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Ganglion cells and (dye-coupled) amacrine cells in the turtle retina that have possible synaptic connection
146
Brain Research, 240 (1982) 146-150 Elsevier Biomedical Press
Ganglion cells and (dye-coupled) amacrine cells in the turtle retina that have possible synaptic connection RALPH J. JENSEN* and ROBERT D. DeVOE Department of Physiology, The Johns Hopkins University School of Medicine, Baltimore, MD 21205 (U.S.A.)
(Accepted February 4th, 1982) Key words: turtle retina -- dye coupling -- amacrine cell -- ganglion cell
A class of ON-OFF amacrine cells and ganglion cells in the turtle retina have been identified from intracellular recordings and stainings. Dye coupling (with Lucifer yellow) occurred among the amacrine cells, which suggests that they may form an electrical network. Evidence is presented that the ganglion cells receive excitatory input from these amacrine cells. Many types of amacrine cells have been identified morphologically in the vertebrate retinal,Is, ~0. Physiologically, amacrine cells respond to flashes of light with either transient O N - O F F depolarizao tionsS, 19, or with sustained depolarizations or hyperpolarizations6,10,12. Amacrine cells appear to be primarily inhibitory to ganglion cells9, although recent studies have indicated that some amacrine cells are excitatory2,s. In this study, a class of monostratified O N - O F F amacrine cells are described in the turtle retina which may be excitatory to a class of bistratified O N - O F F ganglion cells. Experiments were performed on the red-eared turtle, Pseudemys scripta elegans. Following decapitation, an eye was removed from the pithed brain and was hemisected. The eyecup was placed in a chamber and was aerated with moistened 95 % 02, 5% CO2 at room temperature (21-24 °C). Glass micropipettes filled with 3 M potassium acetate (electrode resistances of 100-250 Mf~)or 4% Lucifer Yellow CH (kindly supplied by W. Stewart) was used for intracellular recording. The eycup was stimulated with light from a 45 W tungsten-iodide lamp (Sylvania). The intensity at the level of the retina was 4 × 10a #W/cm 2 (410-730 nm), measured with a silicon photodiode (Model 40A Optometer, United Detector Technology, Inc.). During experi-
ments, the ambient room light was 10-3 to 10-2 #W/ cm 2. Fig. 1 shows the responses of an O N - O F F amacrine cell to flashes of various spot sizes. Spike activity in these cells (n = 4) was absent or disappeared soon after electrode impalement. With 0.7 s flashes of unattenuated light, these amacrine cells responded with O N - O F F depolarizing potentials to small spots (centered on the electrode tip) and only ON to large spots. These transient potentials were less than 60 ms in duration. Occasionally, with large spot stimulation a depolarizing hump followed the initial ON transient (Fig. 1). At times the cells also produced small (less than 2 mV) sustained depolarizations. These amacrine cells had very small cell bodies (about 8/~m in diameter). Fluorescence photomicrographs of a dye-filled flat-mount and radial view of one amacrine cell are shown in Fig. 1. Each cell had a single descending dendrite. Upon reaching the middle (stratum 3, as defined by CajaP) of the inner plexiform layer, it divided into 9-11 thin (1 # m diameter) straight processes. These long processes were frequently either cut off in histology or the fluorescence gradually faded out at the ends. Some processes could be followed, however, for up to 1 mm from the cell body. Amacrine cells of similar morphology
* Present address: Department of Physiology and Biophysics, Washington University School of Medicine, 660 South Euclid Avenue, St. Louis, MO 63110, U.S.A.
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Fig. 1. Photoresponses and morphology of an amacrine cell. On the left are responses to various spot sizes (~m). A 50 Hz low-pass filter was used to reduce noise in the responses. On the right are photomicrographs of a flat-mount (above) and a radial (below) view of the cell. Open arrowhead (in the radial view) marks a dendritic process. Scale bar, 100/~m. have been described in other vertebrate retinasZ, 13, 14,20.
