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Do retinal ganglion cells project bilaterally in ground squirrels?
BRAIN RESEARCH ELSEVIER
Brain Research 673 (1995) 161-164
Short communication
Do retinal ganglion cells project bilaterally in ground squirrels? Nidza Lugo *, Earl Kicliter Institute of Neurobiology and Department of Anatomy, Universityof Puerto Rico Medical Sciences Campus, San Juan, PR, PuertoRico
Accepted 13 December 1994
Abstract
Bilaterally projecting retinal ganglion cells have recently been reported in the Japanese monkey Macaca fuscata and albino rat. In the present experiments, we sought to determine whether ganglion cells project bilaterally in the 13-lined ground squirrel Spermophilus tridecernlineatus. We injected rhodamine-conjugated latex microspheres (red beads) into the right optic tract, superior colliculus and the dorsal lateral geniculate nucleus and FITC-conjugated latex microspheres (green beads) into these structures on the left side. In retinal wholemounts of all subjects, we found cells filled with red beads throughout the left retina and cells with green beads throughout the right retina. Ipsilaterally projecting ganglion cells were confined to the temporal retina where they were mixed with cells projecting contralaterally. In no case did we observe a doubly labeled cell. We interpret these observations as indicating that ganglion cells in this species do not project bilaterally. Keywords: Retina; Ganglion cell; Visual system; Spermophilus tridecemlineatus; Collateral
Collaterals of retinal ganglion cell axons may project bilaterally a n d / o r to more than one target on the same side of the brain. Earlier studies using Golgi- and other axon-staining techniques provided evidence that retinal ganglion cells innervated more than one target on the same side of the brain, e.g., the dorsal lateral geniculate and superior colliculus (e.g., [4,5]). Studies, such as that by Cunningham and Freeman [3], suggested that retinal ganglion cell axons might project bilaterally as well. More recently, retrograde tracers with differing optical properties have allowed investigation of collateral innervation of widely separated targets. Bilaterally projecting retinal ganglion cells have recently been reported in the Japanese monkey Macaca fuscata [8] and albino rat [9]. These recent reports confirm previous findings in the rats [3,6] and raise the question of in which mammals individual retinal ganglion cells project bilaterally. In a previous study of 13-lined ground squirrels, after injecting horseradish peroxidase (HRP) into the lateral geniculate and superior colliculus, we observed
* Corresponding author. Institute of Neurobiology,201 Boulevard del Valle, San Juan, PR 00901, Puerto Rico. Fax: (1) (809) 725-3804. 0006-8993/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved SSDI 0006-8993(94)01462-0
labeled ganglion cells in both ipsilateral and contralateral temporal retina [1,11]. While it is possible that some retinal ganglion cells in the temporal retina of ground squirrels may project bilaterally, we were not able to address this question in the previous experiments in which only a single tracer was used. For this reason, we have studied the possibility of bilateral retinal projections in ground squirrels using two tracers, rhodamine- or fluorescein- conjugated latex microspheres or beads [7]. These particular retrograde tracers offer some distinct advantages owing to their high stability, resistance to fading under illumination and absence of obvious cytotoxicity or phototoxicity. The beads are taken up by cut axons but not by undamaged fibers of passage. A portion of our results have been communicated in abstract form [10]. Three 13qined ground squirrels Spermophilus tridecemlineatus were anesthetized with sodium pentobarbital (40 m g / k g ) and then placed in a stereotaxic apparatus. The overlying skin was reflected and a dental drill used to perform a craniotomy over the lateral geniculate and superior colliculus. Latex microspheres conjugated to a fluorescent dye (Luma Fluor, New York, NY) were pressure injected into the superior colliculus, lateral geniculate and optic tract with a 5-txl Hamilton syringe. Rhodamine- conjugated micro-
162
N. Lugo, E. Kicliter /Brain Research 673 (1995) 161-164
spheres (red beads) were injected on the right side, fluorescein-conjugated microspheres (green beads) on the left side. Several injections were made on each side. A total of 1.2 izl were injected on each side of TA-105; a total of 2.3 izl were injected on each side of TA-114 and TA-115. After completion of the injection procedure, the skin was sutured and a survival period of 10 days (TA-114), 3 months (TA-105) and 4 months (TA-115) allowed for retrograde transport of the beads. The squirrels were then given a lethal dose of sodium pentobarbital and perfused through the heart with 0.1 M phosphate buffer followed by a fixative consisting of
A
4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.2). The retinas were removed and prepared as wholemounts. Preparations were examined with a Nikon Optiphot microscope equipped for epifluorescence. Injection sites were confirmed in coronal sections made through the diencephalon and midbrain. The injections were located mainly in the superior colliculus, dorsal lateral geniculate nucleus and optic tract (Fig. 1A), although some microspheres were also located in the pretectal area. In all of the animals, we observed cells filled with red beads throughout the left retina and cells with
TA-115
F--4 lmm
B
TA-
115,2.3pl,4 m
S
T
S ..
