THE JOURNAL OF COMPARATIVE NEUROLOGY 294:418-430 (1990)

ANNE C. RUSOFF AND SHARON J. HAPNER Department of Biology, Montana State University, Bozeman, Montana 59717

ABSTRACT We have studied the position and numbers of retinopetal axons in the rainbow cichlid fish, Herobilapia multispinosa, to determine the response of related parts of the brain of fish to the continual addition of new neurons in the retina. The retinopetal axons were traced by using the retrograde tracers HRP and cobaltous lysine and an immunocytochemical probe, antibodies to FMRFamide, the molluscan cardioexcitatory peptide. One population of cells with retinopetal axons was found in the telencephalon (in the nucleus olfactoretinalis) and the other was scattered in the diencephalon. Some of the cells in the nucleus olfactoretinalis with retinopetal axons were FMRFamide positive; antibodies were used to trace the axons of these cells into the retina. All the retinopetal axons, from the nucleus olfactoretinalis and the diencephalon, were confined to the portion of the optic nerve that contains axons from the central retinal ganglion cells, that is, the oldest ganglion cells. This result suggests that the retinopetal axons grow into the optic nerve and retina early in the life of the fish, and no new ones are added later in life despite the extensive addition of cells in the retina. Counts of the cells in the nucleus olfactoretinalis that project to the retina in 3-month-old and adult fish support this interpretation. We conclude that retinopetal axons grow into the retina early in the life of the fish and respond to the formation of new retina by extending their arbors toward the new retina. K e y words: retinal efferents, centrifugal fibers

The retinotectal system of fish is noted for its ability to add new neurons into adult life (Muller, '52; Johns, '77; Meyer, '78). However, little is known about the adaptations that the rest of the brain makes to these changes in the retina and tectum. Presumably, the projections of the brain to the retina, referred to as retinopetal projections, centrifugal projections, or efferents to the retina, are affected by the growth of their target. This study was undertaken to determine how the efferent projection changes as the retina grows. Retinopetal projections have been found in a variety of fish, including both teleosts and elasmobranchs. For reviews of the species studied before 1986 see Uchiyama and Ito ('84) and Crapon de Caprona e t al. ('86). Since then retinopetal axons have been found in additional species: an anabantid fish, Colisa lalia (Oka et al.,'86); an anguillid fish, Anguilla rostrata (Grober et al., '87); and a channid fish, Channa micropeltes (von Bartheld and Meyer, '88). Often several regions of the brain project to the retina (Ebbesson and Meyer, '81; for a recent review, see von Bartheld and Meyer, '88). In those fish studied so far, the retinopetal axons project to the retina via the optic nerve and project across most of the retina from the optic disk toward the retinal margin. Since the retina grows a t its peripheral Q

1990 WILEY-LISS, INC.

margin as the fish grows, the efferent axons must change as the retina grows in order to innervate the newly formed retina. We began our attempt to understand these changes by looking at the position of the retinopetal axons in the optic nerve. The positions of retinofugal axons in the optic nerves of a cyprinid fish, the goldfish, and of several perciform fish have been extensively studied and are known to be highly organized. The nerve consists of layers of axons that grew out along slightly older axons. At the junction of the eye and optic nerve, ganglion cell axons in goldfish are arrayed with the oldest axons dorsally and the youngest axons ventrally with an age gradient between them (Rusoff and Easter, '80; Easter et al., '81). Although the optic nerve is ribbon-shaped rather than round in perciform fish, axons are arranged in it much as in the goldfish nerve-the oldest (and largest) axons are at one edge of the ribbon, and the youngest axons are a t the other edge with an age gradient between them (Scholes, '79). Since no one has noted a separate fascicle of axons projecting to the retina, we assumed that retinopetal axons are layered with retinofugal axons that were present in the optic nerve when the retinopetal axons grew into the Accepted November 28,1989.

retina. Therefore, to begin to understand how the brain deals with continuous addition of new neurons in the retina, we studied the position of the retinopetal axons in the optic nerve of a perciform fish, the rainbow cichlid (Herotilapia multispinosa), by using the tracers H R P and cobaltous lysine and immunocytochemistry. We found that the retinopetal axons are confined to the oldest region of the optic nerve, suggesting that all retinopetal axons grew out to the retina early in the life of the fish. The following paper (Rusoff and Hapner, '90) details the development of the retinopetal projection in the rainbow cichlid. Preliminary results from these experiments have been presented in abstract form (Rusoff, '86; Rusoff and Hapner, '87).

the tracer studies. The standard length of the 3-month-old fish was 2.6 cm 0.08 (mean SEM) and an average weight was 0.7 g; the average standard length of the 10-month-old fish was 4.6 cm and their weight varied from 2.9 to 4.8 g; the standard length of the adult fish was 4.5 cm i 0.33 (mean i SEM) and their weight varied from 15 to 25 g, dependent on age and sex. Two additional fish were used as controls for possible transport of HRP by nerves innervating the extraocular muscles. One optic nerve in each of these fish was exposed as if it was going to be cut; however, the optic nerve and eye were not damaged. The orbit was packed with gel-foam soaked in HRP. After a suitable survival time, each brain was prepared as described above.

MATERIALS AND METHODS Rainbow cichlids, Herotilapia multispinosa, (Order Perciformes, Family Cichlidae) raised in our fish-breeding facility were used for all experiments.

