the retina, around the receptor cell bodies, positive responses are recorded. The following experiments studied only the deep (normally positive) responses (in
Lateral Spread in the Octopus Retina
255
section 13the surface and deep responses are compared). The deep responses were recorded at a depth from which maximum amplitude positive responses were obtained when the light spot was centered on the recording electrode. A, Equivalency of the moving spot and moving electrode procedures Previous cxpcrimcnts on the spread of potentials in the octopus retina employed only the moving spot procedure (TASAKI et al., 1963b, 1963c). Since regional differences were observed (see below), it was necessary to determine whether the same spread of potential could be recorded when the electrode rather than the light spot was moved,
-
-v
v-V-7
FIG. 1. Moving electrode (above) and moving spot Wow) procedures. Recordings along the mid-hori~n~ axis on the right side of the retina (around 3:00 o’clock); traces represent the deep response at successive 1 mm displacements across the retina; circles indicate the diameter of the light spot (2 mm). Vertical bars {right), 2 mV; positivity downward in these and following records.
One series of records was taken with the electrode position fixed while the stimulus spot was moved. In the same area of the retina, a second series was taken with the position of the light spot fixed while the electrode was moved. After each displacement the electrode was reinserted in the retina to record maximum amplitude positive responses when the light spot was centered on the electrode, records being taken from a constant functional (rather than physical) depth. With careful procedure, comparable deep responses could be obtained with each penetration in a series. The results of a typical experiment are shown in Fig. 1; these records were taken from the right side of the retina around 3:oO o’clock. The upper line of black traces represents the moving electrode series, while the lower line of white traces represents the moving spot series. The circles indicate the area of the stimulus spot in relation to the distance the electrode (or spot) was moved; the traces represent the response at 1 mm intervals. With both procedures relatively high amplitude positive responses were recorded from within the illuminated area. Outside the illuminated area negative responses were recorded as the electrode was moved to the right (top row) and as the spot was moved R
256
ALLEN C. NORTON, YOSHIRO FUKADA, Korrr
MOTOKAWA
AND
KYOJI TASAKI
to the left (bottom row). Thus, although the actual direction of movement was opposite in the two cases, the orientation of the spot and electrode with respect to each other remained constant when negative responses appeared. It was concluded that, at least for smali areas of the retina, the moving electrode or moving spot procedures would yield equivalent results with respect to the lateral distribution of the deep potentials. Since the depth of the electrode in the retina has considerable influence on the form and amplitude of the deep response recorded (TASAKIet al., 1963c), the moving spot procedure was employed in most of the following experiments, thus assuring a constant vertical depth from which responses were recorded.
FIG. 2. Two-dimensionexperimentaround 9:00 o’clockwith movingspot procedure. Horizontal series A-A’, left; and vertical series B-B’, right. Co-ordinates: Amplitude of the deep response, and distance light spot was moved betweensuccessiverecordings. Insets top and right indicate oscilloscoperecords at position indicated; lower traces (top and left) show
stimulus and time (10 c/s) marks, vertical bars, 1 mV. Dotted line shows diameter of the spot. Lower insert shows the retinal position in which thb electrode was inserted and the lines along which the light spot was moved.
B. Directionaldifferences in the distributionof the deep response The previous experiments considered the distribution of potentials along only one line passing over the active electrode. The following experiments investigate more fully the distribution of potentials around locally iiluminated areas of the octopus retina. Figure 2 illustrates a typical two-dimension experiment. The light spot was first moved along the mid-horizontal line from A to A’; the distribution of response recorded is shown on the horizontal coordinates. Next the light spot was moved vertically aiong a second line passing over the electrode from B to B’; the distribution of responses is shown on the vertical coordinates. Although the horizontal experiment showed the asymmetrical distribution of potentials described above, the vertical experiment showed a symmetrical distribution with positive responses appearing for short distances on either side of the illuminated area and negative responses at still more distant points. A symmetrical distribution of potentials along lines perpendicular to the line of maximum asymmetry was a consistent finding. It will be noted in Fig. 2 (and following figures) that there are slight deviations from strict
Lateral Spread in the Octopus Retina
257
symmetry in distributions
which are called symmetr~~l. Since the mechanical manipulations could only approximate the texture of the actual potential gradients, the present records can Experimental error is particularly show only approximately symmetrical distributions, obvious with respect to centering the light spot on the electrode and determining the edge of the light spot.
