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Cholecystokinin-like immunoreactive retinal ganglion cells project to the ventral lateral geniculate nucleus in pigeons
322
Brain Research, 557 (1991) 322-326 © 1991 Elsevier Science Publishers B.V. All rights reserved. 0006-8993/91/$03.50 A D ONIS 000689939124791 P
BRES 24791
Cholecystokinin-like immunoreactive retinal ganglion cells project to the ventral lateral geniculate nucleus in pigeons Luiz R.G. Britto and Dfinia E. Hamassaki-Britto Neurosciences and Behavior Research Nucleus and Department of Physiology and Biophysics, Institute of Biomedical Sciences, Sdo Paulo State University, Sdo Paulo (Brazil)
(Accepted 7 May 1991) Key words: Cholecystokinin; Neuropeptide; Neurotransmitter; Retinal ganglion cell; Subcortical visual pathway; Ventral geniculate nucleus
A subpopulation of retinal ganglion cells projecting to the pigeon ventral lateral geniculate nucleus was shown to contain cholecystokinin-like immunoreactivity. These ganglion cells were mainly distributed in the peripheral retina, and their somata sizes were medium to large (14-23 am). Taken together with previous findings, these results indicate that the retinal input to the ventral geniculate is chemically heterogeneous. Considerable evidence has accumulated in the past few years for the existence of different neuroactive substances in specific populations of ganglion cells in the vertebrate retina 16 (RGCS). Indeed, there are reports of RGCS that contain G A B A 5'12'31'32, glutamate 14'26, glycine 9'3°, catecholamines 3,4,18, serotonin 27, substance p1,4-7,20, neurotensin-related peptide LANT-61°, corticotropin-releasing hormone 29 and somatostatin 28 in different vertebrates. There is also evidence, mostly indirect, that different subsets of RGCS in the frog retina contain enkephalins, bombesin and cholecystokinin 19'21'22. However, the RGCS containing these neuroactive peptides have not been clearly identified in the retina of any vertebrate. As part of a general effort to characterize neuroactive substances in RGCS, we report here the detection of a population of RGCS that exhibit cholecystokinin-like immunoreactivity (CCK-LI) in the pigeon retina. These cells were shown to project to the thalamic retinorecipient ventral lateral geniculate (GLv) nucleus 13. Retrograde tracing and retinal ablation techniques were combined with immunohistochemistry in this study. Seventeen adult pigeons (Colurnba livia) of either sex were employed in two different sets of experiments. Twelve animals were anesthetized with ketamine (5 mg/100 g of b. w., i.m.) and xylazine (1 mg/100 g, i.m.) and unilateral stereotaxic 15 injections of rhodamine beads 17 were placed into GLv. A 25% solution of rhodamine beads in 0.1 M phosphate buffer (PB) was pressure-injected into GLv through a glass micropipette (tip diameter 12-15/*m). Injection volumes ranged from 0.05 to 0.2/.1, and survival times ranged from 4 to 6 days.
Five additional pigeons were anesthetized with ketamine and xylazine and had the neural retina removed from one eye. Lidocaine was injected into the vitreous before removing the retina with a cotton swab. These animals were allowed to survive for 7-35 days. Following the experimental period, the pigeons were deeply anesthetized with ketamine and xylazine and perfused through the left ventricle with phosphate-buffered saline and ice-cold 2% paraformaldehyde in PB. The eyes and brains were post-fixed for 2-5 h and then transferred to a 30% sucrose solution in PB. After 48 h, the retinae were frozen and sectioned either parallel (at 20/*m) or perpendicular (at 10 /*m) to the vitreal surface. The brains were frozen and cut (at 30/*m) in the coronal plane. The retinal and brain sections were then reacted for CCK 8 immunohistochemistry as described in detail elsewhere 2. Briefly, a commercially available rabbit antiserum against CCK 8 (Incstar Corp.) was used in these experiments. Dilutions in PB containing 1.0% Triton X-100 ranged from 1:15,000 (avidin-biotin-peroxidase technique, ABC) to 1:750 (immunofluorescence technique). Secondary goat anti-rabbit antisera (Jackson Labs.) were diluted 1:200-1:100 in PB and were either biotinylated or labeled with fluorescein isothiocyanate (FITC). Controls for specificity of labeling included (1) omission of the primary antiserum, (2) substitution of the primary antiserum with normal rabbit serum, and (3) preadsorption of the primary antiserum with 10 mM of CCK8 (Peninsula Labs). Specific staining was abolished under any of these conditions. Sizes and distribution of RGCS described below were determined on retinal
Correspondence: L.R.G. Britto, Department of Neurosciences, University of California, San Diego, La Jolla, CA 92093-0608, U.S.A.