Immunocytochemistry

Since some of the cells in the nucleus olfactoretinalis that project to the retina in other teleosts are immunoreactive for luteinizing hormone releasing hormone (LHRH) (Munz et Tracer studies al., '81, '82) and for the molluscan cardioexcitatory peptide Either horseradish peroxidase (HRP) or cobaltous lysine FMRFamide (Stell et al., '84; Bonn and Konig, '88), we used (Springer and Prokosch, '82) was used to fill both the axons antibodies to LHRH and FMRFamide to visualize this of retinopetal cells in the retina and their cell bodies population of cells and their axons in the brain, optic tract elsewhere in the brain. and nerve, and retina. Prior to application of either tracer, each fish was anestheSeven adult rainbow cichlid fish were used for the LHRHtized with 0.1 % MS222 (tricaine methanesulfonate). Then immunoreactivity study and 18 three-month-old t o adult the temporal conjunctiva was cut and the eye rotated fish were used for the FMRFamide-immunoreactivity study. nasalward until the optic nerve was visible. The connective Fish were perfused with washing solution and then with 4% tissue sheath encircling the optic nerve was cut open and paraformaldehyde plus 3% sucrose in 0.05 M phosphate either a portion of the optic nerve or the entire nerve was buffer. Their brains, optic nerves and tracts, and retinae cut; then either a crystal of cobaltous lysine made as were removed and soaked an additional 2 hours in the described in Springer and Prokosch ('82) and dried or a fixative. The tissue was then rinsed in phosphate buffer and small piece of gel-foam soaked in H R P and dried was placed cryoprotected in 30% sucrose in phosphate buffer. Some on the cut (Rusoff, '84). Fish survived 1to 2 days, depending retinae were prepared as whole-mounts (Rusoff and Easter, '80) as were some optic nerves. T o whole-mount an optic on their size, and were then reanesthetized. HRP tracer studies. Fish were perfused first with a nerve the connective tissue sheath surrounding it was washing solution of 1% sodium nitrite, 0.8% sucrose, 0.8% removed and the ribbon-shaped nerve allowed to unfold; it sodium chloride, and 0.4% dextrose in phosphate buffer and was then flattened onto a gelatinized slide. Other tissue was then with 2% glutaraldehyde in phosphate buffer. The sectioned at 10 to 40 pm in a crgostat and placed directly on brains, optic tracts, optic nerves, and retinae were dissected freshly prepared subbed slides. The slides were then stored out of the head intact and fixed further in the glutaralde- in a refrigerator for a maximum of 2 days. The slides were bathed in each of the solutions required to hyde fixative. Retinae from 60 fish were prepared as whole mounts (Rusoff and Easter, '80); these retinae were also reveal immunoreactivity in the tissue. The peroxidaseused for other studies. Tissue to be sectioned was cryopro- antiperoxidase procedure of Sternberger ('74) was used. All tected by immersion in 30% sucrose in phosphate buffer washes were phosphate-buffered saline (PBS) plus 0.3% until it sank, sectioned in a cryostat at 10-50 pm, and placed Triton X-100, unless otherwise stated. Sections were incudirectly on slides. The sections and retinae were processed bated in wash plus 10% DMSO for 30 minutes, washed, with o-dianisidine to reveal the H R P (Easter et al., %1), incubated in normal goat serum for 30 minutes, and incudehydrated in graded alcohols, and coverslipped with Per- bated overnight in the primary antibody at room temperature. mount (Fisher). Several different antibodies were used. Antibodies to Cobaltous lysine tracer studies. Tissue was prepared basically as described by Bazer and Ebbesson ('84). Fish mammalian LHRH (Chemicon),antibodies to salmon LHRH were perfused first with washing solution (above) and then (prepared by N. Sherwood, a gift of Wm. Stell), and with 2.5 5% ammonium sulfide solution; their brains, optic antibodies to FMRFamide (Immunonuclear and/or Camnerves and tracts, and retinae were then removed intact, bridge Research Biologicals) were used. Antibodies to mamsoaked an additional 10 minutes in the ammonium sulfide malian LHRH were used at a dilution of 1500 or 1:1,000. solution, rinsed in phosphate buffer, and fixed in 2 % The antiserum to salmon LHRH was used a t a dilution of glutaraldehyde in phosphate buffer. This tissue was cryopro- 1:500. The antibodies to FMRFamide from the two sources tected and sectioned as described above; the cobalt was were used either alone a t a dilution of 1:1,000 or mixed with intensified as described by Bazer and Ebbesson ('84). Sec- that from Immunonuclear used a t a dilution of 1:1,000 and tions were counterstained with cresyl violet, dehydrated, that from Cambridge used at 1:5,000. This mixture yielded and the slides coverslipped. optimal staining versus background. Control slides were l'wenty-one 3-month-old fish, three 10-month-old fish incubated with normal rabbit serum at a dilution of 1:1,000. (not yet sexually mature), and nineteen adults were used for Slides were then washed, incubated with goat antirabbit

A.C. RUSOFF AND S.J. HAPNER

420

Fig. 1. A A photograph of axons (solid arrow) from retinal ganglion cells crossing part of a flat-mounted retina. The axons are filled with HRP reaction product; the HRP was introduced into the axons through a cut in the dorsal optic nerve. Peripheral retina is at the top of the photograph and central retina is a t the bottom. Dendrites of central ganglion cells also filled with the reaction product are visible in the lower

right corner (open arrow). B A photograph of the identical region of retina; however, the focus is a t the level of the inner plexiform layer. The visible processes are HRP-filled retinopetal axons or “efferents.” They course across the retina in a seemingly random array. The scale bar indicates 100 pm and applies to both A and B.

