THE JOURNAL OF COMPARATIVE NEUROLOGY 2 9 3 6 5 5 4 6 4 (1990)

Electron Microscopic Study of ImmunocytochemicallyLabeled Centrifugal Fibers in the Goldfish Retina KUNIHIKO KAWAMATA, TERUYA OHTSIJKA, AND WIIALIAM K. STELL National Institute for Physiological Sciences, Okazaki 444, Japan (K.K., T.O.); Department of Anatomy and Lions' Sight Centre, The IJniversity of Calgary Faculty of Medicine, Calgary, Alberta, Canada T2N 4Nl (W.K.S.)

ABSTRACT The centrifugal fibers innervating the goldfish retina were studied quantitatively by light and electron microscopy. These fibers originating from cell bodies in the olfactory bulb were labeled by antiserum to the tetrapeptide Phe-Met-Arg-Phe-NH, (FMRFamide). The number of FMRFamide-immunoreactive (ir) centrifugal fibers in each eye of the adult goldfish (body length: 12-15 cm) was 66 + 14 (mean t S.D., n = 7). All of these fibers in the optic nerve and the retina were unmyelinated. Each FMRFamide-ir centrifugal fiber runs along the optic fibcr layer and gives several terminal arborizations in the outermost layer (layer 1)of the inner plexiform layer. Layer 1 is, therefore, densely covered by a plexus of terminal arborizations. Along these terminal arborizations, we found output synapses characterized by a cluster of small clear vesicles (40 nm in diameter) at the presynaptic site and a thickened membrane in the apposed retinal cell processes. In a sample area of 2.000 Fm2, such synapses occurred at a density of one per 105 Wm2, or about 13,000 per centrifugal fiber. Thus, the FMRFamide-ir centrifugal fibers are likely to modulate retinal cell activity through an estimated total of 840,000 output synapses per retina. Key words: FMRFamide-immunoreactivity, synapse, terminal nerve (nervus terminalis), optic nerve, serial reconstruction, quantitative analysis

Centrifugal fibers innervating the retina have been shown in various species of vertebrates (see review by Stell, '72; Stell et al., '87). In the goldfish, retrograde labeling demonstrated that these fibers originate from ganglion cells adjacent to the olfactory bulb. This group of neurons is known as the terminal nerve (nervus terminalis) (Demski and Northcutt, '83; Springer, '53;Stell et al., '84). Immunocytochemical studies showed that the teleostean terminal nerve contains peptides similar to gonadotropin-releasing hormone (GnRH) (Stell et al., '82; Munz et al., '82) and molluscan cardioexcitatory peptide (FMRPamide) (Stell et al., '84). The course of the centrifugal fibers within the retina was also investigated immunocytochemically: they travel in the optic fiber layer, cross through the inner plexiform layer. and form a meshwork a t the border between the inner plexiform and inner nuclear layers (Stell et al., '82, '84; Muske et al., '87). Recently the retinal targets of these centrifugal fibers were identified by double immunocytochemical or immuno-

0 1990 WILEY-LISS, INC.

cytochemical-autoradiographical labeling as GABAergic

and glycinergic amacrine cells (Ball and St. Denis, '87; Stell et al., '87; Ball e t al., '89) as well as dopaminergic interplexiform cells (Stell et al., '87; Zucker and Dowling, '87). Although these immunocytochemiral studies clarified the details of this "olfactoretinal" projection to the teleostean retina, the functional role of centrifugal innervation is still speculative. Recently a neurophysiological study of the goldfish retina provided some evidence for the mechanism of centrifugal modulation (Walker arid Stell, '86); the ganglion cell discharges evoked by light stimuli are modulated by the application of GnRH and FMRFamide, which are released presumably from the presynaptic sites of the centrifugal fibers. 'I'o clarify the functional role of these retinal efferent fibers, we need more quantitative morphological studies on their synaptic structure in the retina. Therefore Accepted November 6,1989.

