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Purpose: To study alterations in different retinal cell types associated with retinal ganglion cell (RGC) death after elevation of intraocular pressure (IOP) in rats., Methods: IOP was elevated by episcleral vein cauterization of the rat left eye. The right unoperated eye was kept as the control. IOP was measured when rats were awake. The animals were euthanized after one week (n=4) and five weeks (n=4). Their eyes were enucleated, postfixed, cryoprotected, and embedded in optimal cutting temperature (OCT) medium. Cryosections of the retina were cut at 14 microm thickness and processed for immunocytochemistry with 15 antibodies that specifically stain different retinal cell types. The distribution and intensity of the label was analyzed by comparing sections of control and glaucomatous retinas obtained from identical locations., Results: The amount of amacrine cells identified by calcium binding proteins and choline acetyltransferase antibodies decreased after five weeks of elevated IOP. By using the anti-protein kinase C-alpha antibody, we were able to label a subpopulation of rod bipolar cells in control retinas but not in retinas that had elevated IOP. No changes were found in RGCs labeled with brain derived neurotrophic factor when comparing control and glaucomatous retinas. Glial fibrillary acidic protein and vimentin expression in glial cells increased after one week of elevated IOP., Conclusions: After one week of elevated IOP and before the onset of RGC death, it was evident that inner retinal cells showed remarkable changes in their molecular expression.

Ciliary neurotrophic factor (CNTF) has been implicated in the survival and regeneration of adult rat retinal ganglion cells (RGCs). Expression of CNTF and its receptors, ciliary neurotrophic factor receptor alpha (CNTFRalpha) and leukemia inhibitory factor receptor (LIFR) was studied in normal, 6-h, 1-, and 3-day optic nerve transected retinas. Optic nerve transection was performed on the right eye in one group. In the other group, it was performed 5 days after intratectal Fluorogold injection. Retinas were studied using immunohistochemical labeling and Western blot analysis. Astrocytes were labeled with GFAP and Müller cells with glutamine synthetase. CNTF was observed in identifiable astrocytes and Müller cells, and its intensity increased in the experimental retinas. CNTFRalpha was localized on RGCs in experimental retinas. LIFR was localized on RGCs and Müller cells in normal and experimental retinas. CNTF and its receptors are differentially expressed after optic nerve transection and could help delay RGC death in such a stressful environment.

Following optic nerve transection, most of the retinal ganglion cells die. Their debris is promptly cleared by phagocytic cells. It is currently not known to what extent peripherally derived macrophages contribute to this activity. Using antibodies OX42 and ED-1, phagocytic cells were labeled in the retinas of optic nerve lesioned adult rats. To distinguish whether the cells were reactive microglial or macrophagic in origin, blood-borne monocytes were labeled with fluorescent microspheres while in the systemic circulation. Macrophages invaded the retina, but only in the nerve fiber layer, sparing the ganglion cell and other layers. These macrophages engulfed only the axonal debris from dying ganglion cells, not their degenerating cell bodies. These results indicate that although peripherally derived monocytic cells are recruited into the retrogradely degenerating retina, their role in clearing debris is limited to the optic fiber layer., (Copyright 1999 John Wiley & Sons, Inc.)

The retinal organization associated with the multiple optic papillae of the catfish Ictalurus punctatus was examined. In each retina from ten to 17 papillae form an oval ring (which is wider dorsoventrally than mediolaterally). The dorsalmost papilla in this ring lies at the center of the retina. In addition, up to seven small papillae lie within the ring. Bundles of fibers leave the neural retina via the papillae. These bundles remain separate as they pass through the distal portions of the neural retina and then merge before passing through the choroid. Bundles running through dorsal papillae receive fibers from a roughly wedge-shaped retinal area; bundles running through ventral papillae receive fibers from a small area of central retina and a disproportionately large area of peripheral retina. A band of high ganglion cell density was observed extending between the nasal and temporal poles of the retina. No correlation was found between the retinal areas contributing fibers to the bundles of axons emerging from individual papillae and the areas of high cell density. Furthermore, no correlation was found between the average area of the retinal ganglion cells and the ganglion cell density. From HRP preparations and axon counts we estimate that each retina of 95-mm catfish contains about 50,000 ganglion cells.