serum for 1 hour, washed, incubated with peroxidaseantiperoxidase complex (DAKO) for 1hour, washed, washed in 0.1 M phosphate buffer, and processed to reveal the peroxidase using diaminobenzidine as the chromogen and glucose-glucose oxidase as the hydrogen peroxide source (Itoh et al., ’79). The tissue was lightly counterstained with cresyl violet, dehydrated, cleared with xylene, and coverslipped with Permount. One optic nerve in each of two additional fish was crushed one week prior to preparation of the retinae for immunocytochemistry. Both retinae of each of these fish were treated to demonstrate FMRFamide immunoreactivity, as described above.

retinal ganglion cells cross the whole-mounted retina essentially parallel to each other. In contrast, as shown in Figure lB, the retinopetal axons form an intricate network in the retina; branches from one axon diverge greatly from each other and may end hundreds of micrometers apart. The retinopetal axons often have a beaded appearance and are visible in approximately the same focal plane as displaced ganglion cells and their dendrites. The position of the retinopetal axons can be more accurately assessed in crosssections of the retina in which only central ganglion cells have been back-filled with tracer. If all the ganglion cells are filled with tracer, it is impossible to separate the retinopetal axons from the dendrites of ganglion cells; however, if only the central retinal ganglion cells are filled with tracer, filled processes are visible peripheral to the extent of the dendrites of displaced ganglion cells (Fig. 2). These processes are near the interface between the inner plexiform layer and the inner nuclear layer and maintain this position for hundreds of micrometers across the retina. Since these filled

RESULTS The retinal location and appearance of retinopetal axons (“efferents”) and retinofugal axons (from retinal ganglion cells) are shown in Figures 1and 2. In Figure 1A axons from

ORGANIZATION I N THE OPTIC NERVE

Fig. 2. A photograph of a 40 pm-thick transverse section through the retina of an old adult fish (over 3 years old). The optic disk is to the left of the region shown and the peripheral margin is to the right. HRP was introduced into the dorsal part of the optic nerve. T h e curved arrow marks the most peripheral ganglion cell filled with HRP reaction product; the open arrow points to the most peripheral processes of displaced ganglion cells; t h e solid arrows point to retinopetal fibers. The scale bar indicates 50 pm. GCF, ganglion cell fiber layer; GCL, ganglion cell layer; INL, inner nuclear layer; IPL, inner plexiform layer; PR, photoreceptors.

processes never approach a cell body of either a normally positioned or displaced ganglion cell, are far from the nearest filled ganglion cell body, and never enter any of the other layers of the inner plexiform layer, we assume that they are part of the terminal arbors of retinopetal axons. In the whole-mounted retinae ganglion cells in different regions were filled with H R P reaction product because a different portion of the optic nerve had been exposed to HRP in each fish. Retinopetal axons were filled with HRP reaction product in only those retinae in which the most central ganglion cells (that is, those with axons in the dorsal portion of the optic nerve) were filled with reaction product. These retinopetal axons were observed in almost all of the retinae with such a fill and coursed across the extent of the retina even if only ganglion cells immediately surrounding the optic disc were filled with HRP reaction product. Since the ribbon-shaped optic nerve of the rainbow cichlid has the same segregation of axons by age typical of other perciform fish (Scholes, '79; Rusoff, '84), this result strongly suggests that the axons of the retinopetal cells are segregated to the portion of the nerve that contains the axons from the central (oldest) retinal ganglion cells. We then used the tracers H R P and cobaltous lysine to determine the source of the retinopetal axons. When either tracer was applied to the dorsal portion of the optic nerve or the entire optic nerve, we found a cluster of axons filled with reaction product that left the contralateral optic tract a t its most rostra1 contact with the brain, ran rostrally through the brain, and endcd in a cluster of cell bodies ventromedially near the transition between the olfactory bulb and telencephalon. (In these fish the olfactory bulb is sessile, that is, directly attached to the telencephalon.) Figure 3 illustrates this region in parasagittal section; Figure 4B is a more highly magnified view of the cell bodies that send axons to the retina. This region has been identified as the

42 1

source of some of the retinopetal axons in other cichlid fish ( M u m and Claas, '81; Ebbesson and Meyer, '81; Crapon de Caprona and Fritzsch, '83; Springer and Mednick, '85) and has, for this reason, been named the nucleus olfactoretinalis (Miinz et al., '81). Figure 4A shows axons from cells in the nucleus olfactoretinalis entering the optic tract. In all cases in which a tracer was applied to at least the dorsal half of the optic nerve, many, but not all, of the cells in the nucleus olfactoretinalis were filled with tracer. No difference was noted in the number of cells filled if the entire optic nerve was filled with tracer. The diameters of the retrogradely filled cells were measured in coronal sections and ranged from 6 to 16 pm. In one fish that was sectioned sagittally, the cells appeared rostrocaudally elongated; the longest dimension of the cells cut in this plane ranged from 6 to 20 pm. When assessed visually, the cells appeared to be divisible into large and small cells (Fig. 4B), as has been reported in other fish (Munz et al., '82). However, histograms of the diameters of the cells in each fish were generally unimodal and appeared very similar to those illustrated by Springer and Mednick ('85) for another cichlid fish (Astronotus ocellatus). In initial experiments using H R P as the tracer and applying it to a cut in the dorsal optic nerve of adult rainbow cichlids more than a year old, only the contralateral nucleus olfactoretinalis obviously contained cell bodies filled with reaction product (Rusoff, '86). However, when HRP was applied in the same way to the optic nerve of juvenile rainbow cichlids (3 months old), cells in both the contralatera1 nucleus olfactoretinalis and diencephalon were filled with the reaction product. Figure 5 shows filled cells in a transverse section through the diencephalon at the level of the habenula. Figure 5A shows the general region and Figure 5B,C shows two of the clusters of cells filled with reaction product. These cells did not form one clear nucleus but were scattered in several retinorecipient regions medial and ventral to the tectum. Nuclei in the diencephalon of the rainbow cichlid have not been mapped, and it is quite difficult to extrapolate between different fish until the anatomical connections of the various nuclei are known. Therefore, we will describe the locations of these cells by the branches of the optic tract with which they are associated and nuclei known to he in that vicinity in goldfish (Braford and Northcutt, '83) and in another cichlid (Fernald and Shelton, '85). Filled cells were found in association with the axial optic tract in the region of the nucleus ventrolateralis (Fig. 7B); they were also found in association with a large bundle off the ventrolateral optic tract in the region of the pretectal nuclei (Fig. 7C). and a few were found in association with the accessory optic tract near its nucleus. No filled cell bodies were seen in the tectum or in the ipsilateral half of the brain. Brains from the control fish in which only the nerves to the extraocular muscles were exposed to the tracer had no filled cells in the diencephalon or olfactory region. The presence of filled cells in an additional part of the brain in juvenile fish suggested either that the cells that send axons to the retina change with age or that the HRP had failed to fill the diencephalic cells adequately in the adult fish. Cobaltous lysine was used as an alternative tracer to attempt to decide between these possibilities. When the dorsal optic nerve of an adult fish was cut and cobaltous lysine was inserted into the cut in place of HRP, cobaltfilled cell bodies were found in both the contralateral