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we used light and electron microscopy to investigate quantitatively the structure of immunocytochemically labeled centrifugal fibers in whole-mounted goldfish retinas. The present results strongly support the idea that activity in the teleostean retina is modulated by efferent activity in the terminal nerve (Walker and Stell, '86).

counterstained with cresyl violet, dehydrated, and covered with a mounting material. FMRFamide-ir fibers in the optic nerve were also studied irnmunocytochemically by the same sectioning method. The optic nerves for this study were obtained from one small goldfish (3.5 cm in body length) and two medium-sized goldfish (12 cm and 14 cm in body length).

MATl3RIALS AND RlETHODS

Electron microscopy

Eyes of adult goldfish (Carassius auratus), body length: 12-15 cm, were used. The fish was anesthetized with 0.05% tricaine methanesulfonate (MS-222: Sigma), both eyes were enucleated, and the fish were killed by decapitat.ion. Then the anterior halves were removed and the remaining eyecup preparations were used for the following studies.

A piece of retina was dissected from the eyecup preparation and placed on the millipore filter vitreal side upward. For fixation and immunocytochemical labeling, we followed the method described by Eldred et, al. ('83). The retinal piece was first immersed in 0.1 M P B containing 4'; paraformaldehyde and 0.1 5; glutaraldehyde for 1 hour a t room temperature, and then transferred to the second fixative Light microscopy 1 M sodium bicarbonate buffer (pH 10.4) conparaformaldehyde for overnight at 4°C. The Whole-mounted retina. The eyecup preparation was immersed immediately in 0.1 M phosphak buffer (PB), pH retina was removed from the filter, washed in PBS, and both 7.4, containing 4 % paraformaldehyde overnight a t 4OC. Af- the pigment epithelium and the distal half of the retina were ter the eyecup was washed in 0.01 M phosphate buffered sa- skimmed ofT. The remaining retina was incubated for 30 line (PBS), pH 7.4, the whole retina was removed from the minutes in 1 rc, sodium borohydride in 0.1 M PR and washed sclera and several radial cuts were made near the rim of the in the same buffer alone until no more bubbles formed. retina lor flat-mounting. Then the retina was spread over a Then the retina was cryoprotected by 30% sucrose and Millipore filter (Type AA, Millipore Corp.) photoreceptor treated by the freeze-and-thaw method (Eldred et al., '83). side upward, and both the pigment epithelium and the dis- Aft,er washing i n PBS, the retina was preincubated for 3 tal retina including photoreceptors and horizontal cells were hours in 1 ?(# normal goat serum in PBS, and immersed for skimmed off. Since this procedure facilitated the infiltra- 24 hours in the same primary antiserum to FMRFamide tion of antisera into the inner plexiforrn layer, the fine described above in PBS containing 0.01% Triton X-100 a t processes of the centrifugal fibers could be seen in the room temperature. Following three 30-min washes in PBS, whole-mounted retina. Nonspecific binding of antibodies the retina was incubated with biotinylated antirabbit IgG was eliminated by soaking in 1R normal goat serum in PBS for 6 hours a t room temperahre and with ABC reagent for 3 hours at room temperature. Subsequently the retina (Vectastain) overnight a t 4°C. After visualization of centrifwas incubated in PBS containing the primary antiserum, a ugal fibers by DAB, the tissues were postfixed for 30 minrabbit antiserum directed to FMRFamide (dilution 1:1000), utes a t 4°C in 1 ''osmium tetroxide in PB. Then the retina for 24 hours a t room temperature (Stell et al., '84). Two anti- was dehydrated in ethanol series and embedded in epoxy sera t o FMRFamide were used, #231 (given by Dr. T. resin. Serial ultrathin sections (silver-gold) stained with O'Donohue) and CA245 (Cambridge Research Biochemi- uranyl acetate and lead citrate were examined with an eleccals). After three 30-minute washes with PBS, the retina tron microscope (H-500H, Hitachi). Some eyes were excised with a stump of the optic nerve. was incubated with biotinylated anti-rabbit TgG for 3 hours and with avidin-biotin-horseradish peroxidase complex The optic nerve attached to the eyeballs was excised (about (ABC kit, Vector Laboratories) for 6 hours a t room tempera- 5 mm in length), immersed in the same fixative solution ture. All these solutions contained 0.3% Triton X-100. After described above, and embedded in 5 9U agarose. Serial transthree 30-minute washes with PBS, the retina was trans- verse slices (thickness 0.5 mm) of optic nerve were made by ferred into 0.05% 3,3'-diaminobenzidine tetrahydrochloride a tissue slicer (Microslicer, DTK3000, Dosaka EM). After (DAB: Sigma) in 0.1 M P B for 15 minutes. Then the retina the centrifugal fibers were labeled immunocytochemically was incubated in the same DAB solution containing 0.015;) by the procedure described above, the thick slice was postH,O, for 10 minutes. Fnllowing three washes in PBS, the fixed by osmium tetroxide, dehydrated, and embedded in retina was dehydrated in a graded ethanol series and epoxy resin. Then ultrathin sections (silver-gold) were embedded in epoxy resin (Ohtsuka, '83). This whole- made. Immunocytochemically labeled fibers were analyzed mormounted retina was studied under an ordinary light microscope. The labeled fibers were drawn with a camera lucida phometrically by using a digitizing device (Mop-Videoplan, Kontron, Munich). and photographed. Cryostat sectioning. A whole eyecup was fixed by the same procedure described above and dissected into several pieces. After several washes by PBS, the retinal pieces with RESULTS pigment epithelium were cryoprotected by immersion in a Centrifugal fibers in the optic nerve series of graded sucrose solutions?0.1 M P B containing 10 The centrifugal fibers labeled by antiserum to FMRFand 30:;. sucrose. Then, each retinal piece was embedded in agarose (Type VII, Sigma), 5 % w/v containing 10% sucrose, amide were scattered in the optic nerve (Fig. IA). In the and sectioned a t about, 20 pm on a cryostat (OTF/AS, Bright light microscope, a t high magnification, we could identify Instruments, England) at, -2OOC. All sections were col- and count individual fibers (Fig. IB). The total numbers of lected on a gelatin-coated glass slide and air-dried. By using FMRFamide-ir fibers detected in one optic nerve a t three the immunocytochemical procedure described above, the different locations (I,3, and 5 mm from the eyeball) were 52, centrilugal fibers were visualized. Then, the sections were 46, and 53 in one medium-sized fish (14 cm), and 7Y,86, and