FIG. 3. Scanning experiment around 9:00 o’clock, moving spot procedure. Traces indicate the deep responses at successive 1 mm intervals within a 13 x 13mm array; electrode was in the center of the array. Stimulus and time mark (10 c/s) lower left, calibration (-2 mV) lower right, millimeter scale lower center.
More detailed scanning experiments were next undertaken. The light spot was moved from right to left and from top to bottom (similar to the scanning on a television screen) around the area in which the electrode was inserted. Responses were recorded at 1 mm intervals; one flash was presented every 10 sec. Figure 3 shows the array of responses obtained from such an experiment; the electrode was in the center of the array, in the area showing high amplitude positive responses. This same data is presented in schematic form in Fig. 4 which shows the response amplitude as proportional to the area of the circles; the
258
ALLEN C. NORTON, YOSHIRO FUKADA, KOITI MOTOKAWA
AND
KYOJI T’ASAKI
filled circles represent negative responses and the open circles positive responses. The inset at the lower left shows the position of the electrode and the area scanned in relation to the mid-horizontal and mid-vertical axes of the retina. Since it was shown above that, at least for small areas of the retina, the moving spot and moving electrode procedures provide equivalent results, the pattern of potentials shown in Fig. 4 may be considered as illustrating the spread of potentials around a locally illuminated region. Considering the actual position of the light spot with respect to the electrode, it is apparent that in this region of the retina (around 903 o’clock) positive potentiaIs spread toward the right and negative potentials to the left of the illuminated area.
0
+SmV
0 -ImV
/--
8 FIG. 4. Schematicrepresentationof data shown in Fig. 3. Response amplitude is shown as proportional to the area of the circles; positive responses open circles, negative responses filled circles. Position of the electrode and area scanned shown at left.
To confirm the previous finding that the asymmetrical distribution of deep responses is not found in the surface (negative) response, simultaneous records of the surface response were also taken. Figure 5 shows the surface responses corresponding to the deep responses shown in Fig. 4 (the two responses were recorded simultaneously); the same schematic representation is employed in both figures. Figure 5 shows that there is no asymmetry in the distribution of the surface responses around a locally illuminated area. This finding, it may be noted, provides additional support to the previous proposition that the surface and deep responses behave differently with respect to stimulus variables and probably reflect two origins of potential within the octopus retina (TASAKI et al., 1963b, 1963~).
259
Lateral Spread in the Octopus Retina
C. Regional differences in the distribution of the deep response
The experiments described in section B were all performed on the left side of the retina (around 9:00 o’clock). Similar directional differences in the distribution of the deep response were observed in other regions of the retina, however, the direction of the asymmetry was found to be different in different regions. Around 3:OO o’clock on the retina the vertical record was symmetrical and the horizontal record asymmetrical. However, unlike the recordings taken at 9:00 o’clock, those taken at 3:00 o’clock revealed negative responses
0
l
-lOmV
-ImV
FIG. 5. Scanning experiment showing distribution of the surface response around 9:OO o’clock. Records taken simultaneously with those shown in Figs. 3 and 4; schematic representation is the same as in Fig. 4.
when the light spot was to the left of the electrode and positive responses when it was to the right (see lower row in Fig. l), the opposite of that seen in Fig. 3. Figure 6 shows the results of a two-dimension experiment performed at 12:00 o’clock. At this position, the horizontal record is symmetrical, but the vertical record shows asymmetry with negative responses occurring toward the bottom. Experiments at 6:00 o’clock showed a similar symmetry of the horizontal records and asymmetry of the vertical records, but in the latter case the negative responses appeared when the light spot was above the electrode. These findings suggest that there is either a horizontal-vertical or a radial organization within the retina. In reference to the first alternative, it may be noted that SUTHERLAND (1960) has proposed a theory of pattern discrimination in the octopus which involves coding of pattern information along the horizontal and vertical extents of the figures. However, since the above experiments were performed only at 3 :OO,6:00,9:00 and 12:OOo’clock, the second alternative cannot be excluded.