For one of the 4 stained amacrine cells, dyecoupling with Lucifer yellow occurred among other amacrine cells of the same type (Fig. 2). This dyecoupling suggests that this cell type may form an electrical syncitium much like the horizontal cells or photoreceptors of the outer plexiform layerIs. Gap junctions, which mediate electrotonic coupling, have been reported interconnecting amacrine cells in the retinas of the cat and salamander3,7,21. Recently, electrotonic coupling has actually been demonstrated between ON-OFF amacrine cells in the catfish retina n. Why dye-coupling occurred only in the one instance is not known for this turtle preparation. In other, non-retinal preparations increased intracellular calcium concentration, an imposed voltage difference across gap junctions, or low intracellular pH are reported to partly or wholly diminish electrotonic coupling 15-17. Intracellular recordings of ganglion cells of the turtle retina (ref. 4; manuscript in preparation) have shown one type of ganglion cell (type III) which had responses to various spot sizes (Fig. 3) similar to those of the amacrine cells. The ganglion cells (n = 17) of this type responded (like the amacrine cells)
O N - O F F to small spots and only ON to large spots of unattenuated light. The duration of the transient depolarizations were short (less than 80 ms). For some cells, a small sustained depolarization was noticeable. The similar photoresponses of these ganglion cells and of the amacrine cells suggest that the latter may be presynaptic to the former. Although extrinsic currents were not used to study the reversal potential(s) of the ON and O F F transient depolarizations, two ganglion cells with initially low resting potentials (as a result of electrode penetration) showed transient O N - O F F hyperpolarizations which, as the cells recovered, reversed together at about the same potential. This finding supports the idea that similar ionic mechanisms may be operating at both the ON and OFF inputs. Morphologically, the ganglion cells were bistratifled (Fig. 3) with dendritic field diameters ranging from 240 to 900 # m (mean of 410 itm). Although Cajal t did not observe this type of ganglion cell in the reptilian retina, he did describe in the frog retina a similar type of ganglion cell which ramified in strata 2 and 4. As seen in radial view, the inner stratum of dendrites of the ganglion cells appears juxtaposed with the dendrites of the amacrine cells,
148
Fig. 2. Photomicrographs of a fiat-mount and a radial view of dye-coupled amacrine cells. A Mueller cell is partially stained at the site of recording (which occasionally occurs with intracellular staining). In the flat mount, the proximal dye-coupled cell bodies (marked by arrows) can be visualized. Although not seen here, at higher magnification faintly-stained dendritic processes of the dye-coupled cells could be seen in fiat mount. Scale bar, 100/tin. Direct synaptic connections between the two cell types is therefore feasible. To investigate the possibility of synaptic interconnection, we looked at the temporal relationship of both the O N and O F F response latencies of the two cell types. The means of the shortest O N and O F F response latencies o f the 4 amacrine cells were 41 -~2.5 ms S.D. and 47 ~ 4.9 ms S.D., respectively. Of 6
ganglion cells, they were 44 ± 3.6 ms S.D. and 53 -~: 14 ms S. D., respectively. The latencies of the two cell types are compatible with the idea that the amacrine cells may be presynaptic to these ganglion cells. A presumptive (unstained) amacrine cell o f this type and one stained ganglion cell were stimulated with light attenuated by l log unit. With large spot
149
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Fig. 3. Photoresponses and morphology of a type III ganglion cell. On the left are photoresponses to various spot sizes (/~m). A 50 Hz low-pass filter was used to reduce noise in the responses. On the right are photomicrographs of a flat-mount and a radial view of the cell. Arrowhead marks the axon. Scale bars, 50 #m.
stimulation, the O F F response a m p l i t u d e s o f b o t h the ganglion a n d a m a c r i n e cells were n o t i c e a b l y larger with the a t t e n u a t e d light t h a n with unatten u a t e d light (not shown). I n s u m m a r y , we have identified in the r e t i n a o f the turtle a p a r t i c u l a r type o f O N - O F F a m a c r i n e cell which m a y f o r m a n electrical synticium. W e have also identified a type o f O N - O F F ganglion cell which m a y receive direct excitatory synaptic i n p u t
f r o m these a m a c r i n e cells. F u r t h e r studies o f these cells m a y c o n t r i b u t e to the u n d e r s t a n d i n g o f the i n p u t - o u t p u t relationships a n d to the function o f possible electrotonic coupling at the inner plexiform layer o f the retina.