323 sections cut parallel to the vitreal surface. T h e p a t t e r n of C C K - L I in the pigeon retina was similar
to the p a t t e r n described in m a n y v e r t e b r a t e retinae 23-25. Neurons containing C C K - L I were o b s e r v e d in the ganglion cell layer ( G C L ) and the inner nuclear layer, whereas stained processes were mainly seen in the innermost part (lamina 5) of the inner plexiform layer (Fig. 1). Only cells in the G C L were studied further. The n u m b e r of C C K - L I cells in the G C L ranged from 3400 to 5600 per retina. Their distribution m a t c h e d the overall distribution of cells in the G C L of the pigeon retina, with higher densities in the fovea and the ' r e d field '11. The C C K - L I neurons in the G C L exhibited a b r o a d range of s o m a t a sizes ( 5 - 2 9 / a m in the largest axis), with the vast majority of t h e m (about 80%) in the small size range ( 5 - 1 2 / ~ m ) . The remaining 20% of C C K - L I neurons in the G C L was comprised of nearly equal numbers of either m e d i u m (13-20 ~ m ) or large-sized (21-29 /~m) neurons. L a b e l e d processes of all these C C K - L I neurons
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Fig. 1. A: photomicrograph of a transverse section of the pigeon retina showing two CCK-LI amacrine cells (arrows) and dense arborizations in the innermost part of the inner plexiform layer (IPL). B: photomicrograph of a transverse retinal section showing one CCK-LI cell in the inner nuclear layer (INL, straight arrow) and one in the ganglion cell layer (GCL, curved arrow). C: photomicrograph of a horizontal retinal section through the GCL/IPL border showing a CCK-LI neuron and its proximal processes. A and B: FITC technique. C: ABC technique. Bars: A, 30 ~m; B, 20/~m; C, 15 ,um.
Fig. 2. Photomicrographs of a horizontal section of the pigeon retina, taken under filters for FITC (A) and rhodamine (B). A large neuron containing both CCK-LI (A, FITC technique) and rhodamine bead fluorescence (B) is indicated by the arrows. There is a marked fluorescence breakthrough of the rhodamine beads (B) through the FITC filter (A). Bar = 20/2m.
324 in the GCL could only be followed to the lamina 5 of the inner plexiform layer. The injections of rhodamine beads filled most of the GLv in 6 cases, with no detectable involvement of other neighboring retinorecipient structures or the optic tract. These injections produced retrograde labeling of 31005300 RGCS in the contralateral retina. The distribution of the RGCS that project to the GLv also matched the distribution of the whole population of cells in the GCL. Somata sizes of the retrogradely labeled neurons varied widely, ranging from 6 to 26 ~m in the largest axis. They could be classified either as small (6-12 ~m), medium (13-19/~m), or large-sized (20-26 ~m). About 23% of the CCK-LI medium and large cells in the GCL of the pigeon retina were clearly identifiable as RGCS, since they were retrogradely labeled following rhodamine beads injections into GLv (Fig. 2). Absolute numbers of the doubly labeled neurons ranged from 134 to 283 per retina. This population of CCK-LI RGCS constituted approximately 5% of the RGCS that were retrogradely labeled after injections into the GLv. The distribution of these CCK-LI RGCS was mainly peripheral, with very few cells of this type occurring in the fovea or in the superior temporal retina (including the 'red field'). The superior nasal retina contained the highest densities of CCK-LI RGCS projecting to GLv (up to 15 celis/mm2), whereas the inferior retina contained only low densities of these cells (about 2 cells/mm2). Somata sizes of CCK-LI RGCS ranged from 14 to 23 ~m in the largest axis. Therefore, the doubly labeled neurons were also classified as medium or large-sized in comparison
with the whole population of RGCS projecting to GLv. The CCK-LI RGCS projecting to GLv were usually characterized by 2-4 primary dendrites that were radially arranged. Labeled processes of CCK-LI RGCS could be followed in lamina 5 of the inner plexiform layer for at least 250 ~m from the cell body. The analysis of brain sections processed for CCK 8 immunohistochemistry revealed a dense plexus of CCKLI terminal-like profiles within a restricted caudomedial portion of the GLv. Labeled fibers and presumptive terminals were also observed within other retinorecipient structures, such as the optic tectum, pretectal nuclei and suprachiasmatic nucleus. In the brains from pigeons that suffered unilateral retinal removal, we observed a complete elimination of the CCK-LI plexus within the contralateral GLv (Fig. 3). This effect was consistently seen from 2 to 5 weeks following