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CENTRIFUGAL FIBERS IN THE GOLDFISH RETINA

another (12 cm). Since these numbers are not signifinI i in ' cantly different from the numbers of axons innervating each retina. our data suggest that t,he centrifugal fibers do not branch within the optic nerve, i.e., that e a c h individual axon entering the retina from the optic nerve may represent a different, terminal nerve ganglion cell. Furthermore, cursory study revealed more than 50 FMRFamide-ir fibers in the optic nerve of a much smaller fish (body length 3.5 cm). Therefore our results suggest also that t,he total number of FMRFamide-ir fibers seen in the optic nerve is not related t,o the body length of the individual fish. Electron micrographs revealed that these FMRFamide-ir optic nerve fibers were 0.2-1.3 ,urn in diameter and had no myelination (Fig. 1C). Details of cytoplasmic structure were nol clear, probably because of heavy staining by the ABC method (see discussion), but several vesicle-like granules could be distinguished. In contrast, the surrounding fibers coming from the ret,inal ganglion cells were all myelinated, and their cytoplasm was filled, not with vesicles, but with numerous neurotubule-like structures.

Centrifugal fibers in the retina In the whole-mounted retina (Fig. a), we perceived that the FMItFamide-ir centrifugal fiber comprises four parts: a radiating fiber, a few ascending fibers, several terminal arborizations, and occasionally a distal process. These subdivisions are described in turn, below. The centrifugal fibers innervated the retina at the optic disk and ran radially toward the peripheral retina along with the optic nerve fibers. In the middle region of the retina. these radiating fibers often turned abruptly to the

..........". .."'ab

._.. ...