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A.C. RUSOFF AND S.J. HAPNER

Fig. 3. A photograph of a 50 wm-thick parasagittal section through the telencephalon of an old adult fish. Rostra1 is to the right and dorsal is up. T h e dorsal part of one optic nerve of this fish was filled with HRP.

The regions enclosed in boxes are shown in higher magnification in Figure 4. The scale bar indicates 300 pm.0. BULB. olfactory bulb; OT, optic tract; TEC, optic tectum; TEL, telencephalon.

nucleus olfactoretinalis and diencephalon. The distribution of cobalt-filled cell bodies in the brains of the adult fish was virtually indistinguishable from the distribution of HRPfilled cell bodies in the brains of the juvenile fish. We conclude, therefore, that the retinopetal projection does not change between juvenile and adult rainbow cichlids but that the HRP is not as useful a tracer for diencephalic efferent cells as is the cobaltous lysine. Since the cobaltous lysine filled more retinopetal cells in adult brains than the HRP did, we used it to determine if there were additional retinopetal axons in the ventral portion of the optic nerve. We compared the distribution of cells filled retrogradely from the dorsal portion of the optic nerve and from the entire nerve. The distribution and number of retrogradely filled cells appeared the same in

both cases. Therefore, we conclude that all the retinopetal axons are in the dorsal portion of the optic nerve. T o verify that all the efferents were present early in the life of the fish, we counted the total number of efferents in both juvenile and adult fish. For these counts we used fish in which the entire optic nerve had been cut and filled with tracer to prevent missing part of the population of retinopeta1 cells. The diencephalic efferents proved difficult to count both because they were often partially hidden among axon terminals, especially in the fish in which HRP was used as a tracer, and because the cobalt precipitate often filled “cells” that had no evidence of axons or dendrites. Therefore, in the preparations visualized with cobalt, only cells with obvious processes were counted. In addition, the filled cells were spread unevenly through the diencephalon so that

ORGANIZATION IN THE OPTIC NERVE

423

Fig. 4. A A more highly magnified view of the region shown in the left box on Figure 3. Solid arrows indicate axons filled with HRP reaction product coursing through the ventral telencephalon and beginning to curve toward the optic tract (lower left); the curved arrow indicates filled axons entering the edge of the optic tract. T h e scale bar

indicates 50 pm. B: A more highly magnified view of the region shown in the right box on Figure 3. Cells in the nucleus olfactoretinalis are filled with HRP reaction product. T h e arrow indicates axons from some of these cells that project caudally through the telencephalon before entering the optic nerve. The scale bar indicates 20 pm.

inability to count cells in a few sections due to loss or folding of the sections might drastically change the results. Only results from fish in which all sections were present and countable are included in Table 1. This table gives both the number of cells counted and a number corrected for counting the same cell in two sections (Abercrombie, '46). This correction overcorrects for counting the same cell twice if the cells are not evenly distributed throughout the sections. Therefore, the actual number of retinopetal cells is between the counted and corrected number. We consider the counts of retinopetal cells in the diencephalon to he rough estimates. Table 1shows that there were approximately twice as many retinopetal cells in the diencephalon of the adult fish as in the 3 month-old fish. However, the corrected count from one of the larger three month-old fish overlaps with that from an above average-sized adult fish. By the time the fish are 10 months old, the numbers fall within the adult ranges. Thus, the population of retinopetal cells in the diencephalon matures between 3 and 10 months of age. Retinopetal cells in the nucleus olfactoretinalis were easier to count since there were no filled axon terminals from retinal ganglion cells surrounding the cells or their axons. When possible, we counted both the filled cell bodies in the nucleus olfactoretinalis and their axons in a more caudal section in which they were cut in cross-section. As shown in Table 1, the number of retinopetal cells in the nucleus olfactoretinalis is essentially identical with that in adults by the time fish are three months old.