+mV
FIG. 6. Two-dimension
@
-mv
experiment around 1290 o’clock. Otherwise same as Fig. 2. Vertical bars, 2 mV.
$ u
4 IOmV
- Imv
Fro. 7. Scanning experiment performed around 4:30 o’clock. Otherwise same as Fig. 4.
261
Lateral Spread in the Octopus Retina
The two alternatives were tested by performing similar experiments at intermediate positions. Figure 7 shows the results of a scanning experiment performed at 4:30; the procedure and schematic representation are the same as above (Figs. 4 and 5). It is seen here that although some asymmetry occurs along the horizontal and vertical lines, the maximum asymmetry is along a diagonal directed toward thecenter of the retina. It was thus concluded that the directional differences of the deep response always occur with respect to the center of the retina. In view of the equivalence of the moving electrode and moving spot procedures, it seems clear that: from any locally illuminated region of the retina there is a spread of positive potentials toward the center of the retina and a spread of negative potentials toward the periphery,
mV -I
l
A
. . . . .
A’
0 +I +2 +3
:~~~
-I 0 +I +2 +3 -I 0 +I +2 +3 -I 0 +I +2 +3 -mV
FIG. 8. At physiological center, 44rection experiment. Inset (Iower right) shows position of electrode and lines along which tight spot was moved. Abscissae: amplitude of the deep response (mv); coordinates: distance light spot was moved (mm). Dotted line (top) shows diameter of stimulus spot.
262
ALLEN C. NORTON, YOSHIRO FUKADA, KOITI MOTOKAWA AND KYOJI TASAKI
D. The physiological center
ofthe octopus retina
ft follows from the above findings that there should be some central region of the retina toward which the maximum asymmet~ is directed. It would be expected that recordings taken from this region would show a symmetrical distribution of potentials along any line passing over the recording electrode. The term physiological center has been adopted to refer to this central region, the region being defined by its physiological properties. The following method was used to locate this central region. With the electrode inserted near the center of the retina to record maximum amplitude positive responses, a two-dimension moving spot experiment was performed. The appearance of asymmetrical negative responses was noted and the electrode was moved in the direction from which the negative responses
w +5mV
-Imv
~ FIG. 9.
allying
cxpcziment ~rform~
at the physiologica ccntcr. Otherwise same as Figs. 4, 5 and 7.
had been recorded. The two-dimension experiment was repeated, and further adjustment of the electrode was made as indicated. In several experiments it was indeed possible to locate a region from which symmetrical distributions were obtained as the light spot was moved along any line passing over the electrode. Since the edges of the retina had been trimmed irregularly in order to flatten the preparation, it could not be determined whether the physiological center coincided with the geometrical center of the whole retina. Examples of the distribution of potentials as the light spot was moved around such a central region are
Lateral Spread in the Octopus Retina
263
shown in Fig. 8 (graphically along four lines passing over the electrode) and Fig. 9 (a scanning experiment shown schematically). In both examples it is to be noted first that there is a symmetrical distribution of intermediate amplitude negative potentials around the central positively responding area, and second that the positively responding region occupies a far more narrow area than is the case in other parts of the retina (compare Fig. 8 with Figs. 2 and 6, or Fig, 9 ~virh Figs. 4 and 7). It was demonstrated for other regions of the retina that the moving spot and moving electrode procedures were equivalent (Section A, above). Since the dir~tionality of potentials observed in other parts of the retina is not seen at the point which the moving spot
FIG. IO. Two-dimension, moving clcctrodo cxpcrimcnt at the physiological ccntcr, Tracts indicate amplitude and wave form of the deep responses at successive one mm intervals along the mid-horizontal and mid-vertical lines of the retina. Verticalbar, 2 mV.