Fig. 1. FMRFamide-ir centrifugal fibers in the optic nerve of the goldfish. A Light micrograph showing a cross-section of the optic nerve in which labeled centrifugal fibers are scattered. Some of the labeled centrifugal fibers are indicated by arrowheads. a: artery. T h e region indicated by the square is enlarged in B. This section was counterstained by cresyl violet. Calibration bar: 100 pm. B: Enlargement of area in A. showing centrifugal fibers (arrowheads) scattered in the optic nerve. Calibration bar: 50 pm. C: Electron micrograph showing an electron-opaque FMRFamide-ir centrifugal fiber with no myelination. Surrounding myelinntcd nxons are presumably afferent optic nerve fibers (retinal ganglion cell axons). This cross section was made 1 mm behind the eyeball. Calibration bar: 0.5 pm.

Fig. 2. Camera lncida drawing of a single FMRFamide-ir centrifugal fiber in a whole-mounted goldfish retina. The centrifugal fiber extends from the optic papilla ( 0 ) as a radiating fiber (rf), which runs along with the optic nerve fibers (onf) toward the peripheral retina. The radiating Iibar abruptly changes direction (arrowhead), turning perpendicular to the optic nerve fibers and becoming a n ascending fiber (af) which rims obliquely in the inner plexiform layer and bifurcates many times. Each branch extends to the border between the inner nuclear layer and inner plexiform layer (layer 1 ) . Each ascending fiber branches to form many fine fibers and terminal arborizations (ab) in layer 1. The centrifugal fiber shown here arborizes in several different regions (encircled by dotted lines) in one quadrant of the retina. T h e peripheral border is delineated by a thick line. Since many terminal arborizations from different centrifugal fibers are interwoven densely in layer 1,only the main trunks of terminal arborizations were drawn (see also Fig. 5). Calibration bar: 1 mm.

658

right or to the left, perpendicular to the optic nerve fibers, and then entered into the inner plexiform layer. Most of the fibers turned within 2.5 mm of the optic papilla; this was well within the central retina, since the average radius of the flat-mounted retinas was about 5.7 mm (Table 1).In the far peripheral retina, therefore, no FMRFamide-ir fihers ran in the optic nerve fiber layer. After changing direction, the centrifugal fihers ran obliquely in the inner plexiform layer toward the peripheral retina and bifurcated into several branches. These ascended to layer 1, the outermost layer of 5 substrata of the inner plexiform layer (Cajal, 1892), and then formed several terminal arhorizations distributed over almost a quadrant of the retina. All FMRFamide-ir fibers were similar in structure, and so layer 1 was covered by a meshwork or plexus of densely interwoven axons from different centrifugal fibers. We have illustrated only the main trunks of a terminal arborization in Figure 2. Numerous varicosities, a t least some of which indicate the presence of synapses en passant (see below), were found along these terminal arborizations. A few FMRFamide-ir fibers were found also in layer 2 and 3. Distal processes, extending from the terminal arborizations through the inner nuclear layer to the horizontal cell layer (Stell e t al., '84; Zucker and nowling, '87),were found only rarely in the present study. We identified only 8 distal processes in an area of 3 mm2 in the middle retina. Thus, we focused lurther investigation on the other three parts of the centrifugal fibers, i.e., the radiating fibers, ascending fibers, and terminal arborizations.

K. KAWAMATA ET AL.

Fig. 3. Light micrograph showing radiating fibers in the optic nerve fiber layer extending toward the peripheral retina (indicated by arrow). There are two types: thick (single arrowhead), and thin with varicosities (double arrowheads). One radiating fiber bifurcates; one of its branches runs among the optic fibers, while the other (small arrow) ascends to the inner plexiform layer. Calibration bar: 50 fim.

Most of these radiating fibers were unbranched. The number of centrifugal fihers running in the optic nerve was similar to the number of fibers radiating around the optic Radiating fibers in the optic fiber layer papilla, and both eyes were innervated by similar numbers Two kinds of smooth radiating fibers were seen in the of these fihers. The total number of centrifugal fibers to one optic nerve fiber layer; one was thick and smooth, while the retina was 65.1 t 14.2 (mean 5 S.D., n = 7 relinas from 4 other was thin and showed frequent varicosities (Fig. 3). goldfish).