The order of production of retinal cells was studied in embryos of the goldfish, Carassius auratus, using 3H-thymidine autoradiography. Cell division ceases first in the neuroepithelium at the fundus of the eye between embryonic stages 19 and 20 and gradually becomes restricted to the retinal margin by stage 24. In the fundus the cells whose nuclei will reside in the inner layer of the retina stop dividing earlier than the cells whose nuclei will reside in the outer retinal layers. Thus the ganglion cells in the fundus of the retina are produced first and the receptors and horizontal cells last.

The retinal projections of the juvenile and adult channel catfish, Ictalurus (Ameiurus) punctatus, were studied by using horseradish peroxidase (HRP) and autoradiography. The contralateral optic tract sends fibers to the suprachiasmatic nucleus (SCN) and divides into lateral (LOT) and medial optic tracts (MOT). In the adult fish, the former is thicker than the latter, whereas in the juvenile form, the reverse is true. The MOT curves laterally and divides into eight to 15 medial fascicles of the optic tract (MFOT). The contralateral optic fibers project to the nucleus opticus dorsolateralis, nucleus of the posterior commissure, nucleus geniculatus lateralis, pretectal nuclear complex, nucleus corticalis, stratum fibrosum et griseum superficiale (SFGS), and a few optic fibers extend into the stratum griseum centrale. The tractus opticus accessorius arises from the posterodorsal margin of the LOT and extends ventromedially to project to the nucleus opticus accessorius. At the optic chiasm a few fibers do not decussate, and these fibers project to almost all ipsilateral sites similar to those of the contralateral side, including the optic tectum. The autoradiographic observations substantiated the analysis of optic fiber projections provided by the HRP technique.

Following optic nerve crush, the precise termination sites of regenerating goldfish optic axons may be influenced by the presence or absence of degenerating axonal debris from the previous projection. We investigated wheter tectal polarity reversal can be induced in the absence of axonal debris. The right optic tectum was denervated by contralateral eye removal. One year later, when no debris was present, a piece of the right tectum was rotated and innervation by the right eye was induced by removal of the left tectum. The new ipsilateral projection to the rotated region was correspondingly rotated. It is concluded that retention of tectal polarity is not dependent upon degenerating axonal debris.

The specification of central connexions of retinal ganglion cells was studied in Xenopus laevis. In one series of experiments, the right eye primordium was rotated 180 degrees at embryonic stages 24-32. In the other series, the left eye was transplanted into the right orbit, and vice versa, with either 0 degrees or 180 degrees rotation. After metamorphosis the visual projections from the operated eye to the contralateral optic tectum were mapped electrophysiologically and compared with the normal retinotectal map. In all cases the visual projection map was rotated through the same angle as was indicated by the position of the choroidal fissure. The left eye exchanged into the right orbit retained its original axes and projected to the contralateral tectum. These results suggest that retinal ganglion cell connexions are specified before stage 24.

The projections of horseradish peroxidase-filled axons from each quadrant of the retina were studied to determine whether retinal projections of goldfish are topographically organized in diencephalic target nuclei. A distinct topography of the dorsal, nasal, ventral and temporal retina exists in the lateral geniculate nucleus and in the dorsolateral optic nucleus of the thalamus. The projections of retinal quadrants show minimal spatial overlap in each of these nuclei. The suprachiasmatic nucleus of the hypothalamus is extensively innervated by ventral retinal fibers, whereas the nucleus is sparsely innervated by fibers from the other three retinal quadrants. A rudimentary topography also exists in the pretectum where the dorsal pretectal area receives projections primarily from the ventral retina and the ventral pretectal area receives projections mostly from the dorsal retina. These data show that retinal projections to some diencephalic nuclei are topographically organized.

The retinal projection to the ipsilateral optic tectum was examined first in normal channel catfish. After unilateral optic nerve section and horseradish peroxidase (HRP) application, a sparse retinal projection was seen in the ipsilateral optic tectum. When HRP was applied to the ipsilateral rostral tectum, labeled ganglion cells were observed in the ventrotemporal quadrant of the ipsilateral eye. Electrophysiological mapping of the ipsilateral projection confirmed that cells in the temporal retina projected to the rostral tectum. When albino catfish were examined using these techniques, the ipsilateral retinotectal projection was found to be much sparser than the ipsilateral projection of normal catfish.