N
T
I RIGHT
LEFT
,~n~n
Fig. 1. A: Camera lucida tracings of coronal sections through brain of subject TA-115 at level of lateral geniculate (left) and superior colliculus (right), illustrating extent of injections. Left side of each section is right side of brain. Triangles represent regions injected with rhodamine-conjugated microspheres, squares regions injected with fluorescein-conjugated microspheres. CTX, cortex; LGd, dorsal lateral geniculate; OT, optic tract; SC, superior colliculus. B: camera lucida tracings of retinas of subject TA-115. Shading shows limits of ipsilaterally projecting cells in each retina. Contralaterally projecting cells were distributed across full extent of each retina. ONH, optic nerve head; I, inferior; N, nasal; S, superior; T, temporal.
N. Lugo, E. Kicliter / Brain Research 673 (1995) 161-164
green beads throughout the right retina (Fig. 1B). Thus, contralaterally projecting ganglion cells were found in both nasal and temporal regions of the retina. Contralaterally projecting cells appeared to be evenly distributed across the retina with no observable maximal or minimal concentration in any particular region. Conversely, ipsilaterally projecting ganglion cells were confined to the temporal retina where they were mixed with cells projecting contralaterally (Fig. 2). Measured in photographs, density of contralaterally projecting cells was 1473-1822/mm 2, ipsilaterally projecting cells in the temporal retina was 2 2 - 2 0 2 / m m 2. The somata of all labeled ganglion cells were 4-16 txm in diameter. Ipsilaterally projecting neurons appeared to be larger on the average, with diameters of 10-16 ~m. Often, primary and secondary dendritic processes contained beads. Thousands of cells were examined, yet in no case did we observe a cell which contained both red and green beads. In a few instances, we saw a cell containing red beads adjacent to another containing green beads. In contrast to recent reports of bilaterally projecting retinal ganglion cells in albino rats and a Japanese monkey, we observed no bilaterally projecting retinal ganglion cells in 13-lined ground squirrels. While contrasting results may indicate a true difference in anatomical organization between these species, we must also consider differences in experimental procedures. The current study employed fluorescent dye-conjugated latex microspheres as retrograde tracers while the studies reporting bilateral projections employed
163
fast blue, diamidino yellow and rhodamine-B-isothiocyanate. Since the latter tracers are molecules, smaller than the latex microspheres which are 0.02-0.2 ~ m in diameter, it is possible that these fluorescent dyes can leak to the extracellular space and be taken up by adjacent cells if survival times are excessive [2]. On the other hand, one could argue that the fluorescent dyes may have been more efficiently transported through smaller axons, thus, revealing a larger quantity of retrogradely labeled cells. However, in previous studies, no selectivity for uptake of the microspheres has been observed [7]. In support of a true difference in anatomical organization, at least between ground squirrels and rats, we point out that single retinal ganglion cells in rats project ipsilaterally to both the dorsal lateral geniculate and the superior colliculus [5,14]. In ground squirrels, electrophysiological evidence indicates that the same retinal ganglion cells do not project to both lateral geniculate and superior colliculus. The ganglion cells which project to these two targets in ground squirrels have differing response characteristics: contrast-sensitive and opponent color cells project to the lateral geniculate [13] while directionally selective cells project to the superior colliculus [12]. Michael [12,13] reported no overlap in these projections: directionally selective cells do not project to the lateral geniculate; contrastsensitive and opponent color cells do not project to the superior colliculus. Thus, rats may exhibit greater collateralization of retinal ganglion ceils than the ground squirrels.
