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Developmental characteristics of receptive organization in the isolated retina-eyecup of the rabbit
Brain Research, 87 (1975) 61-65 ,i:~ Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
61
Short Communications
Developmental characteristics of receptive organization in the isolated retina-eyecup of the rabbit
CONSTANCE BOWE-ANDERS*, ROBERT F. MILLER AND RAMON DACHEUX Neurosensory Ltlboratory, Department of Physiology, State UniversiO' of New York at Buffido, Buffalo, N. Y. 14214 (U.S.A.)
(Accepted December 19th, 1974)
The retina of the rabbit, like that of many other mammals, is immature at birth but develops rapidly during the first weeks of neonatal life. Light responsive ganglion cells are present at 8-9 days after birth s , but little is known about the maturation rates of the different neuronal pathways which underlie ganglion cell receptive field organization. For instance, when on- and off-center ganglion cells become light responsive, do they have the center-surround organization characteristic of adult cells'? Experimental approaches to this question are complicated by the poorly developed optics of the newborn eye and the generally poor tolerance of the neonatal mammal to anesthesia. The isolated retina represents a possible method of avoiding these problems if the preparation can be maintained in a normal or near normal condition. In this arrangement the optics of the eye would be replaced by a clear perfusate, and except for the anesthesia required for the initial surgery, the retina would not experience prolonged exposure to anesthetizing agents. During the past 3 years this laboratory has developed an isolated retina-eyecup preparation using the adult rabbit eye. This preparation has proved suitable for extracellular recordings from ganglion cells as well as intracellular recordings from all the major cell types of the retina 3,7. The stability of this preparation is evident by ganglion cell recordings which show constant thresholds for several hours and display both center and surround organization. Other types of ganglion cells such as motion selective'-' and local edge detecting neurons 6 have also been recorded from this prepa,~ ation. These results encouraged us to explore the possible use of this technique for examining the maturation sequence of center surround organization in on- and off: center ganglion cells. Our results indicate that the retina of the neonate, like that of the adult, can be maintained in a reasonably stable condition for at least 8 h, and in this report we describe a study of 122 ganglion cells recorded from 11 rabbits ranging in * Present address: 1098 Vernier Place, Stanford, Calif. 94305, U.S.A.
62 age from 9 to 24 days after birth. The isolation and maintenance methods used tbr the neonate are similar to those previously described for the adulta,L Albino rabbits were obtained from a commercial dealer who brought the doe and litter to our animal facilities about 6 days after birth. Animals selected for experimentation are anesthetized (urethane, 0.9 g/kg) after which the eye is surgically excised. The cornea, iris and lens are dissected away and the remaining eyecup is inverted and secured in a special chamber designed to accommodate the small size of the neonatal eye. A temperaturecontrolled (35 °C), meter-regulated (40 ml/min) perfusate flows over the vitrea/surface and is continually recirculated by means of a pump. The pH is maintained at 7.4-7.5 by constant aeration of the perfusate with a 95 ° o 0.)-5 °,~I COz gas mixture. Perfusate is made fresh for each experiment and consists of the following, slightly modified from that used by Ames and Pollen1: NaCI 120 mM; KCI 5.0 mM; NaHCO~ 25 raM: glucose 10 mM; Na2HPO4 0.8 mM; NaH2PO4 0.1 mM; CaClz 2 mM: MgCI,_, 1.0 raM; horse serum 2.5 3(,. The chamber is mounted in a light-tight shielded cage. White light stimuli are provided by shuttered tungsten-iodine lamps, each regulated by a DC power supply. One beam provides focal and diffuse light stimulation (maximum intensity 0.0 log units, 4.6 mW/sq, cm) while a second beam provides annuli or a moving slit or edge (maximum intensity 3.7 mW/sq, cm). The moving slit or edge can be rotated through 360 ° by means of a dovetail prism. No diffuse background illumination was employed. Ganglion cell recordings are obtained with glass insulated tungsten electrodes a. Illustrations are reproduced from polaroid photographs of the amplified impulse activity displayed on a storage oscilloscope (Tektronix 5103). The ERG was recorded between chlorided silver electrodes placed between the front and back of the eye and served as a monitor to evaluate the condition of the preparation. ERG recordings at different ages have shown a developmental sequence similar to