the surgery. A marked reduction of CCK-LI fibers/terminals was also observed in the contralateral optic tectum and pretectum, but no change was seen in the suprachiasmatic nucleus. These results provide evidence for the existence of a population of CCK-LI RGCS in the pigeon retina. Both the double-labeling and retinal ablation experiments demonstrated that these cells project to the GLv. The higher densities of CCK-LI RGCS in the superior nasal retina and the location of the CCK-LI plexus within the caudomedial GLv clearly fit the highly organized retinotopic projection upon the avian GLv 8. There are no previous reports of identified RGCS containing CCK-LI in the vertebrate retina, although the occurrence of this type of cell in the frog retina has
Fig. 3. Darkfield photomicrograph of a coronal section of the pigeon brain processed for CCK-LI. The right retina of this animal had been removed 3 weeks before it was sacrificed. In the GLv ipsilateral to the removed retina (right side of the figure), a CCK-LI plexus is visible in a restricted portion of that nucleus (arrow). No CCK-LI is observed in the contralateral, deafferented GLv (left side). The arrow pointing to the left GLv indicates approximately the same region indicated by the arrow pointing to the right GLv. GLv, ventral lateral geniculate nucleus; LH, lateral hypothalamus; PMH, posteromedial hypothalamic nucleus; PVM, periventricular hypothalamic nucleus; SCN, suprachiasmatic nucleus; TrO, optic tract; V, ventricle. Bar = 800/~m.
325 medium- to large-sized. Their somata sizes did not overlap with the sizes of substance P-positive R G C S 4 and overlapped only minimally with the tyrosine hydroxylasecontaining R G C S 4'18. Furthermore, both the substance P and the tyrosine hydroxylase-containing R G C S that project to GLv were mainly distributed in the central retina 4, whereas C C K - L I R G C S that project to GLv appear to be concentrated in the retinal periphery. Double-labeling experiments are needed, however, to verify the possibility that some of the medium-sized CCK-LI R G C S could also contain tyrosine hydroxylase. In summary, the pigeon G L v appears to be the recipient of retinal projections derived from several populations of chemically-specific R G C S : small cells containing substance P, small and medium cells containing tyrosine hydroxylase, medium and large cells containing C C K - L I and one or more populations of chemically unidentified R G C S spanning a wide range of somata sizes. Therefore, the heterogeneity of transmitters/peptides in R G C S 16 is reflected even within a single retinorecipient nucleus of a single species, such as the pigeon GLv.
already been suggested 21. That suggestion was based on the changing pattern of C C K - L I in the frog optic tectum following retinal deafferentation 19 and also on the fact that some optic axons exhibited C C K - L I after optic nerve disruption 22. A few C C K - L I cells in the ganglion cell layer of the cat retina have been described which are presumptive R G C S , since some axons contain CCK-LI in the same retina 25. Thus, the population of CCK-LI R G C S found in the present study in the pigeon may represent a consistent feature of the vertebrate retina. The results of the experiments in which the retina was removed also indicated that other retinorecipient structures, such as the optic rectum, may receive retinal input from C C K - L I RGCS. Studies employing retrograde labeling are needed to confirm these findings and to verify the possibility that the same population of CCK-LI R G C S which project to GLv also project to other visual structures. This is a likely possibility, in view of the fact that many retinal axons entering the GLv are collaterals of axons that project to other retinal targets 4"13. The pigeon GLv has been previously shown to receive retinal input derived from small and medium-sized tyrosine hydroxylase-positive R G C S 4A8 and small-sized substance P-positive R G C S 4. These two types of cells comprised about 33% of the whole population of R G C S which were retrogradely labeled following rhodamine beads injections into GLv. The CCK-LI R G C S projecting to G L v comprised only about 5% of the whole population of R G C S projecting to GLv and were
This study was supported by Grants from Brazilian Agencies FAPESP (88/3969-9; 90/2255-2), CNPq (30.0826/81), and FINEP (4.3.89.0268.00), and by a USP/BID Contract. D.E.H.B. was the recipient of a postdoctoral fellowship from FAPESP (90/2526-6). Thanks are also due to Drs. Kent Keyser, Thomas Hughes and Toru Shimizu (UCSD, La JoUa, CA) for helpful comments during the preparation of this manuscript.