Fig. 4. FMRFamide-ir centrifugal fibers in the retina. A Light micrograph showing radial fibers in the optic fiber layer (OFL) and an ascending fiber (arrowheads) running obliquely through the inner plexiform layer (IPL). Terminal arhorizations (arrows) are seen in layer 1.

ONL = outer nuclear layer. Calibration bar: 50 fim. B: Electron micrograph showing a cross-section of a n ascending fiber containing small clear vesicles ( c ) .No postsynaptic structures are found in the apposed processes. Calibration bar: 0.5 Gm.

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CENTRIFUGAL FIBERS IN THE GOLDFISH RETINA TABLE 1. FMRFamide-ir Centrifugal Fibers Innervuting the Goldfish Retina 17-15 cm

Body length (n = 10)

LM analye& Whole retinal area (n = 3 ) Diameter of flat-mounted retina (n = 3) No. of centrifiigalfibersinnervatingone eye (n = 7) Length of terminal arborkationa per unit area Average length of arbori7~tionsper terminal' EM analpis Terminal arborizationaper unit area Presynapticsites along terminal arborivltiorla in layer 1 One preynaptic site per area Prffiynapticsites of whole retina' Prffiynapticsites in one centrifugal fib? Average distance between synaptic sites'

88 I 1.4mm' 11.3 = 1.4mm 65 t 14 109 + 9.7 rnm/mm'

148mm 205 mm/mmZ

l / l o s ~ ~ &M,m 13,N 11.4fim

'Preynaptic sites of whole retina (S40,oOb)lckn~fugd fibersin one eye (65) 4Lengthof fiber (148,000fim)/No. presynapticsitesperfiber (13,000)

Ascending fibers in the b i e r plexiform layer The FMRFamide-ir ascending fibers were found to run obliquely through the inner plexiform layer toward the peripheral retina, and finally reach layer 1 (Fig. 4A). These ascending unmyelinated fibers had a few varicosities containing small clear vesicles, but neit,her accumulations of vesicles nor synaptic memhrane thickenings were seen in the apposed processes (Fig. 4B). Although Witkovsky ('71) claimed finding myelinated efferent fibers in the inner plexiform layer of the teleost retina, we could not find any myeliiiated FMRFamide-ir fibers.

Plexus of terminal arborizations in the inner plexiform layer Since each centrifugal fiber extends several terminal arborizations over almost a quadrant of the retina, layer 1 of the inner plexiform layer is covered by numerous terminal arborizations coming from different fibers. As shown in Figure 5 , we found that the densely interwoven FMR.Famide-ir fibers make a meshwork sheet or plexus. In the middle retina (i.e., midway between the center and periphery) the orientation of the axonal terminals was random (Fig. 5A). The smallest mesh encircled an area about the size of one neuronal soma (about 10 prri in diameter). In the far periphery (near the ora serrata) the centrifugal fibers tended to run along circular lines, perpendicular to the course of t,he opt.ic nerve fibers (Fig. 5B). The length of terminal arborizat,ions was measured a t different regions chosen randomly in the middle retina. In each region, we drew all terminal arhorizations within a square (150 x 150 pm) by means of a camera lucida, and then the t,otal 1engt.h of these fibers was measured with the aid of a digitizing tablet. The total length was 109 i 9.7 mm/mm' (mean t S.D., n = 5 ) . From this result, we were able to estimate the total length of all terminal arborizations in one eye as roughly 9,600 mm (see Table 1).Thus, the average length of the entire terminal arborization originating from one centrifugal fiber was calculated to be 148 mm. In the camera lucida drawing shown in Figure 2, the length of the centrifugal fiber from the optic papilla to layer 1 was about 8 mm. 'I'herefore the length o f the terminal arborizations forming the plexus in layer 1 comprised about 95% of the total length of one centrifugal fiber. Because these terminal arborizat,ions are so interwoven (Fig. 5), only the main trunks of the t,erminal arborizations could be identified and illustrated (Fig. 2).