1 Cajal, S. Ram6n y, The Structure of the Retina (translated by S. A. Thorpe and M. Glickstein), Charles C. Thomas, Springfield, IL, 1972, 196 pp. 2 Dick, E. and Miller, R. F., Peptides influence retinal ganglion cells, NeuroscL Lett., 26 (1981) 131-135. 3 Famigiietti, E. V., Jr. and Kolb, H., A bistratified amacrine cell and synaptic circuitry in the inner plexiform layer of the retina, Brain Research, 84 (1975) 293-300. 4 Jensen, R. J. and DeVoe, R. D., Identification of specific ganglion cell types in the turtle retina, lnvest. OphthaL, Visual ScL, Suppl. 20 (1981) 184. 5 Kaneko, A., Physiological and morphological identification of horizontal, bipolar and amacrine cells in goldfish retina, J. PhysioL (Lond.), 207 (1970) 623-633. 6 Kaneko, A. and Hashimoto, H., Electrophysiological study of single neurons in the inner nuclear layer of the carp retina, Vision Res., 9 (1969) 37-55. 7 Kolb, H. and Famigiietti, E. V., Rod and cone pathways in the inner plexiform layer of cat retina, Science, 186 (1974) 47-49.
8 Masland, R. H., Acetylcholine in the retina. In N. Bazan and R. Lolley (Eds.), Neurochemistry of the Retina, Pergamon Press, New York, 1980, pp. 501-518. 9 Miller, R. F., The neuronal basis of ganglion-cell receptive-field organization and the physiology of amacrine cells. In F. O. Schmitt and F. G. Worden (Eds.), The Neurosciences: Fourth Study Program, M.I.T. Press, Cambridge, MA, 1979, pp. 227-245. 10 Murakami, M. and Shimoda, Y., Identification of amacrine and ganglion cells in the carp retina, J. Physiol. (Lond.), 264 (1977) 801-818. 11 Naka, K.-I. and Christensen, B. N., Direct electrical connections between transient amacrine cells in the catfish retina, Science, 214 (1981) 462-464. 12 Naka, K.-I. and Ohtsuka, T., Morphological and functional identifications of catfish neurons. II. Morphological identification, J. NeurophysioL, 38 (1975) 72-91. 13 Oliveira Castro, G. de, Branching pattern of amacrine cell processes, Nature (Lond.), 212 (1966) 832-834. 14 Perry, V. H. and Walker, M., Amacrine cells, displaced
Research sponsored by NIH Research Grant EY00008 a w a r d e d to R. D. DeVoe.
150
15
16
17
18
amacrine cells and interplexiform cells in the retina of the rat, Proc. roy. Soc. B, 208 (1980) 415431. Rose, B., Simpson, I. and Loewenstein, W. R., Calcium ion produces graded changes in permeability of membrane channels in cell junction, Nature (Lond.), 267 (1977) 625-627. Spray, D. C., Harris, A. L. and Bennett, M. V. L., Voltage dependence of junctional conductance in early amphibian embryos, Science, 204 (1979) 432~,34. Spray, D. C., Harris, A. L. and Bennett, M. V. L., Gap junctional conductance is a simple and sensitive function of intracellular pH, Science, 211 (1981) 712-715. Werblin, F. S., Integrative pathways in local circuits
between slow-potential cells in the retina. In F. O. Schmit t and F. G. Worden (Eds.), The Neurosciences: Fourth Stud), Program, M.I.T. Press, Cambridge, MA, 1979, pp. 193-211. 19 Werblin, F. S. and Dowling, J. E., Organization of the retina of the mudpuppy, Necturus maculosus. I 1. Intracellular recording, J. Neurophysiol., 32 (1969) 339-355. 20 West, R. W., Light and electron microscopy of the ground squirrel retina: functional considerations, J. comp. Neurol., 168 (1976) 355-378. 21 Wong-Riley, M. T. T., Synaptic organization of the inner plexiform layer in the retina of the tiger salamander, J. NeurocytoL, 3 (1974) 1-33.