Fig. 2. Photomicrographs of labeled cells in temporal retina of subject TA-115. A: contralaterally projecting ganglion cells retrogradely labeled
with red microspheres. B" same field of view as in A but using a fluorescein filter set which allows some of rhodamine fluorescence to be visualized. Arrowhead points to ipsilaterally projecting ganglion cell labeled with green microspheres. Arrows in A and B point to same cells filled with red microspheres. Note that ipsilaterally projecting cell is larger than typical contralaterally projecting cell. Scale bar in B applies to both photomicrographs,20 ~m.
164
N. Lugo, E. Kicliter / Brain Research 673 (1995) 161-164
We thank I. Santiago and C. del Cueto for technical assistance. This work was supported by ONR Grant N00014-89-J-3070 (to N. Lugo) and NIH Grant MH48190. [1] Abreu, M., Kicliter, E. and Lugo-Garcia, N., Displaced amacrine cells in the ganglion cell layer of the ground squirrel retina, Puerto Rico Health Sci. J., 12 (19931 137-141. [2] Bentivoglio, M., Kuypers, H.G.J.M., Catsman-Berrevoets, C.E., Loewe, H. and Dann, O., Two new fluorescent retrograde neuronal tracers which are transported over long distances, Neurosci. Lett., 18 (19801 25-3(/. [3] Cunningham, T.J. and Freeman, J.A., Bilateral ganglion cell branches in the normal rat: a demonstration with electrophysiological collision and cobalt tracing methods, J. Comp. Neurol., 172 (19771 165-176. [4] Dong, K., Rahman, H.A. and Yamadori, T., A retrograde fluorescence double-labeling study of the cat's optic nerve cell which has a bifurcating axon, Acta Anat. Nippon. 67 (19921 207-213. [5] Giolli, R.A. and Towns, L.C., A review of axon collateralization in the mammalian visual system, Brain Behar. Erol., 17 (1980) 364-390. [6] Jeffery, G., Cowey, A. and Kuypers, H.G.J.M., Bifurcating retinal ganglion cell axons in the rat, demonstrated by retrograde labelling, Exp. Brain Res., 44 (19811 34-40.
[7] Katz, L.C., Burkhalter, A. and Dreyer, W.J., Fluorescent latex microspheres as a retrograde marker to in vivo and in vitro studies of visual cortex, Nature (London), 310 (19841 498-500. [8] Kondo, Y., Takada, M., Kayahara, T., Yasui, Y., Nakano, K. and Mizuno, N., Single retinal ganglion cells sending axon collaterals to the bilateral superior colliculi: a fluorescent retrograde double-labeling study in the Japanese monkey (Macaca fuscata), Brain Res., 597 (19921 155-161. [9] Kondo, Y., Takada, M., Honda, Y. and Mizuno, N., Bilateral projections of single retinal ganglion cells to the lateral geniculate nuclei and superior colliculi in the albino rat, Brain Res., 608 (19931 204-215. [10] Lugo, N. and Kicliter, E., Do retinal ganglion cells project bilaterally in ground squirrels? Neurosci. Abstr., 19 (1993) 528. [I 1] Lugo-Garcla, N. and Kicliter, E., Morphology of ganglion cells which project to the dorsal lateral geniculate and superior colliculus in the ground squirrel, Brain Res., 454 (1988) 67-77. [12] Michael, C.R., Visual receptive fields of single neurons in superior colliculus of the ground squirrel, J. Neurophysiol., 35 (19721 815-832. [13] Michael, C.R., Opponent color and opponent-contrast cells in the lateral geniculate nucleus of the ground squirrel, J~ Neurophysiol., 36 (1973) 536-550. [14] Yamadori, T., Nakamura, T. and Takami, K., A study on the retinal ganglion cell which has uncrossed bifurcating axon in the albino rat, Brain Res., 488 (1989) 143-148.