Synapses along the terminal arborizations Along the terminal arborizations, numerous varicosities (0.3- I .5pm in diameter) were connected by thin axon segments (U.1-0.5 pm). We observed two types of varicosities (Fig. 6A,B). Varicosities of one type were filled with a large aggregation of small clear vesicles, about 40 nm in diameter (Fig. 6A). The memhrane of the postsynaptic process opposite such a varicosity was straight and thickened by electron-dense material. Even when this contact site was sectioned obliquely, as in Fig. 6A (arrowhead), it could be recognized by the rectilinearity and dense thickening of the postsynaptic membrane. Thus, the aggregation of small clear vesicles seemed to be located a t the anatomical site of syriapt,ic output from the centrifugal fibers. Varicosities containing mainly or only small clear vesicles comprised 83 ',(> of all the identified centrifugal fiber varicosities identified in serial electron micrographs (Fig. 8). Varicosities of the other type were filled with large vesicles (Fig. 6B) which were n o t imaged clearly, probably because heavy staining in these preparations obscured details of the cytoplasmic contents. However, these seemed to be identical to the large dense-cored vesicles shown in the terminal nerve of the white perch (Zucker and Dowling, '87). We could not find any form of membrane specialization in the processes facing varicosities filled with large vesicles. Varicosities containing mainly o r only large dense-cored vesicles comprised 17'; of all the varicosities identified in serial electron micrographs (Fig. 8). Direct axosomatic contacts by FMRFamide-ir axons were extremely rare. Axonal varicosities in layer 1 were often close to somata in the inner nuclear layer, but in most, cases such close pairs were separated by other intervening processes. We observed only two examples of direct contact between a FMRFamide-ir varicosity and a soma in the inner nuclear layer, one of which is shown in Figure 6C. It is not certain that this contact represents an anatomical synapse. If it, is a synapse, the results of previous studies suggest that the postsynaptic element is an amacrine cell (Ball et al., '87) or a dopaminergic iriterplexiform cell (Stell et al., '87; Zucker and Dowling, '87). On Ihe basis of these quantitative studies, we concluded that the centrifugal fibers of the goldfish retina make synaptic contacts mainly with retinal cell processes (neurit,es) running in layer I of the inner plexiform layer. Contacts with cell bodies in the amacrine cell layer are very rare.

Processes postsynaptic to the centrifugal fibers Since the processes postsynaptic to the FMRFamide-ir centrifugal fibers were thin (1-2 pm in diameter) and very long, we could not trace them to their somata without extensive serial sections. These processes were characterized by neurotubule-rich cytoplasm as shown by paraxial sections along them (Fig. 7A-C) or transverse sections across them (Fig. 7D-F).

Output synapses along the terminal arborizations We investigated quantitatively the output synapses along the terminal arborizalions by using the clusters of small Clpar .~.. vesicles in the varicosities as morDholoeical indicators.

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Fig. 5. Terminal arborization meshwork of FMRFamide-ir centrifugal fi hers in the whole-mounted retina. Numerous terminal arborizations are interwoven with each other to make a plexus in layer 1 of the inner plexiform layer. A In the central retina, the axons run in random directions. B: A t the far periphery, near the ora serrata, the axons run in parallel arcs concentric with the optic papilla (direction indicated by arrow). A and B are the same magnification. Calihration bar: 50 pm.

We made a series of about 200 ultrathin vertical sections (corresponding to about 20 Fm) in the middle retina, and reconstructed the terminal arborizations in this 20 x 100 fim patch (Fig. 8). In this 2,000 fim' area there were eight terminal FMRFamide-ir fibers, seven in layer 1 and one in layer 3. We found 20 output synapses along these fibers, 19 in layer 1 and one in layer 3. By using this result, and knowing that about 65 centrifugal fibers innervate one eye, we calculated that the terminal arborizations make a total of 840,000 output synapses in layer 1 (Table 1). The total length of 7 terminal arborizations in laver 1was 409 Fm, which corresponds to 205 mm/mm2. This is about twice the mean length of terminal arborizations obtained by light microscopy. This difference was probably due to sampling error; viz., the area we studied by electron microscopy might have been unusually rich in terminal arborizations, or the finest fibers might not have been as readily detectable by light microscopy as by electron microscopy.