Brain Research, 240 (1982) 146-150 Elsevier Biomedical Press
Ganglion cells and (dye-coupled) amacrine cells in the turtle retina that have possible synaptic connection RALPH J. JENSEN* and ROBERT D. DeVOE Department of Physiology, The Johns Hopkins University School of Medicine, Baltimore, MD 21205 (U.S.A.)
(Accepted February 4th, 1982) Key words: turtle retina -- dye coupling -- amacrine cell -- ganglion cell
A class of ON-OFF amacrine cells and ganglion cells in the turtle retina have been identified from intracellular recordings and stainings. Dye coupling (with Lucifer yellow) occurred among the amacrine cells, which suggests that they may form an electrical network. Evidence is presented that the ganglion cells receive excitatory input from these amacrine cells. Many types of amacrine cells have been identified morphologically in the vertebrate retinal,Is, ~0. Physiologically, amacrine cells respond to flashes of light with either transient O N - O F F depolarizao tionsS, 19, or with sustained depolarizations or hyperpolarizations6,10,12. Amacrine cells appear to be primarily inhibitory to ganglion cells9, although recent studies have indicated that some amacrine cells are excitatory2,s. In this study, a class of monostratified O N - O F F amacrine cells are described in the turtle retina which may be excitatory to a class of bistratified O N - O F F ganglion cells. Experiments were performed on the red-eared turtle, Pseudemys scripta elegans. Following decapitation, an eye was removed from the pithed brain and was hemisected. The eyecup was placed in a chamber and was aerated with moistened 95 % 02, 5% CO2 at room temperature (21-24 °C). Glass micropipettes filled with 3 M potassium acetate (electrode resistances of 100-250 Mf~)or 4% Lucifer Yellow CH (kindly supplied by W. Stewart) was used for intracellular recording. The eycup was stimulated with light from a 45 W tungsten-iodide lamp (Sylvania). The intensity at the level of the retina was 4 × 10a #W/cm 2 (410-730 nm), measured with a silicon photodiode (Model 40A Optometer, United Detector Technology, Inc.). During experi-
ments, the ambient room light was 10-3 to 10-2 #W/ cm 2. Fig. 1 shows the responses of an O N - O F F amacrine cell to flashes of various spot sizes. Spike activity in these cells (n = 4) was absent or disappeared soon after electrode impalement. With 0.7 s flashes of unattenuated light, these amacrine cells responded with O N - O F F depolarizing potentials to small spots (centered on the electrode tip) and only ON to large spots. These transient potentials were less than 60 ms in duration. Occasionally, with large spot stimulation a depolarizing hump followed the initial ON transient (Fig. 1). At times the cells also produced small (less than 2 mV) sustained depolarizations. These amacrine cells had very small cell bodies (about 8/~m in diameter). Fluorescence photomicrographs of a dye-filled flat-mount and radial view of one amacrine cell are shown in Fig. 1. Each cell had a single descending dendrite. Upon reaching the middle (stratum 3, as defined by CajaP) of the inner plexiform layer, it divided into 9-11 thin (1 # m diameter) straight processes. These long processes were frequently either cut off in histology or the fluorescence gradually faded out at the ends. Some processes could be followed, however, for up to 1 mm from the cell body. Amacrine cells of similar morphology
* Present address: Department of Physiology and Biophysics, Washington University School of Medicine, 660 South Euclid Avenue, St. Louis, MO 63110, U.S.A.
147
-J E v
Q) N
, m
2oo
.#,.
0
340 i=h~J~':.
~.'~:,~-'~,~ :-~ 4,~.'-:
JSmV
1 4 6 0 ~q~
::~---:4"~-:.'~-: - ' - 1
0 . 7 s e c "1
Fig. 1. Photoresponses and morphology of an amacrine cell. On the left are responses to various spot sizes (~m). A 50 Hz low-pass filter was used to reduce noise in the responses. On the right are photomicrographs of a flat-mount (above) and a radial (below) view of the cell. Open arrowhead (in the radial view) marks a dendritic process. Scale bar, 100/~m. have been described in other vertebrate retinasZ, 13, 14,20.