that observed in the intact neonatal rabbit 8. All ganglion cell recordings in this study were obtained within the first 4 h following the initial isolation, though an E R G can be maintained for more than 8 h. Fig. 1E illustrates the receptive field organization of an off-center cell recorded from a 24-day animal. This cell showed adult characteristics in its receptive field properties. A centrally located 160 l~m light flash (upper trace) resulted in a decrease in the spontaneous activity followed by a discharge at the termination of the light stimulus. The lower trace shows both on- (85 msec latency) and off- (35 msec latency) activity to a 5 mm light stimulus of the same intensity. The long latency on-discharge is due to activation of the surround while the short latency off-discharge results from stimulation of the center. Fig. 1D shows the receptive field organization of an offcenter cell recorded from a 15 day rabbit. A diffuse light stimulus evoked an offdischarge but no surround discharge was evident regardless of stimulus intensity. However, a surround discharge could be evoked by selective adaptation of the center. A centrally located spot of light (240 #m) was flashed for 5 sec with an interstimulus interval of 30 sec; 1 sec after the onset of the center spot, a l-sec annulus (0.6 mm I.D., 5 mm O.D.) was flashed to see if a surround discharge could be evoked by this procedure. When the intensity of the annulus was relatively low (upper trace) no surround discharge was observed. However, an increase in the intensity of the annulus
63
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Fig. 1. The upper trace of A shows a prolonged discharge of an on-cell recorded from an I l-day rabbit in response to a high intensity diffuse light stimulus. A low intensity light flash (lower trace) produced a sustained discharge which lasted the duration of the stimulus. B is a plot of the percentage of on- and off-cells with demonstrable surrounds v e r s u s age after birth. The off-cell in C was recorded from an I 1-day animal. The upper trace shows an off-discharge to a center light stimulus with a weak annulus superimposed (240/~m spot; annulus I.D. 600 Hm, O.D. 5 mm). The lower trace shows a response to the same center light with a high intensity annulus. The annulus failed to evoke an 'on'discharge but resulted in a single impulse at 'off' and subsequently suppressed the off-discharge to the center spot. D shows the activity of an off-cell recorded from a 15-day animal, using the identical stimulation parameters of C. The high intensity annulus (lower trace) resulted in 'on'- and "off'discharge but did not subsequently cause a suppression of the off-response to the center stimulus. The records of E were obtained from a 24-day animal. A 160 pm spot of light (upper trace) induced an off-discharge, but a diffuse light stimulus (lower trace) of the same intensity caused a long latency (85 msec) on-discharge and a short latency (35 msec) off-discharge characteristic of adult cells. Intensity values are logarithmic reductions of maximum intensity. Time calibrations are indicated. Negativity downward.
( l o w e r trace) resulted in o n - a n d o f f - d i s c h a r g e t h e r e b y d e m o n s t r a t i n g the p r e s e n c e o f a s u r r o u n d d i s c h a r g e w i t h this m e t h o d . T h e r e c o r d s o f Fig. 1C were o b t a i n e d f r o m an off-cell s t u d i e d in an 11-day a n i m a l . T h e s t i m u l u s p a r a m e t e r s w e r e i d e n t i c a l to t h o s e d e s c r i b e d in Fig. ID. H o w e v e r , in this case, a low i n t e n s i t y ( u p p e r trace) a n d high intensity ( l o w e r trace) a n n u l u s failed to e v o k e any s u r r o u n d discharge. N o s u r r o u n d d i s c h a r g e was o b s e r v e d in this cell a l t h o u g h the i n t e n s i t y o f the s p o t a n d a n n u l u s was i n d e p e n d e n t l y v a r i e d o v e r a 5 l o g u n i t r a n g e in 1 log u n i t i n c r e m e n t s . Cells which h a d a s u r r o u n d d i s c h a r g e to e i t h e r a diffuse light s t i m u l u s o r the c e n t e r a d a p t i o n m e t h o d were classified as cells ' w i t h s u r r o u n d s ' . I f the a b o v e p r o c e d u r e s failed to e v o k e a s u r r o u n d d i s c h a r g e , it was classified as a cell w i t h o u t an a n t a g o n i s t i c s u r r o u n d . Fig. I B g r a p h i c a l l y illustrates t h e p e r c e n t a g e o f cells w i t h s u r r o u n d s o b s e r v e d in t h e age g r o u p s studied. A t age 9 days, the y o u n g e s t a n i m a l studied, n o n e o f the cells fulfilled the c r i t e r i a for a s u r r o u n d , t h o u g h e a c h cell was t h o r o u g h l y studied. A t age 21 days and o l d e r all o f the on- a n d off-cells studied had a n t a g o n i s t i c s u r r o u n d s . Fig. 1C, D