Brain Research 673 (1995) 161-164
Short communication
Do retinal ganglion cells project bilaterally in ground squirrels? Nidza Lugo *, Earl Kicliter Institute of Neurobiology and Department of Anatomy, Universityof Puerto Rico Medical Sciences Campus, San Juan, PR, PuertoRico
Accepted 13 December 1994
Abstract
Bilaterally projecting retinal ganglion cells have recently been reported in the Japanese monkey Macaca fuscata and albino rat. In the present experiments, we sought to determine whether ganglion cells project bilaterally in the 13-lined ground squirrel Spermophilus tridecernlineatus. We injected rhodamine-conjugated latex microspheres (red beads) into the right optic tract, superior colliculus and the dorsal lateral geniculate nucleus and FITC-conjugated latex microspheres (green beads) into these structures on the left side. In retinal wholemounts of all subjects, we found cells filled with red beads throughout the left retina and cells with green beads throughout the right retina. Ipsilaterally projecting ganglion cells were confined to the temporal retina where they were mixed with cells projecting contralaterally. In no case did we observe a doubly labeled cell. We interpret these observations as indicating that ganglion cells in this species do not project bilaterally. Keywords: Retina; Ganglion cell; Visual system; Spermophilus tridecemlineatus; Collateral
Collaterals of retinal ganglion cell axons may project bilaterally a n d / o r to more than one target on the same side of the brain. Earlier studies using Golgi- and other axon-staining techniques provided evidence that retinal ganglion cells innervated more than one target on the same side of the brain, e.g., the dorsal lateral geniculate and superior colliculus (e.g., [4,5]). Studies, such as that by Cunningham and Freeman [3], suggested that retinal ganglion cell axons might project bilaterally as well. More recently, retrograde tracers with differing optical properties have allowed investigation of collateral innervation of widely separated targets. Bilaterally projecting retinal ganglion cells have recently been reported in the Japanese monkey Macaca fuscata [8] and albino rat [9]. These recent reports confirm previous findings in the rats [3,6] and raise the question of in which mammals individual retinal ganglion cells project bilaterally. In a previous study of 13-lined ground squirrels, after injecting horseradish peroxidase (HRP) into the lateral geniculate and superior colliculus, we observed
* Corresponding author. Institute of Neurobiology,201 Boulevard del Valle, San Juan, PR 00901, Puerto Rico. Fax: (1) (809) 725-3804. 0006-8993/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved SSDI 0006-8993(94)01462-0
labeled ganglion cells in both ipsilateral and contralateral temporal retina [1,11]. While it is possible that some retinal ganglion cells in the temporal retina of ground squirrels may project bilaterally, we were not able to address this question in the previous experiments in which only a single tracer was used. For this reason, we have studied the possibility of bilateral retinal projections in ground squirrels using two tracers, rhodamine- or fluorescein- conjugated latex microspheres or beads [7]. These particular retrograde tracers offer some distinct advantages owing to their high stability, resistance to fading under illumination and absence of obvious cytotoxicity or phototoxicity. The beads are taken up by cut axons but not by undamaged fibers of passage. A portion of our results have been communicated in abstract form [10]. Three 13qined ground squirrels Spermophilus tridecemlineatus were anesthetized with sodium pentobarbital (40 m g / k g ) and then placed in a stereotaxic apparatus. The overlying skin was reflected and a dental drill used to perform a craniotomy over the lateral geniculate and superior colliculus. Latex microspheres conjugated to a fluorescent dye (Luma Fluor, New York, NY) were pressure injected into the superior colliculus, lateral geniculate and optic tract with a 5-txl Hamilton syringe. Rhodamine- conjugated micro-
162
N. Lugo, E. Kicliter /Brain Research 673 (1995) 161-164
spheres (red beads) were injected on the right side, fluorescein-conjugated microspheres (green beads) on the left side. Several injections were made on each side. A total of 1.2 izl were injected on each side of TA-105; a total of 2.3 izl were injected on each side of TA-114 and TA-115. After completion of the injection procedure, the skin was sutured and a survival period of 10 days (TA-114), 3 months (TA-105) and 4 months (TA-115) allowed for retrograde transport of the beads. The squirrels were then given a lethal dose of sodium pentobarbital and perfused through the heart with 0.1 M phosphate buffer followed by a fixative consisting of
A
4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.2). The retinas were removed and prepared as wholemounts. Preparations were examined with a Nikon Optiphot microscope equipped for epifluorescence. Injection sites were confirmed in coronal sections made through the diencephalon and midbrain. The injections were located mainly in the superior colliculus, dorsal lateral geniculate nucleus and optic tract (Fig. 1A), although some microspheres were also located in the pretectal area. In all of the animals, we observed cells filled with red beads throughout the left retina and cells with
TA-115
F--4 lmm
B
TA-
115,2.3pl,4 m
S
T
S ..