1 Brecha, N., Johnson, D., Bolz, J., Sharma, S., Parnavelas, J.G. and Lieberman, A.R., Substance P-immunoreactive retinal ganglion cells and their central axon terminals in the rabbit, Nature, 327 (1987) 155-158. 2 Britto, L.R.G., Hamassaki, D.E., Keyser, K.T. and Karten, H.J., Neurotransmitters, receptors, and neuropeptides in the accessory optic system: an immunohistochemical survey in the pigeon (Columba livia), Vis. Neurosci., 3 (1989) 463-475. 3 Britto, L.R.G., Keyser, K.T., Hamassaki, D.E. and Karten, H.J., Catecholaminergic subpopulation of retinal displaced ganglion cells projects to the accessory optic nucleus in the pigeon ( Columba livia), J. Comp. Neurol., 269 (1988) 109-117. 4 Britto, L.R.G., Keyser, K.T., Hamassaki, D.E., Shimizu, T. and Karten, H.J., Chemically specific retinal ganglion cells collateralize to the pars ventralis of the lateral geniculate nucleus and optic tectum in the pigeon (Columba livia), Vis. Neurosci., 3 (1989) 477-482. 5 Caruso, D.M., Owczarzak, M.T. and Pourcho, R.G., Colocalization of substance P and GABA in retinal ganglion cells: a computer-assisted visualization, VIS. Neurosci., 5 (1990) 389394. 6 Cuenca, N. and Kolb, H., Morphology and distribution of neurons immunoreactive for substance P in the turtle retina, J. Comp. Neurol., 290 (1989) 391-411. 7 Ehrlich, D., Keyser, K.T. and Karten, H.J., The distribution of substance P-like immunoreactive retinal ganglion cells and their pattern of termination in the optic tectum of chick (Gallus gallus), J. Comp. NeuroL, 266 (1987) 220-233. 8 Ehrlich, D. and Mark, R.E, Retinal topography of primary visual centres in the brain of the chick (Gallus gallus), J. Comp.
Neurol., 223 (1984) 611-625. 9 Eldred, W.D. and Cheung, K., Immunocytochemical localization of glycine in the retina of the turtle (Pseudemys scripta), Vis. Neurosci., 2 (1989) 331-338. 10 Eldred, W.D., Isayama, T., Reiner, A. and Carraway, R., Ganglion cells in the turtle retina contain the neuropeptide LANT-6, J. Neurosci., 8 (1988) 119-132. 11 Hayes, B.P., The structural organization of the pigeon retina, Prog. Retin. Res., 1 (1982) 197-226. 12 Hurd, L.B. and Eldred, W.D., Localization of GABA- and GAD-like immunoreactivity in the turtle retina, Vis. Neurosci., 3 (1989) 9-20. 13 Jones, E.G., The Thalamus, Plenum, New York, 1985, pp. 723-733. 14 Kageyama, G.H. and Meyer, R.L., Glutamate immunoreactivity in the retina and optic tectum of goldfish, Brain Research, 503 (1989) 118-127. 15 Karten, H.J. and Hodos, W., A Stereotaxic Atlas of the Brain of the Pigeon (Columba livia), Johns Hopkins, Baltimore, 1967, 185 pp. 16 Karten, H.J., Keyser, K.T. and Brecha, N.C., Biochemical and morphological heterogeneity of retinal ganglion cells. In B. Cohen and I. Bodis-Wollner (Eds.), Vision and the Brain: The Organization of the Central Visual System, Raven, New York, 1990, pp. 19-34. 17 Katz, L.C., Burkhalter, A. and Dreyer, W.J., Fluorescent latex microspheres as a retrograde neuronal marker for in vivo and in vitro studies of visual cortex, Nature, 310 (1984) 498-500. 18 Keyser, K.T., Britto, L.R.G., Woo, J.-I., Park, D.H., Joh, T.H. and Karten, H.J., Presumptive catecholaminergic ganglion cells