Fig. 6. Electron micrographs showing varicosities along the terminal arhorization of FMRFamide-ir centrifugal fibers. A Varicosity filled with densely aggregated small clear vesicles, 40 nrn in diameter. A membrane thickening (arrows) is seen in t h e postsynaptic process facing the varicosity. The arrowhead indicates another probable postsynaptic density, obliquely sectioned. B: Varicosity filled with large vesicles. Note t h a t no membrane specialization is seen in the apposed processes. C: Direct contact between a terminal arborization and an unidentified cell soma (s) located in the inner nuclear layer. A-C are the same magnification. Calihration bar: 0.5 pm.

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CENTRIFUGAL FIBERS IN THE GOLDFISH RETINA

Fig. 7. Electron micrographs showing serial sections of synapses between the terminal arborization of FMRFamide-ir centrifugal fiber and postsynaptic processes. A-C: Postsynaptic process (p) was seetioned paraxially, parallel to the microtubules. A membrane thickening (arrows) is seen in the postsynaptic process, facing a presynaptic varicosity filled with small clear vesicles. D - F Another series of sections

showing microtubules in a cross-sectioned postsynaptic process (p). In E,the postsynaptir memhrane thickening is hardly seen, because the section series is leaving the area of apposition and the sectioning plane is oblique to the synapse. Both presynaptic and postsynaptic processes contain mitochondria (m). A-F are all the same magnification. Calihration bar: 0.5 wm.

DISCUSSION Centrifugal fibers in the optic nerve

gin o f the goldfish optic nerve. This expectation has been contradicted by our observation that the FMRFamide-ir fibers are distributed diffusely throughout the entire optic nerve in both young and old goldfish. Still, in goldfish no direct evidence has been obtained that new terminal nerve ganglion cells (or new retinopetal axons from existing cells) are in fact generated throughout life. Such evidence is beyond the scope of the present investigation, but the apparent difference between centrifugal fiber distributions in the optic nerves of goldfish and higher teleosts is certainly worthy of further study.

In the goldfish, new retinal ganglion cell axons are added continuously throughout life. These retinal afferent fibers are arranged in regular order, the oldest being in the dorsal part of the nerve and the youngest in the ventral part (Kusoff and Easter, '80; Easter et al., '81; Springer and Mednick, '86). Similarly, studies in several higher teleosts have suggested that the full adult complement of terminal nerve cells is achieved early in development (Crapon de Caprona and Fritzsch, '83; Halpern-Sebold and Schreibman, '83), and that the immunoreactive "olfactoretinal" fibers are concentrated in the oldest portion of the optic nerve ( M u m and Claas, '81; Munz et al., '81, '82; Crapon de Caprona and Fritzsch, '83; Uchiyama and Ito, '84; Uchiyama, '89). If all the terminal nerve ganglion cells were to become postmitotic and send their axons into the optic nerve early in development, in goldfish as in these higher teleosts, then the FMRFamide-ir axons should be confined to the dorsal mar-

Centrifugal fibers in the retina By conventional histological study, Springer ('83) found that 65 ganglion cells of' the terminal nerve in the goldfish were associated with the olfactory bulb on each side. But only one-fourth of them were labeled retrogradely by application of cobaltous lysine to the optic nerve. In the present study, however, 65 FMRFamide-ir fibers (on average) were

~

Fig. 8. Synapses along the terminal arborizations of FMRFamide-ir centrifugal fibers. A Reconstruction of synaptic sites along axons of terminal arborizations. This projection upon the horizontal plane was made from a series of 200 transverse ultrathin sections in the middle retina. Terminal arborizations were localized within layer 1, except for one a t the far right (dotted line) in layer 3. Arrows indicate varicosities filled with small clear vesicles, while arrowheads indicate varicosities filled with large vesicles. A radial section along the broken line in A is shown in B. B: Electron micrograph showing a radial section of INLiIPL junction. Arrows indicate the terminal arborizations of FMRFamide-ir centrifugal fibers in layer 1. Sublayers 1to 4 (of 5 suhlayers) are shown in the inner plexiform layer (IPL).H axon terminals of horizontal cells. A, B are at the same magnification. INL = inner nuclear layer. Calibration bar: 10 pm.