For one of the 4 stained amacrine cells, dyecoupling with Lucifer yellow occurred among other amacrine cells of the same type (Fig. 2). This dyecoupling suggests that this cell type may form an electrical syncitium much like the horizontal cells or photoreceptors of the outer plexiform layerIs. Gap junctions, which mediate electrotonic coupling, have been reported interconnecting amacrine cells in the retinas of the cat and salamander3,7,21. Recently, electrotonic coupling has actually been demonstrated between ON-OFF amacrine cells in the catfish retina n. Why dye-coupling occurred only in the one instance is not known for this turtle preparation. In other, non-retinal preparations increased intracellular calcium concentration, an imposed voltage difference across gap junctions, or low intracellular pH are reported to partly or wholly diminish electrotonic coupling 15-17. Intracellular recordings of ganglion cells of the turtle retina (ref. 4; manuscript in preparation) have shown one type of ganglion cell (type III) which had responses to various spot sizes (Fig. 3) similar to those of the amacrine cells. The ganglion cells (n = 17) of this type responded (like the amacrine cells)
O N - O F F to small spots and only ON to large spots of unattenuated light. The duration of the transient depolarizations were short (less than 80 ms). For some cells, a small sustained depolarization was noticeable. The similar photoresponses of these ganglion cells and of the amacrine cells suggest that the latter may be presynaptic to the former. Although extrinsic currents were not used to study the reversal potential(s) of the ON and O F F transient depolarizations, two ganglion cells with initially low resting potentials (as a result of electrode penetration) showed transient O N - O F F hyperpolarizations which, as the cells recovered, reversed together at about the same potential. This finding supports the idea that similar ionic mechanisms may be operating at both the ON and OFF inputs. Morphologically, the ganglion cells were bistratifled (Fig. 3) with dendritic field diameters ranging from 240 to 900 # m (mean of 410 itm). Although Cajal t did not observe this type of ganglion cell in the reptilian retina, he did describe in the frog retina a similar type of ganglion cell which ramified in strata 2 and 4. As seen in radial view, the inner stratum of dendrites of the ganglion cells appears juxtaposed with the dendrites of the amacrine cells,
148
Fig. 2. Photomicrographs of a fiat-mount and a radial view of dye-coupled amacrine cells. A Mueller cell is partially stained at the site of recording (which occasionally occurs with intracellular staining). In the flat mount, the proximal dye-coupled cell bodies (marked by arrows) can be visualized. Although not seen here, at higher magnification faintly-stained dendritic processes of the dye-coupled cells could be seen in fiat mount. Scale bar, 100/tin. Direct synaptic connections between the two cell types is therefore feasible. To investigate the possibility of synaptic interconnection, we looked at the temporal relationship of both the O N and O F F response latencies of the two cell types. The means of the shortest O N and O F F response latencies o f the 4 amacrine cells were 41 -~2.5 ms S.D. and 47 ~ 4.9 ms S.D., respectively. Of 6
ganglion cells, they were 44 ± 3.6 ms S.D. and 53 -~: 14 ms S. D., respectively. The latencies of the two cell types are compatible with the idea that the amacrine cells may be presynaptic to these ganglion cells. A presumptive (unstained) amacrine cell o f this type and one stained ganglion cell were stimulated with light attenuated by l log unit. With large spot
149
60 ~
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.
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E
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Fig. 3. Photoresponses and morphology of a type III ganglion cell. On the left are photoresponses to various spot sizes (/~m). A 50 Hz low-pass filter was used to reduce noise in the responses. On the right are photomicrographs of a flat-mount and a radial view of the cell. Arrowhead marks the axon. Scale bars, 50 #m.
stimulation, the O F F response a m p l i t u d e s o f b o t h the ganglion a n d a m a c r i n e cells were n o t i c e a b l y larger with the a t t e n u a t e d light t h a n with unatten u a t e d light (not shown). I n s u m m a r y , we have identified in the r e t i n a o f the turtle a p a r t i c u l a r type o f O N - O F F a m a c r i n e cell which m a y f o r m a n electrical synticium. W e have also identified a type o f O N - O F F ganglion cell which m a y receive direct excitatory synaptic i n p u t
f r o m these a m a c r i n e cells. F u r t h e r studies o f these cells m a y c o n t r i b u t e to the u n d e r s t a n d i n g o f the i n p u t - o u t p u t relationships a n d to the function o f possible electrotonic coupling at the inner plexiform layer o f the retina.