326 in the pigeon retina, Vis. Neurosci., 4 (1990) 225-235. 19 Kuljis, R.O. and Karten, H.J., Modifications in the laminar organization of peptide-like immunoreactivity in the anuran optic tectum following retinal deafferentation, J. Cornp. Neurol., 217 (1983) 239-251. 20 Kuljis, R.O. and Karten, H.J., Substance P-containing ganglion cells become progressively less detectable during retinotectal development in the frog (Rana pipiens), Proc. Natl. Acad. Sci. U.S.A., 83 (1986) 5736-5740. 21 Kuljis, R.O. and Karten, H.J., Neuroactive peptides as markers of retinal ganglion cell populations that differ in anatomical organization and function, Vis. Neurosci., 1 (1988) 73-81. 22 Kuljis, R.O., Krause, J.E. and Karten, H.J., Peptide-like immunoreactivity in anuran optic nerve fibers, J. Comp. Neurol., 226 (1984) 222-237. 23 Marshak, D.W., Aldrich, L.B., Del Valle, J. and Yamada, T., Localization of immunoreactive cholecystokinin precursor to amacrine cells and bipolar cells of the macaque monkey retina, J. Neurosci., 10 (1990) 3045-3055. 24 Osborne, N.N., Cholecystokinin in the retina of vertebrates, Ann. N.Y. Acad. Sci., 448 (1985) 157-166. 25 Thier, P. and Bolz, J., Cholecystokinin in the cat retina. Action of exogenous CCK8 and localization of cholecystokinin-like immunoreactivity, Invest. Ophthalmol. Vis. Sci., 26 (1985) 266-272.
26 Van Haesendonck, E. and Missotten, L., Glutamate-like immunoreactivity in the retina of a marine teleost, the dragonet, Neurosci. Lett., 111 (1990) 281-286. 27 Weiler, R. and Ammermuller, J., Immunocytochemical localization of serotonin in intracellularly analyzed and dye-injected ganglion cells of the turtle retina, Neurosci. Lett., 72 (1986) 147-152. 28 White, C.A., Chalupa, L.M., Johnson, D. and Brecha, N.C., Somatostatin-immunoreactive cells in the adult cat retina, J. Comp. Neurol., 293 (1990) 134-150. 29 Williamson, D.E. and Eldred, W.D., Amacrine and ganglion cells with corticotropin-releasing-factor-like immunoreactivity in the turtle retina, J. Comp. Neurol., 280 (1989) 424-435. 30 Yang, C.-Y. and Yazulla, S., Light microscopic localization of putative glycinergic neurons in the larval tiger salamander retina by immunocytochemical and autoradiographical methods, J. Comp. Neurol., 272 (1988) 343-357. 31 Yang, C.-Y. and Yazulla, S., Localization of putative GABAergic neurons in the larval tiger salamander retina by immunocytochemical and autoradiographic methods, J. Comp. Neurol.. 277 (1988) 96-108. 32 Yu, B.C., Watt, C.B., Lam, D.M. and Fry, K.R., GABAergic ganglion cells in the rabbit retina, Brain Research, 439 (1988) 376-382.