In channel catfish the ganglion cell axons leave the retina via a ring of approximately 13 separate optic papillae. Each papilla serves an area of retina extending from the central zone of the retina to the periphery. Papillae located at a dorsal position in the ring serve exclusively dorsal retina. Ventrally located papillae, however, have an exaggerated peripheral retinal representation, so that they serve mostly ventral retina but also some areas of peripheral retina dorsal to the nasal and temporal poles. The ganglion cell axon bundles departing from the retina via individual papillae were labelled with horseradish peroxidase, and sections of the optic pathway were examined to reveal the topographic organization of the fibers. The topographic order of the optic nerve was dissimilar to that of cichlids and goldfish. Fibers from individual papillae remained together throughout the optic nerve. Close to the optic nerve head, the papillae were arranged as a continuum around the U-shaped optic nerve, without the discontinuity in the representation of the ventral retina seen in other fish. Fibers associated with the dorsal papillae were located at the tip of the caudolateral arm of the U, and fibers from ventral papillae were on the rostromedial arm. Fibers from nasally and temporally located papillae were found on the base of the U. By the level of the optic chiasm the U shape had flattened out but retained the relative ordering of the papillae. Rotation of the nerve as it became the optic tract brought the representation of the ventral papillae to the dorsal pole of the tract, and the dorsal papillae to the ventral tract. It was only in the optic tract that rearrangement of fibers became apparent. As described above, the axons of some ganglion cells in dorsal, peripheral retina left the retina and travelled through the optic nerve with axons from extreme ventral retina. In the optic tract, these dorsal fibers joined the main body of fibers from the dorsal retina. The significance of these observations for theories of fiber rearrangement is discussed.

In order to determine the topography of retinal axons in diencephalic nuclei of Ictalurus punctatus, horseradish peroxidase was applied to the optic papillae. The topography was found to be similar in all nuclei in that the terminal endings of ventral retinal axons innervated rostrodorsally whereas dorsal retinal axons innervated caudoventrally. In general, the retinal topography in the diencephalon corresponded with the topography of retinal axons in the optic tracts. These data suggest a relation between retinal axon ordering within a target and the pathway by which axons enter the target.

The winter flounder shifts the orinetation of its body 90 degrees at metamorphosis so that its left side is functionally ventral and its right side functionally dorsal. Concomitantly the left eye migrates onto the right side. The net result of these complex metamorphic changes is that the dorsoventral axes of the visual fields are perpendicular to the body rather than parallel as in most other teleosts. The developing flatfish may provide a resource for studying the formation of neural connections, for the change in orientation may necessitate some shift in connections in visuomotor pathways. As a baseline for developmental studies, we have established the retinotectal projection in adult winter flounder by means of anatomical tracing techniques (autoradiography and degeneration staining) and electrophysiological mapping techniques. The histological pattern of retinal afferents to the tectum is similar to that of other teleosts; afferents are confined to the superficial white and gray zone, with a few fibers coursing in the deep white zone. Electrophysiological mapping shows that the visuotectal projection is complete over the entire extent of the tectum, symmetrical for right and left fields and patterned normally.

Although several monoclonal antibodies (MAbs) have been reported that recognize antigens present in ganglion cells of the mammalian retina, these MAbs do not cross-react in the goldfish. In the current study we have identified a MAb that recognized a 14.4 kDa antigen that is present on the ganglion cells in the retinae of goldfish but is absent from the retinae of all other species that were tested. No other cell type in the goldfish retina or optic nerve was labeled with this MAb. Furthermore, the axons in the optic nerve, optic tract and the terminal layers in the optic tectum were labeled.