64 TABLE 1 On- and off-cells are separately classified for each according to whether or not an antagonistic surround discharge was observed. Motion selective cells (9 on, 5 on-off) are separately tabulated in the bottom row. Age (days) No. cells studied On-cells with surrounds no surrounds Off-cells with surrounds no surrounds of on- and off-cells with surrounds Motion selective
9 8 4 0 4 4 0 4
1l 28 13 _~ 11 13 0 13
12 9 4 ,_~ ,.~ 5 1 4
13 4 2 ,.~ 0 2 0 ~_
14 13 7 6 I 3 I -~
15 8 3
0 0
8 2
33 0
50 0
70 3
50 0
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16 14 7 5 ~ 5 4 I
17 14 4 4 0 8 6 ,:"
19 5 2 2 0 2 1
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24 t4 3
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69 2
83 2
75 1
100 2
0 9 9 100 2
and E suggest that a quantitative difference existed between the surrounds observed in the y o u n g e r animals c o m p a r e d to those studied in older age groups. The following comparison illustrates this point. At 14 days 3 of the 7 cells which had surrounds showed surround discharge to a diffuse light stimulus, while the other 4 cells required the center adaptation procedure; at 17 days, the diffuse to center adaptation ratio was 7/10 and at 24 days this ratio was 10/12. Thus, progressing from the 9- to 24-day animals revealed an increasingly higher percentage o f cells with surrounds and the surrounds themselves became easier to demonstrate. Table 1 tabulates the data obtained for each age group. There was a tendency for the on-cells to mature somewhat faster in their surround acquisitions c o m p a r e d to the off-cells. It should be emphasized that our classification criteria required the presence of a surround discharge in order to be included in the s u r r o u n d category. It is possible that some surround influence is present at the early ages, but is too weak to produce a surround discharge; other techniques not used in this study might have revealed a weak s u r r o u n d mechanism in the 9-day age group. Some interesting trigger features were evident in the on- and off-cells studied in the 9- and 11-day animals. Only 2 of the 36 cells studied in this g r o u p fulfilled our criteria for having an antagonistic surround; the receptive fields at this age were dominated by the center. Fig. I A illustrates a c o m m o n feature o f cells studied in the 9- and I l-day age group. A diffuse, low intensity light stimulus (:lower trace) resulted in a sustained discharge of the on-cell which lasted the duration of the flash. Increasing the intensity of the flash (upper trace) by 3 log units produced a prolonged discharge which outlasted the stimulus duration by several seconds. A n equivalent behavior observed in off-cells can be appreciated by comparing the lower traces of Fig. 1C, D, In the I 1-day recording a high intensity annulus prevented the off-discharge f r o m occurring at the termination of the small spot light stimulus. Identical stimulation procedures in the 15-day animal did not have this effect. This 'high intensity effect' was less evident in older animals, particularly those with surrounds and seemed to be especially characteristic o f the cells studied in the 9- and 11-day animals.
65 On-off-cells and on-cells which r e s p o n d e d p o o r l y to diffuse i l l u m i n a t i o n were tested for m o t i o n selectivity by using a b a c k w a r d - f o r w a r d m o v i n g slit rotated t h r o u g h 180 '~. Thirteen o f the 122 cells (9 on, 14 on off) showed m o t i o n selective properties and these cells are separately t a b u l a t e d in Table I ( b o t t o m row). Two m o t i o n selective cells were observed at age I l days, 1 d a y before eye opening. Thus, the neuronal netw o r k which underlies this mechanism is well developed at an early age and is p r o b a b l y different f r o m the mechanism responsible for c e n t e r - s u r r o u n d o r g a n i z a t i o n o f on- and off-center cells. This is consistent with intracellular r e c o r d i n g experiments in the m u d p u p p y which indicate that horizontal cells underlie the a n t a g o n i s t i c s u r r o u n d o r g a n i z a t i o n 1°, and m o t i o n selective mechanisms are a function of the inner retina and involve a m a c r i n e cells ~,9. Thus, the a p p l i c a t i o n of intracellular recording techniques, particularly in the 9- and 11-day age group, should provide a d d i t i o n a l insights into the pathways o f c e n t e r - s u r r o u n d o r g a n i z a t i o n c o m p a r e d to those involved in m o t i o n selectivity. The isolated retina-eyecup p r e p a r a t i o n is ideally suited for this continuing study. We t h a n k W. K. Noell for constructive advice. This work was s u p p o r t e d by N . I . H . G r a n t EY-00844.