Brain Research, 557 (1991) 322-326 © 1991 Elsevier Science Publishers B.V. All rights reserved. 0006-8993/91/$03.50 A D ONIS 000689939124791 P
BRES 24791
Cholecystokinin-like immunoreactive retinal ganglion cells project to the ventral lateral geniculate nucleus in pigeons Luiz R.G. Britto and Dfinia E. Hamassaki-Britto Neurosciences and Behavior Research Nucleus and Department of Physiology and Biophysics, Institute of Biomedical Sciences, Sdo Paulo State University, Sdo Paulo (Brazil)
(Accepted 7 May 1991) Key words: Cholecystokinin; Neuropeptide; Neurotransmitter; Retinal ganglion cell; Subcortical visual pathway; Ventral geniculate nucleus
A subpopulation of retinal ganglion cells projecting to the pigeon ventral lateral geniculate nucleus was shown to contain cholecystokinin-like immunoreactivity. These ganglion cells were mainly distributed in the peripheral retina, and their somata sizes were medium to large (14-23 am). Taken together with previous findings, these results indicate that the retinal input to the ventral geniculate is chemically heterogeneous. Considerable evidence has accumulated in the past few years for the existence of different neuroactive substances in specific populations of ganglion cells in the vertebrate retina 16 (RGCS). Indeed, there are reports of RGCS that contain G A B A 5'12'31'32, glutamate 14'26, glycine 9'3°, catecholamines 3,4,18, serotonin 27, substance p1,4-7,20, neurotensin-related peptide LANT-61°, corticotropin-releasing hormone 29 and somatostatin 28 in different vertebrates. There is also evidence, mostly indirect, that different subsets of RGCS in the frog retina contain enkephalins, bombesin and cholecystokinin 19'21'22. However, the RGCS containing these neuroactive peptides have not been clearly identified in the retina of any vertebrate. As part of a general effort to characterize neuroactive substances in RGCS, we report here the detection of a population of RGCS that exhibit cholecystokinin-like immunoreactivity (CCK-LI) in the pigeon retina. These cells were shown to project to the thalamic retinorecipient ventral lateral geniculate (GLv) nucleus 13. Retrograde tracing and retinal ablation techniques were combined with immunohistochemistry in this study. Seventeen adult pigeons (Colurnba livia) of either sex were employed in two different sets of experiments. Twelve animals were anesthetized with ketamine (5 mg/100 g of b. w., i.m.) and xylazine (1 mg/100 g, i.m.) and unilateral stereotaxic 15 injections of rhodamine beads 17 were placed into GLv. A 25% solution of rhodamine beads in 0.1 M phosphate buffer (PB) was pressure-injected into GLv through a glass micropipette (tip diameter 12-15/*m). Injection volumes ranged from 0.05 to 0.2/.1, and survival times ranged from 4 to 6 days.
Five additional pigeons were anesthetized with ketamine and xylazine and had the neural retina removed from one eye. Lidocaine was injected into the vitreous before removing the retina with a cotton swab. These animals were allowed to survive for 7-35 days. Following the experimental period, the pigeons were deeply anesthetized with ketamine and xylazine and perfused through the left ventricle with phosphate-buffered saline and ice-cold 2% paraformaldehyde in PB. The eyes and brains were post-fixed for 2-5 h and then transferred to a 30% sucrose solution in PB. After 48 h, the retinae were frozen and sectioned either parallel (at 20/*m) or perpendicular (at 10 /*m) to the vitreal surface. The brains were frozen and cut (at 30/*m) in the coronal plane. The retinal and brain sections were then reacted for CCK 8 immunohistochemistry as described in detail elsewhere 2. Briefly, a commercially available rabbit antiserum against CCK 8 (Incstar Corp.) was used in these experiments. Dilutions in PB containing 1.0% Triton X-100 ranged from 1:15,000 (avidin-biotin-peroxidase technique, ABC) to 1:750 (immunofluorescence technique). Secondary goat anti-rabbit antisera (Jackson Labs.) were diluted 1:200-1:100 in PB and were either biotinylated or labeled with fluorescein isothiocyanate (FITC). Controls for specificity of labeling included (1) omission of the primary antiserum, (2) substitution of the primary antiserum with normal rabbit serum, and (3) preadsorption of the primary antiserum with 10 mM of CCK8 (Peninsula Labs). Specific staining was abolished under any of these conditions. Sizes and distribution of RGCS described below were determined on retinal
Correspondence: L.R.G. Britto, Department of Neurosciences, University of California, San Diego, La Jolla, CA 92093-0608, U.S.A.