The present study was aimed at determining how transformations in fiber order establish a retinal topography in the optic tract of adult Carassius auratus. Horseradish peroxidase was applied to the optic nerve or retina, and the pathways of labeled axons originating from retinal annuli or wedges were analysed in reconstructed serial-sections and wholeamounts of the optic pathway. The age-related fiber order of the optic tract involves a rotation of the optic pathway that begins near the chiasm, continues through the optic tract as it wraps around the brain, and extends through the brachia. The relative order of laminae, in which each lamina is composed of age-related axons, is maintained in the optic pathway. The laminae add systematically onto the optic tract in a mediolateral direction with the oldest lamina forming the medial margin. Retinal sector order in the optic tract is established by the rearrangement of axons from each lamina. These rearrangements begin at the chiasm and, in part, involve transposition of axons originating from the ventrotemporal and dorsonasal sectors of the retina. The transformations achieve a fiber order in the optic tract that is appropriate for entry into the tectum. It is proposed that the final retinal topography of the optic tract is determined by the combined influences of selective affinities along the neural axis and substrate guidance mechanisms, the latter being mediated largely by the oldest axons of the fasciculus medialis tractus opticus.

Through anatomical and physiological studies of the regenerating retinotectal projection of goldfish, we sought to determine whether the establishment of a topographic projection is attained through a refinement of an initially less precise pattern of innervation. A 1-mm-wide mediolateral strip of caudal tectum was removed so that a small island of tectal tissue was spared at the caudal pole, and the contralateral nerve was either crushed (TIX) or left intact (TI). The presence of regenerated axons in the ablated zone and the reinnervation of the caudal island were assessed with anterograde and retrograde labeling methods in the following postoperative intervals: early, 20-50 days; middle, 50-110 days; and late, more than 170 days. The anterograde radioautographic method revealed that the appropriate layers of the tectal island became reinnervated by optic axons during the early period. During the middle and late periods, one to several large, discrete bundles bridging the lesion zone along the surface of exposed subtectal structures were readily identified both by radioautography and by anterograde or retrograde labeling following application of horseradish peroxidase to the transected optic nerve or tectal island, respectively. In contrast, the anterograde horseradish peroxidase method did not reveal axon bundles extending caudal to the half-tectum in the absence of a tectal island. Among TIX cases, retrograde horseradish peroxidase labeling of the contralateral nasal retina was more widespread in the middle period than in the late period, a result we interpret as reflecting an improvement in topographical precision with time. The area of retinal labeling among TIX cases in the late period was similar to that following caudal tectal injection in cases with simple nerve crush, although it was still elevated above normal control values. Physiological maps indicated a focal representation of the nasal retina in the tectal island in both periods and did not reveal a transient extreme convergence of retinal input. These findings are discussed in relation to Sperry's chemoaffinity theory.

Horseradish peroxidase was applied to lesions in the optic nerve of catfish (Ictalurus punctatus). The retinae were processed to reveal HRP-labelled ganglion cells. The histochemical techniques employed allowed fine details of the dendritic arbor to be resolved. Flat-mounted retinae were examined and the following characteristics were noted in individual ganglion cells: Soma area, shape, and depth; number and diameter of major dendrites; shape, area, and depth(s) within the inner plexiform layer (ipl) of the dendritic arbor; origin of the axon (from the soma or a dendrite). On the basis of these characteristics, eleven classes of ganglion cells were delineated: four classes of giant cells (G1-G4) and seven classes of smaller cells (S1-S7). G1 cells had dendrites arborizing in the most distal sublamina of the ipl. G1 cells in the dorsal retina had nasotemporally elongated dendritic arbors. G2 cells had dendrites in the proximal portion of the ipl. G3 cells were almost completely confined to a band running between the nasal and temporal retinal poles, through the center of the retina. In this location, the cells had dorsoventrally elongated dendritic arbors, which were bistratified in the ipl. G4 cells were displaced into the inner nuclear layer. S1 and S4 cells had axons arising from their somata, and dendrites arborizing in the distal and the proximal ipl, respectively. S2 cells were typified by their unstratified dendritic arbors. Similarly, S3 cells were characterised by their bistratified arbors. S5 cells arborized in the most proximal ipl sublamina. S6 cells were small ganglion cells with their somata lying in the inner nuclear layer. S7 cells tended to have complex dendritic arbors, and their axons arose from dendrites.

In teleosts, the fasciculus medialis tractus optici departs the optic tract and follows an aberrant path towards the midbrain tectum. To determine the retinal origin of the fascicle, horseradish peroxidase was injected into localized lesions of either the optic nerve or the retina. The fasciculus medialis axons of adult goldfish derive selectively from ganglion cells residing in the dorsotemporal portion of the central retina. The fasciculus medialis appears to be an integral part of the chronotopic ordering of fibers in the optic tract.