procedure indicates to be the physiological center, it is to be expected that similar findings would be shown by the moving electrode procedure. This expectation was tested directly. The light spot was fixed at the point indicated as the physiological center and the electrode was moved along orthogonal lines passing over this point. The results of this procedure are shown in Fig. 10. As was demonstrated with the moving spot procedure, there is a symmetrical distribution of intermediate amplitude negative responses around the illuminated area, and the positively responding area is relatively narrow (compare with Fig. 1) with the positive responses limited to the iIluminated area. The fun~tiona1 si~ificance of this central part of the retina is considered below.
264
ALLEN
C. NORTON, YOSHIRO FIIKAUA,
Ken-I MOTOKAWA
AND KYOJI ‘TASAKI
DISCUSSION The present study reveals a highly organized directionality in the spread of the deep responses in the octopus retina. Around any locally illuminated area in the retina there is a spread of positive potentials toward a unique central part of the retina. Negative responses appear in the opposite direction, radially toward the near periphery. Thus an asymmetrica distribution of positive and negative potentials is observed along lines passing between an illuminated area and the unique central area. Given this basic organization of the spread of potentials it follows that there is a symmetrical distribution of potentials along lines per~ndicular to the line of maximum asymmetry. The unique central part of the retina has been termed the~~y~~o~Qg~~fff cenfer; this area is defined by its physiological properties: that there is a symmetrical distribution of potentials along any tine passing over it. It seems highly unlikely that the organization reported above is due to artifacts in the experimental procedure. The phenomena reported were consistent, predictable and easily replicated. The possibility that the deep negative responses, for example, arose from the scattering of stray light from the recording electrode can be rejected by the finding that the ncgativc rcsponscs appear in relation to the radial organization of the retina (described above), while the electrode was always inserted from the right of the preparation (3:OO o’clock). The orthogonal arrangement of the experimental apparatus, in contrast to the radial organization found in the spread of the deep potentials, excludes the possibility of many other procedural errors. The possibility that radial gradients arose from the placement of the indifferent electrode may also be rejected; the same results were obtained whether the indifferent electrode was placed in the center or near the periphery of the preparation. Furthermore the black cloth soaked with vitreous fluid between the sclera and the indifferent electrode served to distribute the potential of the reference electrode over the entire surface of the sclera. Attempts to measure the conduction velocity of the spread of potentials were unsu~essful. Because of the small distance in which responses of the same polarity could be recorded and the relativeiy slow rise time of the responses, conduction velocities as slow as a few meters per second could not be proved. It should be noted, however, that the conduction velocity of the slow potentials of the carp retina was found to be as low as 100 mmjsec (MOTOKOWA et al., 1959b). [Our thanks are expressed to Prof. J. Z. YOUNG (personal communication) for pointing out the significance of the latter two factors.] Although there was considerable variabjIjty in amplitude of response between preparations, little variability of response amplitude was observed in different parts of the same retina. The origin of the surface (negative) and deep (normally positive) potentials of the octopus retina has been considered previously (TASAKI et al., 1963b, 1963~). It was tentatively suggested that the surface responses arose from the pigment cells and the deep responses from the receptor cells; the likelihood that the recorded responses actually represented an interaction of these two potentials was also noted. The origin of the negative potentials in the deep portions of the retina is unclear. The previous findings (TASAKI et al., 1963b), first that the deep negative responses do not occur when the stimulus spot is very small and second that the deep responses are entirely negative when the stimulus spot is very large, suggest a complex spatial interaction in the generation of the deep responses, both positive and negative. At present, experimental findings are not available to account for the origin of the deep responses, their dependence upon the area of the stimulus spot, and the basis of their radial organization (as reported in the present paper). These three factors are obviously interrelated, but a clearer understanding must await further experimental study. The mechanism by which responses appear to spread through the octopus retina is also
Lxtcral Sprcact in the Oclopus Rclina
265