Since there are two very different sources of retinopetal axons in these fish, one would like to be able to separate their axons in the optic nerve and retina to determine if they distribute separately in the nerve and across the retina. In some species of fish some of the cells in the nucleus olfactoretinalis that project to the retina are immunoreactive for luteinizing hormone releasing hormone (LHRH) (Munz et al., '82; Stell et al., '84) and for the molluscan cardioexcitatory peptide FMRFamide (Stell et al., '84; Bonn and Konig, '88). Therefore, we used antibodies to these compounds to allow us to trace this population of efferents separately. (Unfortunately, no procedure has yet been devised to selectively label the diencephalic efferents without also labeling axons of retinal ganglion cells.) The commercially available antiserum to LHRH that we tried (Chemicon) was made against a mammalian LHRH (rabbit) and did not specifically label any cells or axons in the nucleus olfactoretinalis in the rainbow cichlid. (Later Wm. Stell gave us an aliquot of antibodies made by N. Sherwood against salmon LHRH. This antibody was immunoreactive with cells in the nucleus olfactoretinalis and their axons in the retina, indicating that the antigen in these cells is similar to that found in other fish [Stell et al., '841.) Fortunately, commercially available antibodies to FMRFamide (Immunonuclear and Cambridge Research Biochemicals) were strongly immunoreactive with cells in the nucleus olfactoretinalis and their axons in the rainbow cichlid fish. We used this immunoreactivity to visualize the cells so that

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Fig. 5. A A photograph of a 40 pm-thick transverse section through the optic tectum (upper left), optic tract (lower right) and diencephalon (center) of a young adult fish. Dorsal is up and the midline is to the right. HRP was introduced into the dorsal third of the optic nerve. Axons filled with HRP-reaction product are visible in the optic tract, tectum, and diencephalon. The areas enclosed by the boxes are shown a t higher

magnification in B and C. Scale bar indicates 200 wm. B: A more highly magnified view of the region in the box a t the right in A. Cells filled with HRP reaction product are evident. This region is part of the nucleus ventrolateralis. Scale bar indicates 20 pm. C: A more highly magnified view of the region in the box a t the left in A. Filled cells are also visible in this region that is part of a pretectal nucleus. Scale bar indicates 20 pm.

we could trace their processes through the telencephalon into the optic tract and nerve and to their terminals in the retina. (No one has isolated the antigen with which FMRFamide reacts in fish. However, a family of FMRFamide-like peptides has been found in the brains of other vertebrates (Yang et al., '85);the amidated C-terminus of these peptides reacts strongly with antisera to FMRFamide; we assume that there is a similar FMRFamide-like peptide in these cells; we have simply utilized this immunoreactivity to trace these processes without interference from ganglion cell axons since they are not immunoreactive for FMRFamide.) Figure 6 shows FMRFamide-immunoreactive (FMRFamide-ir) cells and processes in the telencephalon. In this parasagittal section cells were so intensely immunoreactive that individual cells and axons can not be discriminated; instead a dense cluster of cells in the nucleus and a thick

cluster of axons leaving it are visible (Fig. 6B). The intense immunoreactivity was present in the nucleus olfactorctinalis regardless of the plane in which the brain was sectioned; individual cells could not be isolated well enough to count them or measure their size. However, not every cell in the nucleus olfactoretinalis was immunoreactive. The inability to visualize clearly individual immunoreactive cells made it impossible to compare the characteristics of immunoreactive versus non-immunoreactive cells. The cluster of FMRFamide-ir axons passed caudally through the telencephalon before dispersing into the caudal brain. Individual processes entered the optic tract a t its extreme margin and formed a cluster a t one edge of the optic tract and nerve. This cluster was visible either in cross section or in whole mounts of the optic nerve (Figs. 7,8). In cross sections the FMRFamide-ir processes were always

ORGANIZATION I N THE OPTIC NERVE

425

Fig. 6. A A photograph of a 20 pm-thick parasagittal section through the telencephalon of a 3-month-old fish. Rostra1 is to the left and up and caudal is to the right and down. This section was reacted to reveal FMRFamide immunoreactivity. The solid arrow points to FMRFamide-ir cells and processes in the nucleus olfactoretinalis. The open arrow points to a FMRFamide-ir process. Scale bar indicates 200 km. OB, olfactory bulb; OT, optic tract; TEL, telencephalon. B: A more highly magnified view of the FMRFamide-ir cells and processes in the nucleus olfactoretinalis. The open arrow points to individual bundles of immunoreactive processes that have separated from the heavy mass of immunoreactivity. Scale bar indicates 20 pm. TABLE 1. Number of CeUs in the Brain Filled With Tracer From Optic Nerve' NOR

Diencephalic cells

Cells No. of fish

Axons

Counted

COunted

Corrected

115'

301 t 30" (217351)

223 + 36 (141-282)

71 * 6' (67-93)

196 + 326 (125-257)

136+30 (79-177)

Correded

Adults 11

3-monthold fish 10 10-month-& fish 2

'Values are the mew plus or minus the standarderrur of the mean. Numbers in parenthews indiicalr the range of values.NOR 2Valuefrom 1fish. 'Values from 4 fiah. 'Values from 4 fish. 'Values from 9 fish. 'Value from 3 fish.

confined to the edge of the ribbon-shaped nerve that contains the largest retinofugal axons. These are the oldest ganglion cell axons and originate in central retina (Scholes, '79). We usually saw 8-10 immunoreactive axons but seldom more than that either in cross-sections of the optic nerve or tract or in whole-mounts of the nerve. FMRFamide-ir axons were never seen throughout the rest of the expanse of the nerve or tract nor were they ever observed entering the optic tract from any other source than the nucleus olfactoretinalis.

268

175

(232-303)

(16M86)

= nucleusdfsctmretinah.