I AMES,A., AND POLLEN, D. A., Neurotransmitters in central nervous system : a study of the isolated
rabbit retina, J. Neurophysiol., 32 (1969) 424-442. 2 BARLOW, H. B., HILL, R. M., AND LEVICK, W. R., Retinal ganglion cells responding selectively
to direction and speed of image motion in the rabbit, J. Physiol. (Lond.), 173 (1964) 377-407. 3 DACHEUX,R. F., DELMELLE,M., MILI.ER, R. F., AND NOELL, W. K., Isolated rabbit retina prepara-
tion suitable for intra- and extracellular analysis, FeEt.Proc., 32 (1973) 327. 4 DOWLINC;,J, E., Organization of vertebrate retinas, Invest. Ophthal., 9 (1970) 665 680. 5 LEVJCK,W. R., Another tungsten microelectrode, Med. biol. Engng., 10 (1972) 510-515. 6 LEVlCK,W. R., Receptive fields and trigger features of ganglion cells in the visual streak of the rabbit's retina, J. Physiol. (Lond.), 188 (1967) 285-307. 7 MILLER,R. F., AND DACHEUX, R. F., Information processing in the retina: importance of chloride ions, Science, 121 (1973)266-268. 8 NOELL, W. K., Differentiation, metabolic organization and viability of the visual cell, Arch. Ophthal. (Chic.), 60 (1958) 702-733. 9 WERBLIN, F. S., Responses of retinal cells to moving spots: intracellular recording in Necturus macnlosus, J. Neurophysiol., 33 (1970) 342-350. 10 WERBLIN, F. S., AND DOWL1NC,J. E., Organization of the retina of the mudpuppy, Necturus ,mculosus. 11. lntracellular recording, J. Neurophysiol., 32 (1969) 339 355.
61
Short Communications
Developmental characteristics of receptive organization in the isolated retina-eyecup of the rabbit
CONSTANCE BOWE-ANDERS*, ROBERT F. MILLER AND RAMON DACHEUX Neurosensory Ltlboratory, Department of Physiology, State UniversiO' of New York at Buffido, Buffalo, N. Y. 14214 (U.S.A.)
(Accepted December 19th, 1974)
The retina of the rabbit, like that of many other mammals, is immature at birth but develops rapidly during the first weeks of neonatal life. Light responsive ganglion cells are present at 8-9 days after birth s , but little is known about the maturation rates of the different neuronal pathways which underlie ganglion cell receptive field organization. For instance, when on- and off-center ganglion cells become light responsive, do they have the center-surround organization characteristic of adult cells'? Experimental approaches to this question are complicated by the poorly developed optics of the newborn eye and the generally poor tolerance of the neonatal mammal to anesthesia. The isolated retina represents a possible method of avoiding these problems if the preparation can be maintained in a normal or near normal condition. In this arrangement the optics of the eye would be replaced by a clear perfusate, and except for the anesthesia required for the initial surgery, the retina would not experience prolonged exposure to anesthetizing agents. During the past 3 years this laboratory has developed an isolated retina-eyecup preparation using the adult rabbit eye. This preparation has proved suitable for extracellular recordings from ganglion cells as well as intracellular recordings from all the major cell types of the retina 3,7. The stability of this preparation is evident by ganglion cell recordings which show constant thresholds for several hours and display both center and surround organization. Other types of ganglion cells such as motion selective'-' and local edge detecting neurons 6 have also been recorded from this prepa,~ ation. These results encouraged us to explore the possible use of this technique for examining the maturation sequence of center surround organization in on- and off: center ganglion cells. Our results indicate that the retina of the neonate, like that of the adult, can be maintained in a reasonably stable condition for at least 8 h, and in this report we describe a study of 122 ganglion cells recorded from 11 rabbits ranging in * Present address: 1098 Vernier Place, Stanford, Calif. 94305, U.S.A.