N
T
I RIGHT
LEFT
,~n~n
Fig. 1. A: Camera lucida tracings of coronal sections through brain of subject TA-115 at level of lateral geniculate (left) and superior colliculus (right), illustrating extent of injections. Left side of each section is right side of brain. Triangles represent regions injected with rhodamine-conjugated microspheres, squares regions injected with fluorescein-conjugated microspheres. CTX, cortex; LGd, dorsal lateral geniculate; OT, optic tract; SC, superior colliculus. B: camera lucida tracings of retinas of subject TA-115. Shading shows limits of ipsilaterally projecting cells in each retina. Contralaterally projecting cells were distributed across full extent of each retina. ONH, optic nerve head; I, inferior; N, nasal; S, superior; T, temporal.
N. Lugo, E. Kicliter / Brain Research 673 (1995) 161-164
green beads throughout the right retina (Fig. 1B). Thus, contralaterally projecting ganglion cells were found in both nasal and temporal regions of the retina. Contralaterally projecting cells appeared to be evenly distributed across the retina with no observable maximal or minimal concentration in any particular region. Conversely, ipsilaterally projecting ganglion cells were confined to the temporal retina where they were mixed with cells projecting contralaterally (Fig. 2). Measured in photographs, density of contralaterally projecting cells was 1473-1822/mm 2, ipsilaterally projecting cells in the temporal retina was 2 2 - 2 0 2 / m m 2. The somata of all labeled ganglion cells were 4-16 txm in diameter. Ipsilaterally projecting neurons appeared to be larger on the average, with diameters of 10-16 ~m. Often, primary and secondary dendritic processes contained beads. Thousands of cells were examined, yet in no case did we observe a cell which contained both red and green beads. In a few instances, we saw a cell containing red beads adjacent to another containing green beads. In contrast to recent reports of bilaterally projecting retinal ganglion cells in albino rats and a Japanese monkey, we observed no bilaterally projecting retinal ganglion cells in 13-lined ground squirrels. While contrasting results may indicate a true difference in anatomical organization between these species, we must also consider differences in experimental procedures. The current study employed fluorescent dye-conjugated latex microspheres as retrograde tracers while the studies reporting bilateral projections employed
163
fast blue, diamidino yellow and rhodamine-B-isothiocyanate. Since the latter tracers are molecules, smaller than the latex microspheres which are 0.02-0.2 ~ m in diameter, it is possible that these fluorescent dyes can leak to the extracellular space and be taken up by adjacent cells if survival times are excessive [2]. On the other hand, one could argue that the fluorescent dyes may have been more efficiently transported through smaller axons, thus, revealing a larger quantity of retrogradely labeled cells. However, in previous studies, no selectivity for uptake of the microspheres has been observed [7]. In support of a true difference in anatomical organization, at least between ground squirrels and rats, we point out that single retinal ganglion cells in rats project ipsilaterally to both the dorsal lateral geniculate and the superior colliculus [5,14]. In ground squirrels, electrophysiological evidence indicates that the same retinal ganglion cells do not project to both lateral geniculate and superior colliculus. The ganglion cells which project to these two targets in ground squirrels have differing response characteristics: contrast-sensitive and opponent color cells project to the lateral geniculate [13] while directionally selective cells project to the superior colliculus [12]. Michael [12,13] reported no overlap in these projections: directionally selective cells do not project to the lateral geniculate; contrastsensitive and opponent color cells do not project to the superior colliculus. Thus, rats may exhibit greater collateralization of retinal ganglion ceils than the ground squirrels.