Figure 9 shows a FMRFamide-ir process in the retina. In the adult fish these processes were confined to the outer boundary of the inner plexiform layer (close to the amacrine cell bodies). Compare the FMRFamide-ir process shown in Figure 9 with the HRP-filled processes indicated by the solid arrows in Figure 2. In young fry the FMRFamide-ir processes were visible entering the retina via the optic disc, penetrating the ganglion cell layer, and crossing the inner plexiform layer before coursing through the inner plexiform layer close to the amacrine cell bodies (Rusoff and Hapner,

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A.C. RUSOFF AND S.J. HAPNER

Fig. 7. A photomosaic of a flat-mount of one optic nerve from a 3-month-old fish. T h e connective tissue sheath around the nerve was removed and the ribbon-shaped nerve was flattened onto the slide and then treated to reveal FMRFamide immunoreactivity. T h e caudal (tectal) end of the nerve is u p and the rostra1 (retinal) end of the nerve is

down. The open arrows indicate the two margins of the nerve. [The right margin of the nerve is folded slightly and, therefore, is not a t the right margin of t h e picture.) T h e solid arrows indicate FMRFamide-ir axon8 near the right margin of the nerve. All other dark profiles are vascular in origin. Scale bar indicates 200 pm.

'90). However, we were unable to see this transition in older fish. With some lots of antibodies, immunoreactive processes also appeared in the inner plexiform layer near the ganglion cell bodies. However, we saw no connections between these processes and those near the amacrine cells, and the processes near the ganglion cell bodies were still immunoreactive 1week after optic nerve crush while the processes near the amacrine cells were no longer FMRFamide-ir. We conclude that the immunoreactive processes near the ganglion cell bodies are not related to the retinopetal fibers and probably reflect a contaminant in certain lots of antisera (Wm. Stell, personal communication). In the white perch, Zucker and Dowling ('87) found branches of centrifugal fibers ascending toward the outer plexifarm layer. We did not observe any such branches, although we did not search systematically for them. Other regions of the brain also contain cells and fibers that are intensely FMRFamide-ir, as also noted recently by Bonn and Konig ('88) in a cyprinidontoform fish, Xenotoca eisenii. There are many FMRFamide-ir fibers in other parts of the telencephalon; these may also originate from the nucleus olfactoretinalis as no other immunoreactive cell bodies are visible in the telencephalon. Several of the tectal

layers also contain fibers that are FMRFamide-ir; the source of these fibers is not clear, as no FMRFamide-ir cells were visible in the tectum. The only other intensely FMRFamideir cells that we have seen in the adult fish are large cells clustered in the dorsal tegmentum. These cells and their projections will be described in a later paper. In addition, in young fry the medulla contains cells that are FMRFamide-ir and send long axons rostrally into the brainstem and caudally into the spinal cord (Rusoff, unpublished observations); we have not yet seen these cell bodies in adult fish, although there are many longitudinally oriented FMRFamide-ir fibers in the brainstem. Since retinapetal fibers have been hypothesized to affect circadian rhythms and/or reproduction, we compared our ability to find FMRFamide-ir cells and fibers in this fish to the sex of each fish, the time of year it was killed and the time of day when it was killed. We obtained good FMRFamide-immunoreactivity in both males and females, in fish killed in all four seasons and in fish killed from 7:30 A.M. to 1O:OO P.M. In short, we found no variability in FMRFamideimmunoreactivity between the sexes or the time of year or day when they were killed.

ORGANIZATION I N THE OPTIC NERVE

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Fig. 8. A more highly magnified view of the right edge of the nerve shown in Figure 7. The arrow points to FMRFamide-ir axons near the margin. Scale bar indicates 100 pm.

DISCUSSION These experiments provide evidence that retinopetal axons exist in the rainbow cichlid fish, Herotilapia multispinosa, as in many other fish. The sources of retinopetal axons were determined using both HRP and cobaltous lysine as retrograde tracers. (We attempted to trace these axons anterogradely from the nucleus olfactoretinalis to the retina since this is the definitive experiment. However, fish did not survive long enough for transport of the tracer after the devastating surgery required to reach the brain in a fish that has massive cranial musculature. This experiment has been done successfully in the goldfish, a fish with almost no cranial musculature and with its olfactory bulbs separated from the rest of the brain [Springer, '831. Retrograde tracing has been considered sufficient evidence in other fish.) The efferents come from two major sources: the transition region between the olfactory bulb and the telencephalon (nucleus olfactoretinalis) and the diencephalon. These sources of retinopetal cells are similar to those found in other cichlid fish (Munz and Claas, '81; Springer and Mednick, '85). Although earlier workers report the presence of retinopetal cells in other regions of the fish brain (reviewed in Springer and Mednick, '85), most recent reports from both cichlid and non-cichlid fish find a source of retinopetal axons in the telencephalon and many find them in the diencephalon. The telencephalic source is given a variety of names depending on the species but is often called the nucleus olfactoretinalis and is probably homologous with the nervus terminalis (Ebbesson and Meyer, '81; Munz and Claas, '81; Munz et al., '82; Demski and Northcutt, '83; Crapon de Caprona and Fritzsch, '83; Springer, '83; Ito et al.,

Fig. 9. A photograph of a 40 pm-thick transverse section through the retina of an adult fish. This section was reacted to reveal FMRFamide immunoreactivity. The arrow indicates a FMRFamide-ir axon. Scale bar indicates 30 pm. GCF, ganglion cell fiber layer; GCL, ganglion cell layer; INL, inner nuclear layer; IPL, inner plexiform layer.