62 age from 9 to 24 days after birth. The isolation and maintenance methods used tbr the neonate are similar to those previously described for the adulta,L Albino rabbits were obtained from a commercial dealer who brought the doe and litter to our animal facilities about 6 days after birth. Animals selected for experimentation are anesthetized (urethane, 0.9 g/kg) after which the eye is surgically excised. The cornea, iris and lens are dissected away and the remaining eyecup is inverted and secured in a special chamber designed to accommodate the small size of the neonatal eye. A temperaturecontrolled (35 °C), meter-regulated (40 ml/min) perfusate flows over the vitrea/surface and is continually recirculated by means of a pump. The pH is maintained at 7.4-7.5 by constant aeration of the perfusate with a 95 ° o 0.)-5 °,~I COz gas mixture. Perfusate is made fresh for each experiment and consists of the following, slightly modified from that used by Ames and Pollen1: NaCI 120 mM; KCI 5.0 mM; NaHCO~ 25 raM: glucose 10 mM; Na2HPO4 0.8 mM; NaH2PO4 0.1 mM; CaClz 2 mM: MgCI,_, 1.0 raM; horse serum 2.5 3(,. The chamber is mounted in a light-tight shielded cage. White light stimuli are provided by shuttered tungsten-iodine lamps, each regulated by a DC power supply. One beam provides focal and diffuse light stimulation (maximum intensity 0.0 log units, 4.6 mW/sq, cm) while a second beam provides annuli or a moving slit or edge (maximum intensity 3.7 mW/sq, cm). The moving slit or edge can be rotated through 360 ° by means of a dovetail prism. No diffuse background illumination was employed. Ganglion cell recordings are obtained with glass insulated tungsten electrodes a. Illustrations are reproduced from polaroid photographs of the amplified impulse activity displayed on a storage oscilloscope (Tektronix 5103). The ERG was recorded between chlorided silver electrodes placed between the front and back of the eye and served as a monitor to evaluate the condition of the preparation. ERG recordings at different ages have shown a developmental sequence similar to that observed in the intact neonatal rabbit 8. All ganglion cell recordings in this study were obtained within the first 4 h following the initial isolation, though an E R G can be maintained for more than 8 h. Fig. 1E illustrates the receptive field organization of an off-center cell recorded from a 24-day animal. This cell showed adult characteristics in its receptive field properties. A centrally located 160 l~m light flash (upper trace) resulted in a decrease in the spontaneous activity followed by a discharge at the termination of the light stimulus. The lower trace shows both on- (85 msec latency) and off- (35 msec latency) activity to a 5 mm light stimulus of the same intensity. The long latency on-discharge is due to activation of the surround while the short latency off-discharge results from stimulation of the center. Fig. 1D shows the receptive field organization of an offcenter cell recorded from a 15 day rabbit. A diffuse light stimulus evoked an offdischarge but no surround discharge was evident regardless of stimulus intensity. However, a surround discharge could be evoked by selective adaptation of the center. A centrally located spot of light (240 #m) was flashed for 5 sec with an interstimulus interval of 30 sec; 1 sec after the onset of the center spot, a l-sec annulus (0.6 mm I.D., 5 mm O.D.) was flashed to see if a surround discharge could be evoked by this procedure. When the intensity of the annulus was relatively low (upper trace) no surround discharge was observed. However, an increase in the intensity of the annulus
63
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Fig. 1. The upper trace of A shows a prolonged discharge of an on-cell recorded from an I l-day rabbit in response to a high intensity diffuse light stimulus. A low intensity light flash (lower trace) produced a sustained discharge which lasted the duration of the stimulus. B is a plot of the percentage of on- and off-cells with demonstrable surrounds v e r s u s age after birth. The off-cell in C was recorded from an I 1-day animal. The upper trace shows an off-discharge to a center light stimulus with a weak annulus superimposed (240/~m spot; annulus I.D. 600 Hm, O.D. 5 mm). The lower trace shows a response to the same center light with a high intensity annulus. The annulus failed to evoke an 'on'discharge but resulted in a single impulse at 'off' and subsequently suppressed the off-discharge to the center spot. D shows the activity of an off-cell recorded from a 15-day animal, using the identical stimulation parameters of C. The high intensity annulus (lower trace) resulted in 'on'- and "off'discharge but did not subsequently cause a suppression of the off-response to the center stimulus. The records of E were obtained from a 24-day animal. A 160 pm spot of light (upper trace) induced an off-discharge, but a diffuse light stimulus (lower trace) of the same intensity caused a long latency (85 msec) on-discharge and a short latency (35 msec) off-discharge characteristic of adult cells. Intensity values are logarithmic reductions of maximum intensity. Time calibrations are indicated. Negativity downward.