323 sections cut parallel to the vitreal surface. T h e p a t t e r n of C C K - L I in the pigeon retina was similar
to the p a t t e r n described in m a n y v e r t e b r a t e retinae 23-25. Neurons containing C C K - L I were o b s e r v e d in the ganglion cell layer ( G C L ) and the inner nuclear layer, whereas stained processes were mainly seen in the innermost part (lamina 5) of the inner plexiform layer (Fig. 1). Only cells in the G C L were studied further. The n u m b e r of C C K - L I cells in the G C L ranged from 3400 to 5600 per retina. Their distribution m a t c h e d the overall distribution of cells in the G C L of the pigeon retina, with higher densities in the fovea and the ' r e d field '11. The C C K - L I neurons in the G C L exhibited a b r o a d range of s o m a t a sizes ( 5 - 2 9 / a m in the largest axis), with the vast majority of t h e m (about 80%) in the small size range ( 5 - 1 2 / ~ m ) . The remaining 20% of C C K - L I neurons in the G C L was comprised of nearly equal numbers of either m e d i u m (13-20 ~ m ) or large-sized (21-29 /~m) neurons. L a b e l e d processes of all these C C K - L I neurons
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Fig. 1. A: photomicrograph of a transverse section of the pigeon retina showing two CCK-LI amacrine cells (arrows) and dense arborizations in the innermost part of the inner plexiform layer (IPL). B: photomicrograph of a transverse retinal section showing one CCK-LI cell in the inner nuclear layer (INL, straight arrow) and one in the ganglion cell layer (GCL, curved arrow). C: photomicrograph of a horizontal retinal section through the GCL/IPL border showing a CCK-LI neuron and its proximal processes. A and B: FITC technique. C: ABC technique. Bars: A, 30 ~m; B, 20/~m; C, 15 ,um.
Fig. 2. Photomicrographs of a horizontal section of the pigeon retina, taken under filters for FITC (A) and rhodamine (B). A large neuron containing both CCK-LI (A, FITC technique) and rhodamine bead fluorescence (B) is indicated by the arrows. There is a marked fluorescence breakthrough of the rhodamine beads (B) through the FITC filter (A). Bar = 20/2m.
324 in the GCL could only be followed to the lamina 5 of the inner plexiform layer. The injections of rhodamine beads filled most of the GLv in 6 cases, with no detectable involvement of other neighboring retinorecipient structures or the optic tract. These injections produced retrograde labeling of 31005300 RGCS in the contralateral retina. The distribution of the RGCS that project to the GLv also matched the distribution of the whole population of cells in the GCL. Somata sizes of the retrogradely labeled neurons varied widely, ranging from 6 to 26 ~m in the largest axis. They could be classified either as small (6-12 ~m), medium (13-19/~m), or large-sized (20-26 ~m). About 23% of the CCK-LI medium and large cells in the GCL of the pigeon retina were clearly identifiable as RGCS, since they were retrogradely labeled following rhodamine beads injections into GLv (Fig. 2). Absolute numbers of the doubly labeled neurons ranged from 134 to 283 per retina. This population of CCK-LI RGCS constituted approximately 5% of the RGCS that were retrogradely labeled after injections into the GLv. The distribution of these CCK-LI RGCS was mainly peripheral, with very few cells of this type occurring in the fovea or in the superior temporal retina (including the 'red field'). The superior nasal retina contained the highest densities of CCK-LI RGCS projecting to GLv (up to 15 celis/mm2), whereas the inferior retina contained only low densities of these cells (about 2 cells/mm2). Somata sizes of CCK-LI RGCS ranged from 14 to 23 ~m in the largest axis. Therefore, the doubly labeled neurons were also classified as medium or large-sized in comparison
with the whole population of RGCS projecting to GLv. The CCK-LI RGCS projecting to GLv were usually characterized by 2-4 primary dendrites that were radially arranged. Labeled processes of CCK-LI RGCS could be followed in lamina 5 of the inner plexiform layer for at least 250 ~m from the cell body. The analysis of brain sections processed for CCK 8 immunohistochemistry revealed a dense plexus of CCKLI terminal-like profiles within a restricted caudomedial portion of the GLv. Labeled fibers and presumptive terminals were also observed within other retinorecipient structures, such as the optic tectum, pretectal nuclei and suprachiasmatic nucleus. In the brains from pigeons that suffered unilateral retinal removal, we observed a complete elimination of the CCK-LI plexus within the contralateral GLv (Fig. 3). This effect was consistently seen from 2 to 5 weeks following the surgery. A marked