unclear. For the slow surface responses in the fish retina, MOTOKAWAet al. (1959a) have demonstrated that there is a negatively responding region which surrounds the local (positive) potentials and have suggested this as involved in visual contrast (MOTOKAWA eral., 1960). The negative potentials in the fish retina, however, are symmetrically distributed around the positively responding (illuminaled) area, in contrast to the asymmetrical distribution found in the octopus. The neural plexus of the vertebrate retina, which is generally held rcsponsiblc for lateral conduction, dots not exist in the cephalopod retina. One possible analog to such connections might be the axon collaterals described by YOUNG(1960, l962a). Another means by which an excited cell might influence the surrounding cells has been revealed by recent studies on the ultrastructure of the octopus retina. Electron-micrographs have shown points at which the unit membranes of two receptor cells are in direct contact. These points of contact appear similar to the apposition of cells at the synapse, but the area of contact is much greater than that of a tight junction (ROBERTSON,1963). The contact occurs between the rhabdome-free portion of the distal segment and the basement membrane. (Further studies on the ultrastructure of the octopus retina are now in progress.) At present, there are not sufficient data on the overall organization of either the axon collaterals or the cell-to-cell contacts to determine whether either could be responsible for the spread of potentials observed in the octopus retina. The significance of the radial spread of potentials to pattern vision in the octopus is uncertain. Behavioral studies on pattern discrimination have shown that the octopusis capable of a high level of pattern vision (SUTHERLAND,1958,1960,1961). It has been shown that not all pairs of patterns can be discriminated with equal ease; some discriminations are learned rapidly, others slowly or not at all. Attempts to analyze those discriminations which are learned rapidly and those which are not have led to the proposal of several theories of pattern vision in the octopus. At present, Sutherland’s theory (SUTHERLAND,1960) appears to account for most of the data on pattern discrimination in the octopus. This theory proposes a coding of pattern information with respect to the horizontal and vertical extents of the pattern. In light of the findings of the present paper it appears unlikely that such a coding occurs within the retina. Indeed SUTHERLAND(1960) himself suggests the optic lobes as a possible locus for such coding; the anatomical complexity of the optic lobes (YOUNG, 1962b) seems compatible with this suggestion. Also of interest with respect to visual function was the finding of a physiologically unique central area of the retina, the physiological center. YOUNG (1960) stressed the structural regularities throughout the cephalopod retina; in a later paper he (YOUNG, 1962a) noted some structural differences between the center and periphery of the retina but concluded that the existence of a differentiated central area remained unsettled. Still later he (YOUNG, 1963) described the differences between the center and periphery of the retina as marked and also noted differences in pigment migration and contraction of the rhabdomes in response to light stimulation between the center and periphery. Thus, although there is the possibility of some homologous relationship between the center of the cephalopod retina and the vertebrate fovea, the structural differences between the center and periphery of the cephalopod retina are subtle. Functional differences, on the other hand, are extreme. The physiological center forms a focus around which the spread of the deep responses of the entire retina is organized. The finding, that at the physiological center the positive responses are sharply limited to the illuminated region, suggests improved visual acuity in this area of the retina. It is of interest to point out these findings as an example of functional specialization preceding structural specialization.
266
ALLEN C. NORTON, YOSHIRO FUKADA, KOITI MOTOKAWA AND KYOJI TASAKI
SUMMARY
A detailed investigation of the lateral spread of the deep potentials of the octopus retina revealed the following: 1. The moving spot and moving electrode procedures yield equivalent results with respect to the distribution of potentials. 2. The deep response, but not the surface response, shows an asymmetrical distribution of potentials around a locally illuminated region. 3. The distribution of the deep response shows maximum asymmetry along lines directed toward the center of the retina. 4. There is a unique contra1 portion of the retina, the physiological center; symmetrical distributions of potentials are recorded along all lines passing over this area. It was concluded that there is a radial organization in the spread of potentials in the octopus retina, positive responses spreading toward the center and negative responses toward the near periphery. ~ck~~o~v/rlgo~tc~~/-lt is a plcasurc to thank the director and staR of the Onagawa Fishcrics Laboratory (Faculty of Agriculture, Tohoku University) for their aid in collecting and maintaining the octopuses used in this study.