( l o w e r trace) resulted in o n - a n d o f f - d i s c h a r g e t h e r e b y d e m o n s t r a t i n g the p r e s e n c e o f a s u r r o u n d d i s c h a r g e w i t h this m e t h o d . T h e r e c o r d s o f Fig. 1C were o b t a i n e d f r o m an off-cell s t u d i e d in an 11-day a n i m a l . T h e s t i m u l u s p a r a m e t e r s w e r e i d e n t i c a l to t h o s e d e s c r i b e d in Fig. ID. H o w e v e r , in this case, a low i n t e n s i t y ( u p p e r trace) a n d high intensity ( l o w e r trace) a n n u l u s failed to e v o k e any s u r r o u n d discharge. N o s u r r o u n d d i s c h a r g e was o b s e r v e d in this cell a l t h o u g h the i n t e n s i t y o f the s p o t a n d a n n u l u s was i n d e p e n d e n t l y v a r i e d o v e r a 5 l o g u n i t r a n g e in 1 log u n i t i n c r e m e n t s . Cells which h a d a s u r r o u n d d i s c h a r g e to e i t h e r a diffuse light s t i m u l u s o r the c e n t e r a d a p t i o n m e t h o d were classified as cells ' w i t h s u r r o u n d s ' . I f the a b o v e p r o c e d u r e s failed to e v o k e a s u r r o u n d d i s c h a r g e , it was classified as a cell w i t h o u t an a n t a g o n i s t i c s u r r o u n d . Fig. I B g r a p h i c a l l y illustrates t h e p e r c e n t a g e o f cells w i t h s u r r o u n d s o b s e r v e d in t h e age g r o u p s studied. A t age 9 days, the y o u n g e s t a n i m a l studied, n o n e o f the cells fulfilled the c r i t e r i a for a s u r r o u n d , t h o u g h e a c h cell was t h o r o u g h l y studied. A t age 21 days and o l d e r all o f the on- a n d off-cells studied had a n t a g o n i s t i c s u r r o u n d s . Fig. 1C, D
64 TABLE 1 On- and off-cells are separately classified for each according to whether or not an antagonistic surround discharge was observed. Motion selective cells (9 on, 5 on-off) are separately tabulated in the bottom row. Age (days) No. cells studied On-cells with surrounds no surrounds Off-cells with surrounds no surrounds of on- and off-cells with surrounds Motion selective
9 8 4 0 4 4 0 4
1l 28 13 _~ 11 13 0 13
12 9 4 ,_~ ,.~ 5 1 4
13 4 2 ,.~ 0 2 0 ~_
14 13 7 6 I 3 I -~
15 8 3
0 0
8 2
33 0
50 0
70 3
50 0
I
,_~ 5 3 ~
16 14 7 5 ~ 5 4 I
17 14 4 4 0 8 6 ,:"
19 5 2 2 0 2 1
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24 t4 3
!
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0
69 2
83 2
75 1
100 2
0 9 9 100 2
and E suggest that a quantitative difference existed between the surrounds observed in the y o u n g e r animals c o m p a r e d to those studied in older age groups. The following comparison illustrates this point. At 14 days 3 of the 7 cells which had surrounds showed surround discharge to a diffuse light stimulus, while the other 4 cells required the center adaptation procedure; at 17 days, the diffuse to center adaptation ratio was 7/10 and at 24 days this ratio was 10/12. Thus, progressing from the 9- to 24-day animals revealed an increasingly higher percentage o f cells with surrounds and the surrounds themselves became easier to demonstrate. Table 1 tabulates the data obtained for each age group. There was a tendency for the on-cells to mature somewhat faster in their surround acquisitions c o m p a r e d to the off-cells. It should be emphasized that our classification criteria required the presence of a surround discharge in order to be included in the s u r r o u n d category. It is possible that some surround influence is present at the early ages, but is too weak to produce a surround discharge; other techniques not used in this study might have revealed a weak s u r r o u n d mechanism in the 9-day age group. Some interesting trigger features were evident in the on- and off-cells studied in the 9- and 11-day animals. Only 2 of the 36 cells studied in this g r o u p fulfilled our criteria for having an antagonistic surround; the receptive fields at this age were dominated by the center. Fig. I A illustrates a c o m m o n feature o f cells studied in the 9- and I l-day age group. A diffuse, low intensity light stimulus (:lower trace) resulted in a sustained discharge of the on-cell which lasted the duration of the flash. Increasing the intensity of the flash (upper trace) by 3 log units produced a prolonged discharge which outlasted the stimulus duration by several seconds. A n equivalent behavior observed in off-cells can be appreciated by comparing the lower traces of Fig. 1C, D, In the I 1-day recording a high intensity annulus prevented the off-discharge f r o m occurring at the termination of the small spot light stimulus. Identical stimulation procedures in the 15-day animal did not have this effect. This 'high intensity effect' was less evident in older animals, particularly those with surrounds and seemed to be especially characteristic o f the cells studied in the 9- and 11-day animals.