1 Cajal, S. Ram6n y, The Structure of the Retina (translated by S. A. Thorpe and M. Glickstein), Charles C. Thomas, Springfield, IL, 1972, 196 pp. 2 Dick, E. and Miller, R. F., Peptides influence retinal ganglion cells, NeuroscL Lett., 26 (1981) 131-135. 3 Famigiietti, E. V., Jr. and Kolb, H., A bistratified amacrine cell and synaptic circuitry in the inner plexiform layer of the retina, Brain Research, 84 (1975) 293-300. 4 Jensen, R. J. and DeVoe, R. D., Identification of specific ganglion cell types in the turtle retina, lnvest. OphthaL, Visual ScL, Suppl. 20 (1981) 184. 5 Kaneko, A., Physiological and morphological identification of horizontal, bipolar and amacrine cells in goldfish retina, J. PhysioL (Lond.), 207 (1970) 623-633. 6 Kaneko, A. and Hashimoto, H., Electrophysiological study of single neurons in the inner nuclear layer of the carp retina, Vision Res., 9 (1969) 37-55. 7 Kolb, H. and Famigiietti, E. V., Rod and cone pathways in the inner plexiform layer of cat retina, Science, 186 (1974) 47-49.
8 Masland, R. H., Acetylcholine in the retina. In N. Bazan and R. Lolley (Eds.), Neurochemistry of the Retina, Pergamon Press, New York, 1980, pp. 501-518. 9 Miller, R. F., The neuronal basis of ganglion-cell receptive-field organization and the physiology of amacrine cells. In F. O. Schmitt and F. G. Worden (Eds.), The Neurosciences: Fourth Study Program, M.I.T. Press, Cambridge, MA, 1979, pp. 227-245. 10 Murakami, M. and Shimoda, Y., Identification of amacrine and ganglion cells in the carp retina, J. Physiol. (Lond.), 264 (1977) 801-818. 11 Naka, K.-I. and Christensen, B. N., Direct electrical connections between transient amacrine cells in the catfish retina, Science, 214 (1981) 462-464. 12 Naka, K.-I. and Ohtsuka, T., Morphological and functional identifications of catfish neurons. II. Morphological identification, J. NeurophysioL, 38 (1975) 72-91. 13 Oliveira Castro, G. de, Branching pattern of amacrine cell processes, Nature (Lond.), 212 (1966) 832-834. 14 Perry, V. H. and Walker, M., Amacrine cells, displaced
Research sponsored by NIH Research Grant EY00008 a w a r d e d to R. D. DeVoe.
150
15
16
17
18
amacrine cells and interplexiform cells in the retina of the rat, Proc. roy. Soc. B, 208 (1980) 415431. Rose, B., Simpson, I. and Loewenstein, W. R., Calcium ion produces graded changes in permeability of membrane channels in cell junction, Nature (Lond.), 267 (1977) 625-627. Spray, D. C., Harris, A. L. and Bennett, M. V. L., Voltage dependence of junctional conductance in early amphibian embryos, Science, 204 (1979) 432~,34. Spray, D. C., Harris, A. L. and Bennett, M. V. L., Gap junctional conductance is a simple and sensitive function of intracellular pH, Science, 211 (1981) 712-715. Werblin, F. S., Integrative pathways in local circuits
between slow-potential cells in the retina. In F. O. Schmit t and F. G. Worden (Eds.), The Neurosciences: Fourth Stud), Program, M.I.T. Press, Cambridge, MA, 1979, pp. 193-211. 19 Werblin, F. S. and Dowling, J. E., Organization of the retina of the mudpuppy, Necturus maculosus. I 1. Intracellular recording, J. Neurophysiol., 32 (1969) 339-355. 20 West, R. W., Light and electron microscopy of the ground squirrel retina: functional considerations, J. comp. Neurol., 168 (1976) 355-378. 21 Wong-Riley, M. T. T., Synaptic organization of the inner plexiform layer in the retina of the tiger salamander, J. NeurocytoL, 3 (1974) 1-33.