'84; Stell et al., '84; Springer and Mednick, '85; Matsutani et al., '86). Although the reports of retinopetal cells in the diencephalon differ, much of the difference is in naming rather than in the real position of the cells in the diencephalon. We found retinopetal cells associated with the axial optic tract in the region of the nucleus ventrolateralis (Braford and Northcutt, '83), associated with a large branch off the ventrolateral optic tract in the region of the pretectal nuclei, and a few associated with the accessory optic tract. Munz and Claas ('81) report retinopetal cells in the pretectal area of eight species of cichlids; their figures do not contain sufficient landmarks to allow comparison with our sections. Ebbesson and Meyer ('81) found retinopetal cells in the dorsomedial optic nucleus and the pretectal complex, as well as in other locations. Their dorsomedial optic nucleus is in

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the location that we have called nucleus ventrolateralis. Springer and Mednick ('85) regarded the diencephalic source of retinopetal cells in another cichlid as one U-shaped nucleus that they called the nucleus thalamoretinalis. Fritzsch et al. ('87) have also used this nomenclature in describing the source of retinopetal cells in another cichlid. Comparison of our sections with the figures in Springer and Mednick ('85) and Fritzsch et al. ('87) suggests that the retinopetal cells are in the same locations in these various cichlid fish; only the names for the locations vary. Comparison of the diencephalic sources of retinopetal cells in more distantly related fish suggests more variability. Retinopetal cells in a channiform fish are confined to a discrete nucleus called the nucleus thalamoretinalis (von Bartheld and Meyer, '88); this nucleus is near the pretectal nuclei and may be a smaller version of the retinopetal cell source near the pretectal nuclei in cichlids. Retinopetal cells in a balistid and a pantodontid fish are found closely associated with the optic tract (Uchiyama et al., '81; Gerwerzhagen et al., '82). These cells have been extensively characterized by Uchiyama and colleagues. They describe a well-defined preoptic retinopetal nucleus. The cells in this nucleus are very different from the retinopetal cells seen in cichlids both in appearance and in location: rostrally, the cells of the preoptic nucleus are small, round, tightly clustered, and closely associated with the optic tract before it begins to bifurcate (Uchiyama et al., '81); the retinopetal cells in the diencephalon of cichlids are large, of irregular morphology, and scattered along the course of parts of the optic tract. However, the preoptic retinopetal nucleus extends as far caudally as the posterior commissure and in its caudal portion is located near the pretectal nuclei (Uchiyama et al., '88a), as in cichlid fish. It is possible that the cells that we have described as near the pretectal nuclei are actually in a preoptic retinopetal nucleus that is much less elaborate than the homologous nucleus in balistid fish. The problem of naming and homology in the diencephalon can not be resolved until the locations of retinopetal cells are plotted with respect to cells in the various nuclei and the anatomical connections of those nuclei are analyzed. Such a study exceeds the scope of the present work. Another difference between the results presented here and the results of others is that some other workers find a small ipsilateral projection from these nuclei to the retina. When present in cichlid fish, the number of cells reported to project to the ipsilateral retina is very small; Springer and Mednick ('85), for example, found only six cells in the ipsilateral nucleus olfactoretinalis that projected to the retina. It is possible that the rainbow cichlid differs slightly from some other cichlid fish and does not have any ipsilatera1 cells that project to the retina; it is also possible that a few such cells have escaped our attention. The two populations of cells with retinopetal axons are in quite different anatomical locations and, presumably, are physiologically different. Many of the cells in the diencephalon that send axons to the retina are themselves surrounded by the axons and terminals of retinal ganglion cells and, thus, in a position to be affected by retinal inputs. In contrast, we did not see any filled axons terminating in the nucleus olfactoretinalis after introducing the tracer into the optic nerve of either adult or developing fish. In addition, the relative agreement between the number of axons counted caudal to the nucleus olfactoretinalis and the number of cell bodies in the nucleus indicates that there are few, if any, axons from retinal cells projecting to the nucleus olfactoret-

A.C. RUSOFF AND S.J. HAPNER inalis. Therefore, the cells in the nucleus olfactoretinalis that project to the retina probably do not receive direct visual input. Thus, these two different sources of efferents themselves probably receive different inputs and present different outputs to the retina. (See Northcutt and Wullimann "881 for a different interpretation; they hypothesize that all the retinopetal cells may be derived from cells of the nervus terminalis, some of which migrated into the diencephalon. The cells might then function in isolation from the retinorecipient regions near them. More work will be required to determine the derivation and physiological properties of these cells.) The immunocytochemistry allowed us to begin to further subdivide the populations of retinopetal cells. Antibodies to FMRFamide demonstrated a subgroup of the retinopetal axons in the nucleus olfactoretinalis. We were able to use this immunoreactivity to trace their axons to the retina. A similar result has been reported previously for the goldfish, a species of the superorder Ostariophysi (Stell et al., '84), and for the cyprinidontoform fish Xenotoca eisenii, a species of the superorder Atherinomorpha (Bonn and Konig, '88). Our experiments extend these results to include a species in the superorder Acanthopterygii. (FMRFamide immunoreactivity has also been found in another Acanthopterygian fish, the whitespotted greenling [Uchiyama, personal communication].) Since FMRFamide-ir retinopeta1 fibers have recently been reported in frogs (WirsigWiechmann and Basinger, '88; Uchiyama et al., '88b) as well as in these various fish, it seems likely that FMRFamide-ir retinal efferents are widely distributed in lower vertebrates. Much of the criss-crossing of efferents on the retina (Fig. 1B) may reflect the distribution of efferents with different functions across the entire retina. The presence of FMRFamide-immunoreactivity in some of the cells that project from the nucleus olfactoretinalis to the retina will allow us to determine their specific pattern of innervation of the retina. Zucker and Dowling ('87) have already begun to exploit this system. Unfortunately, we do not yet have specific markers for the other subpopulations of retinopetal axons. Determination of their specific synaptic contacts in the retina and unraveling their overlapping pattern on the retina will have to wait. The major thrust of these experiments was to determine how nuclei that are affected by changes in the number of cells in the retina cope with the changes. We present evidence here that retinopetal cells are born and send their axons to the retina early in the life of the fish and do not add new neurons to compensate for the growth of the retina. First, all the retinopetal axons are confined to the dorsal region of the optic nerve that contains the axons of only the oldest retinal ganglion cells. Second, the number of cells that project to the retina from the nucleus olfactoretinalis in the three month old fish is the same as in the adult fish, despite the large increase in size after 3 months of age. The number of cells found in the diencephalon appears to be approaching maturation a t 3 months of age and to be a t the adult value before the fish reaches 10 months of age, when it still is much smaller than an adult fish. Third, the FMRFamide-ir axons from the nucleus olfactoretinalis are found a t the margin of the optic tract and nerve, indicating that they grew through the tract and nerve to the retina very early in the life of the fish. Fourth, the retinopetal axons are typically seen a t the boundary between the inner plexiform and inner nuclear layers. Axons may be traced a t this boundary for long distances across the retina. They are only