65 On-off-cells and on-cells which r e s p o n d e d p o o r l y to diffuse i l l u m i n a t i o n were tested for m o t i o n selectivity by using a b a c k w a r d - f o r w a r d m o v i n g slit rotated t h r o u g h 180 '~. Thirteen o f the 122 cells (9 on, 14 on off) showed m o t i o n selective properties and these cells are separately t a b u l a t e d in Table I ( b o t t o m row). Two m o t i o n selective cells were observed at age I l days, 1 d a y before eye opening. Thus, the neuronal netw o r k which underlies this mechanism is well developed at an early age and is p r o b a b l y different f r o m the mechanism responsible for c e n t e r - s u r r o u n d o r g a n i z a t i o n o f on- and off-center cells. This is consistent with intracellular r e c o r d i n g experiments in the m u d p u p p y which indicate that horizontal cells underlie the a n t a g o n i s t i c s u r r o u n d o r g a n i z a t i o n 1°, and m o t i o n selective mechanisms are a function of the inner retina and involve a m a c r i n e cells ~,9. Thus, the a p p l i c a t i o n of intracellular recording techniques, particularly in the 9- and 11-day age group, should provide a d d i t i o n a l insights into the pathways o f c e n t e r - s u r r o u n d o r g a n i z a t i o n c o m p a r e d to those involved in m o t i o n selectivity. The isolated retina-eyecup p r e p a r a t i o n is ideally suited for this continuing study. We t h a n k W. K. Noell for constructive advice. This work was s u p p o r t e d by N . I . H . G r a n t EY-00844.
I AMES,A., AND POLLEN, D. A., Neurotransmitters in central nervous system : a study of the isolated
rabbit retina, J. Neurophysiol., 32 (1969) 424-442. 2 BARLOW, H. B., HILL, R. M., AND LEVICK, W. R., Retinal ganglion cells responding selectively
to direction and speed of image motion in the rabbit, J. Physiol. (Lond.), 173 (1964) 377-407. 3 DACHEUX,R. F., DELMELLE,M., MILI.ER, R. F., AND NOELL, W. K., Isolated rabbit retina prepara-
tion suitable for intra- and extracellular analysis, FeEt.Proc., 32 (1973) 327. 4 DOWLINC;,J, E., Organization of vertebrate retinas, Invest. Ophthal., 9 (1970) 665 680. 5 LEVJCK,W. R., Another tungsten microelectrode, Med. biol. Engng., 10 (1972) 510-515. 6 LEVlCK,W. R., Receptive fields and trigger features of ganglion cells in the visual streak of the rabbit's retina, J. Physiol. (Lond.), 188 (1967) 285-307. 7 MILLER,R. F., AND DACHEUX, R. F., Information processing in the retina: importance of chloride ions, Science, 121 (1973)266-268. 8 NOELL, W. K., Differentiation, metabolic organization and viability of the visual cell, Arch. Ophthal. (Chic.), 60 (1958) 702-733. 9 WERBLIN, F. S., Responses of retinal cells to moving spots: intracellular recording in Necturus macnlosus, J. Neurophysiol., 33 (1970) 342-350. 10 WERBLIN, F. S., AND DOWL1NC,J. E., Organization of the retina of the mudpuppy, Necturus ,mculosus. 11. lntracellular recording, J. Neurophysiol., 32 (1969) 339 355.