reduction of CCK-LI fibers/terminals was also observed in the contralateral optic tectum and pretectum, but no change was seen in the suprachiasmatic nucleus. These results provide evidence for the existence of a population of CCK-LI RGCS in the pigeon retina. Both the double-labeling and retinal ablation experiments demonstrated that these cells project to the GLv. The higher densities of CCK-LI RGCS in the superior nasal retina and the location of the CCK-LI plexus within the caudomedial GLv clearly fit the highly organized retinotopic projection upon the avian GLv 8. There are no previous reports of identified RGCS containing CCK-LI in the vertebrate retina, although the occurrence of this type of cell in the frog retina has
Fig. 3. Darkfield photomicrograph of a coronal section of the pigeon brain processed for CCK-LI. The right retina of this animal had been removed 3 weeks before it was sacrificed. In the GLv ipsilateral to the removed retina (right side of the figure), a CCK-LI plexus is visible in a restricted portion of that nucleus (arrow). No CCK-LI is observed in the contralateral, deafferented GLv (left side). The arrow pointing to the left GLv indicates approximately the same region indicated by the arrow pointing to the right GLv. GLv, ventral lateral geniculate nucleus; LH, lateral hypothalamus; PMH, posteromedial hypothalamic nucleus; PVM, periventricular hypothalamic nucleus; SCN, suprachiasmatic nucleus; TrO, optic tract; V, ventricle. Bar = 800/~m.
325 medium- to large-sized. Their somata sizes did not overlap with the sizes of substance P-positive R G C S 4 and overlapped only minimally with the tyrosine hydroxylasecontaining R G C S 4'18. Furthermore, both the substance P and the tyrosine hydroxylase-containing R G C S that project to GLv were mainly distributed in the central retina 4, whereas C C K - L I R G C S that project to GLv appear to be concentrated in the retinal periphery. Double-labeling experiments are needed, however, to verify the possibility that some of the medium-sized CCK-LI R G C S could also contain tyrosine hydroxylase. In summary, the pigeon G L v appears to be the recipient of retinal projections derived from several populations of chemically-specific R G C S : small cells containing substance P, small and medium cells containing tyrosine hydroxylase, medium and large cells containing C C K - L I and one or more populations of chemically unidentified R G C S spanning a wide range of somata sizes. Therefore, the heterogeneity of transmitters/peptides in R G C S 16 is reflected even within a single retinorecipient nucleus of a single species, such as the pigeon GLv.
already been suggested 21. That suggestion was based on the changing pattern of C C K - L I in the frog optic tectum following retinal deafferentation 19 and also on the fact that some optic axons exhibited C C K - L I after optic nerve disruption 22. A few C C K - L I cells in the ganglion cell layer of the cat retina have been described which are presumptive R G C S , since some axons contain CCK-LI in the same retina 25. Thus, the population of CCK-LI R G C S found in the present study in the pigeon may represent a consistent feature of the vertebrate retina. The results of the experiments in which the retina was removed also indicated that other retinorecipient structures, such as the optic rectum, may receive retinal input from C C K - L I RGCS. Studies employing retrograde labeling are needed to confirm these findings and to verify the possibility that the same population of CCK-LI R G C S which project to GLv also project to other visual structures. This is a likely possibility, in view of the fact that many retinal axons entering the GLv are collaterals of axons that project to other retinal targets 4"13. The pigeon GLv has been previously shown to receive retinal input derived from small and medium-sized tyrosine hydroxylase-positive R G C S 4A8 and small-sized substance P-positive R G C S 4. These two types of cells comprised about 33% of the whole population of R G C S which were retrogradely labeled following rhodamine beads injections into GLv. The CCK-LI R G C S projecting to G L v comprised only about 5% of the whole population of R G C S projecting to GLv and were
This study was supported by Grants from Brazilian Agencies FAPESP (88/3969-9; 90/2255-2), CNPq (30.0826/81), and FINEP (4.3.89.0268.00), and by a USP/BID Contract. D.E.H.B. was the recipient of a postdoctoral fellowship from FAPESP (90/2526-6). Thanks are also due to Drs. Kent Keyser, Thomas Hughes and Toru Shimizu (UCSD, La JoUa, CA) for helpful comments during the preparation of this manuscript.
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