REFERENCES MOTOKAWA,K., OIKAWA, T. and OGAWA, T. (1959a). Slow potentials induced from the illuminated part into the surrounding area of the retina. Japan J. Physiol. 9, 218-227. MOTOKAWA, K., OIKAWA, T., TASAKI, K. and OGAWA, T. (1959b). The spatial distribution of electric responses to focal illumination of the carp’s retina. Tohoku J. exp. Med. 70, 151-164. MOTOKAWA, K., YAMASHITA,E. and OGAWA, T. (1960). The physiological basis of simultaneous contrast in the retina. Neurophysiologie und Psychophysik des Visuellen Systems, pp. 31-43 (Ed. JUNG, R. and KORNHUBER, H.), Springer, Berlin, Giittingen, Heidelberg. ROBERTSON,J. D. (1963). The occurrence of a subunit pattern in the unit membranes of club endings in Mauthner cell synapses in goldfish brains. J. ceN. Biol. 19.201-221. SUTHERLAND,N. S. (1958). Visual discriminations of shape by Octopus: squares and triangles. Quart. J. exp. Psychol. 10,40-47. SUTHERLAND,N. S. (1960). Theories of shape discrimination in Octopus. Nature, Land. 186, 840-844. SUTHERLAND.N. S. (1961). Discrimination of horizontal and vertical extents by Octopus. J. camp. physial. Psychol. 54,434. TASAKI, K., NORTON, A. C. and FUKADA, Y. (1963a). Regional and directional differences in the lateral spread of retinal potentials in the octopus. Nature, Load. 198, 12064208. TASAKI, K., NORTON, A. C., FUKADA, Y. and MOTOKAWA,K. (1963b). Further studies on the dual nature of the octopus ERG. Tohoku J. exp. Med. 80.7548. TASAKI, K., OIKAWA, T. and NORTON, A. C. (1963~). The dual nature of the octopus electroretinogram. Vision Res. 3, 61-73. WELLS, M. J. (1962). Brain and behaviour in Cephalopods. Heinemann, London. YOUNG, J. Z. (1960). Regularities in the retina and optic lobes of Octopus in relation to form discrimination. Nature, Land. 186,836-839. YOUNG, J. Z. (1962a). The retina of cephalopods and its degeneration after optic nerve section. Phil. Trans. Roy. Sot. Lond. B 245, 1-18. YOUNG, J. Z. (1962b). The optic lobes of Octopus vulgaris. Phil. Trans. Roy. Sot. Lond. B 245, 19-58. YOUNG, J. Z. (1963). Light- and dark-adaptation in the eyes of some cephalopods. Proc. Zool. Sm. Land. 140,255-272.
Abstract-A radial organization was found in the spread of the normally positive responses rccordcd from deep (near the sclcra) portions of the octopus retina. Around a locally illuminated region, positive rcsponscs arc rccordcd toward the ccntcr of the retina and negative responses toward the near periphery. A unique central area of the retina was also found.
Lateral Spread in the Octopus Retina R&snn&-On trouve une organisation radiale dans l’extension des reponses normalement positives enregistr&s dans des regions profondes (p&s de la sclerotique) de la n5tine de l’octopus. Autour dune region localement eclairee, on enregistre des resonses positives vers le centre de la &tine et des reponses negatives vers la p&ipherie. On trouve aussi une unique aire cent&e dans la r&tine. Zusammenfassung-In der Verteilung der normalerweise positiven Antworten, die in tiefen Schichten (nahe der Sklera) der Octopus Netzhaut registriert wurden, wurde eine radiale Organisation gefunden. In der Umgebung einer lokal beleuchteten Netzhautstelle wurden positive Antworten gegen das Netzhautzentrum hin und negative Antworten gegcn die Peripherie hin gemessen. Es ergab sich such eine einzelne zentrale Netzhautstelle.
267