Fig. 2. Photomicrographs of labeled cells in temporal retina of subject TA-115. A: contralaterally projecting ganglion cells retrogradely labeled
with red microspheres. B" same field of view as in A but using a fluorescein filter set which allows some of rhodamine fluorescence to be visualized. Arrowhead points to ipsilaterally projecting ganglion cell labeled with green microspheres. Arrows in A and B point to same cells filled with red microspheres. Note that ipsilaterally projecting cell is larger than typical contralaterally projecting cell. Scale bar in B applies to both photomicrographs,20 ~m.
164
N. Lugo, E. Kicliter / Brain Research 673 (1995) 161-164
We thank I. Santiago and C. del Cueto for technical assistance. This work was supported by ONR Grant N00014-89-J-3070 (to N. Lugo) and NIH Grant MH48190. [1] Abreu, M., Kicliter, E. and Lugo-Garcia, N., Displaced amacrine cells in the ganglion cell layer of the ground squirrel retina, Puerto Rico Health Sci. J., 12 (19931 137-141. [2] Bentivoglio, M., Kuypers, H.G.J.M., Catsman-Berrevoets, C.E., Loewe, H. and Dann, O., Two new fluorescent retrograde neuronal tracers which are transported over long distances, Neurosci. Lett., 18 (19801 25-3(/. [3] Cunningham, T.J. and Freeman, J.A., Bilateral ganglion cell branches in the normal rat: a demonstration with electrophysiological collision and cobalt tracing methods, J. Comp. Neurol., 172 (19771 165-176. [4] Dong, K., Rahman, H.A. and Yamadori, T., A retrograde fluorescence double-labeling study of the cat's optic nerve cell which has a bifurcating axon, Acta Anat. Nippon. 67 (19921 207-213. [5] Giolli, R.A. and Towns, L.C., A review of axon collateralization in the mammalian visual system, Brain Behar. Erol., 17 (1980) 364-390. [6] Jeffery, G., Cowey, A. and Kuypers, H.G.J.M., Bifurcating retinal ganglion cell axons in the rat, demonstrated by retrograde labelling, Exp. Brain Res., 44 (19811 34-40.
[7] Katz, L.C., Burkhalter, A. and Dreyer, W.J., Fluorescent latex microspheres as a retrograde marker to in vivo and in vitro studies of visual cortex, Nature (London), 310 (19841 498-500. [8] Kondo, Y., Takada, M., Kayahara, T., Yasui, Y., Nakano, K. and Mizuno, N., Single retinal ganglion cells sending axon collaterals to the bilateral superior colliculi: a fluorescent retrograde double-labeling study in the Japanese monkey (Macaca fuscata), Brain Res., 597 (19921 155-161. [9] Kondo, Y., Takada, M., Honda, Y. and Mizuno, N., Bilateral projections of single retinal ganglion cells to the lateral geniculate nuclei and superior colliculi in the albino rat, Brain Res., 608 (19931 204-215. [10] Lugo, N. and Kicliter, E., Do retinal ganglion cells project bilaterally in ground squirrels? Neurosci. Abstr., 19 (1993) 528. [I 1] Lugo-Garcla, N. and Kicliter, E., Morphology of ganglion cells which project to the dorsal lateral geniculate and superior colliculus in the ground squirrel, Brain Res., 454 (1988) 67-77. [12] Michael, C.R., Visual receptive fields of single neurons in superior colliculus of the ground squirrel, J. Neurophysiol., 35 (19721 815-832. [13] Michael, C.R., Opponent color and opponent-contrast cells in the lateral geniculate nucleus of the ground squirrel, J~ Neurophysiol., 36 (1973) 536-550. [14] Yamadori, T., Nakamura, T. and Takami, K., A study on the retinal ganglion cell which has uncrossed bifurcating axon in the albino rat, Brain Res., 488 (1989) 143-148.