ORGANIZATION IN THE OPTIC NERVE seen to cross the ganglion cell layer and the inner plexiform layer in young fry and then do so only near the optic nerve head (Rusoff and Hapner, '90). Together these pieces of evidence suggest that the cells that will send axons to the retina in both the nucleus olfactoretinalis and the diencephalon are born and extend axons very early in the life of the fish. These axons grow toward the retina in the optic tract and nerve over axons of retinal ganglion cells that grew out to the brain early in the life of the fish. As the retinopetal axons reach the retina, they grow for a short distance over the ganglion cell layer and then penetrate it and grow across the inner plexiform layer. When the axons reach the boundary between the inner nuclear layer and the inner plexiform layer, the axons begin to form terminals near amacrine (Witkovsky, '71) and interplexiform cells (Zucker and Dowling, '87). These axons continue to grow toward the peripheral margin of the retina along the boundary between the inner plexiform and inner nuclear layers as long as the retina continues to grow. No other workers have commented specifically on the location of the retinopetal axons in the optic nerve with respect to the specific arrangement of retinofugal axons (although Uchiyama [personal communication] has recent evidence for an arrangement similar to that shown here in the whitespotted greenling). However, Munz et al. ('82) illustrate the bundle of axons from the nucleus olfactoretinalis passing through the telencephalon and into the optic tract as a single bundle. Springer ('83) and Stell e t al. ('84) also comment that in goldfish the axons that project to the retina from the olfactory bulb remain together in the optic nerve. Our observation that the retinopetal axons are in a restricted portion of the optic nerve with the axons from the oldest ganglion cells fits together with and extends their observations. Additional evidence that the cells that project to the retina are born early comes from the work of Crapon de Caprona and Fritzsch ('83). They used two other cichlid fish and counted the number of cells in the nucleus olfactoretinalis that sent axons to the retina a t various times in development and concluded that all of the cells in the nucleus olfactoretinalis that project to the retina are present by 3 weeks of age. Their result, except for the exact dates, agrees closely with the result that we present in the following paper (Rusoff and Hapner, '90). We conclude, therefore, that cells that have retinopetal axons are born and extend their axons very early in the life of the fish. This result means that these nuclei do not respond to the growth of their terminal field by adding more cells; instead, a fixed number of cells projects to the retina and each cell enlarges its terminal field as the retina grows. An analogous situation exists elsewhere in the fish visual system and also in that of amphibians. As the eye enlarges, the extraocular muscles hypertrophy; presumably, a larger muscle is needed to move a heavier eye. The hypertrophy of the muscles is accomplished partially by an increase in number of muscle fibers; this has been quantified for the superior oblique muscle in goldfish (Easter, '79) and in Xenopus (Fritzsch and Sonntag, '87). However, the number of axons in the trochlear nerve does not increase in the goldfish (Easter, '79) and the number of motoneurons does not increase in Xenopus (Fritzsch and Sonntag, '87). Each motoneuron must innervate more muscle fibers in the old animal than in the young one and must, therefore, increase the size of its terminal arbor with age. Thus, increasing

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terminal arbor size may be a common solution to the problems created by continuous growth of target areas. The early appearance of the retinopetal system raises a number of intriguing questions. What stimulus causes the retinopetal axons to enlarge their terminals so that they continue to cover the retina when it is many times larger than the retina that they originally innervated? Are the retinopetal axons growing toward the retina in the same region of the optic nerve that contains growing axons from ganglion ceIls? If so, what guides the two groups of axons as they grow in opposite directions? Finally, the presence of two groups of axons intermingled in the optic nerve one from a group of nuclei that are postmitotic and the other from a population that continues to enlarge as the fish grows makes the optic nerve an even more interesting system for studying the parameters that allow regeneration than was previously thought.

ACKNOWLEDGMENTS We thank Dr. William Stell for the gift of an aliquot of anti-LHRH, Dr. Stephen S. Easter, Jr. for critically reading the manuscript, and Dr. R. Glenn Northcutt for helpful discussions of the neuroanatomy. This work was supported by National Institutes of Health grant EY06495.

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Organization of retinopetal axons in the optic nerve of the cichlid fish, Herotilapia multispinosa.

We have studied the position and numbers of retinopetal axons in the rainbow cichlid fish, Herotilapia multispinosa, to determine the response of rela...
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