CENTRIFUGAL FIBERS IN THE GOLDFISH RETINA found to innervate the retina. Since in goldfish only the terminal nerve has been shown to produce centrifugal fibers innervating the retina (Stell et al., '84), we concluded that all 65 centrifugal fibers in the retina are from the terminal nerve. We may propose two possible explanations for such a large discrepancy. One is that terminal nerve fibers innervating the retina could bifurcate (Stell et al., '84; von Bartheld and Meyer, '86) so that the number of centrifugal projections to the retina would be much larger than the number of terminal nerve ganglion cells. Our observations rule out a large degree of axonal bifurcation in the optic nerve, but still allow the possibility that it occurs intracranially. Another possibility is that insufficient retrograde transport resulted in labeling of only a minoriLy of the retinopetal cell bodies in the terminal nerve ganglion. A recent quantitative study indicated that this is so for centrifugal fibers to the frog ret,ina (Uchiyama et al., '88); and it may be true also in goldfish, since our results are otherwise consistent with a 100% projection of terminal nerve ganglion cells as retinal efferent fibers. In goldfish and carp retinas, myelinated axons were found in the inner plexiform layer (Witkovsky, '71). Since these fihers are similar to the myelinated centrifugal fibers found in the avian retina, Witkovsky ('71) concluded that they come from the central nervous system. Although the intraretinal course of the myelinated fihers described by Witkovsky was very similar to that of the FMRFamide-ir centrifugal fibers shown in the present study, we have observed that all of the FMRFamide-ir fibers are unmyelinated. Furthermore, the myelinated fibers described by Witkovsky contained only small vesicles, while the FMRFamide-ir centrifugal fibers have both large and small vesicles. Since retrograde labeling has shown only one source of retinopetal cells in the goldfish (Munz et al., '82; Springer, '83; Stell et al., '841, the myelinated fibers in the inner plexiform layer are unlikely to be efferent nerves originating in some other part of the brain (although, as just pointed out, inefficient retrograde transport could produce a false negative result). It might be concluded that the myelinated fibers in the inner plexiform layer are axons of intrinsic retinal neurons, or of displaced ganglion cells (as discussed by Stell, '72).

Presynaptic sites of the centrifugal fibers In the varicosities along the terminal arborization, we found two different kinds of vesicles: large ones and small clear ones. These two types of vesicles were also shown in white perch (Zucker and Dowling, '87); t,he large densecored vesicles showed immunoreactivity to GnRH as well as FMRFamide, while the interiors (or contents) of the small clear vesicles were not immunoreactive for these peptides. In the goldfish retina, as in the white perch retina (Zucker and Dowling, '87), we found that the cluster of large vesicles is always away from the presynaptic site occupied by the cluster of small clear vesicles. Similar presynaptic structures were found also in synapses onto the interplexiform cells of the goldfish (Yazulla and Zucker, '88). These observations in two difTerent teleost species suggest that the terminal nerve transmits information to retinal cells by some unidentified substance released from the small vesicles at conventional synapses, while the release of peptides from the large dense-cored vesicles at unknown sites might modulate functions at structurally undifferentiated but "chemically addressed" targets (Iversen, '84; Stell et al., '86). This speculation must be confirmed by physiological experiments to show the mechanism of transmission and sites of action of

663 the centrifugal fibers. I t should be noted, however, that LJmino and Dowling ('88) have reported effects of GnRH in the retina of white perch, consistent with action through dopaminergic interplexiform cells.

Retinal cell targets of centrifugal fibers The processes postsynaptic to the FMRFamide-ir centrifugal fibers are characterized by microtubule-rich cytoplasm. Generally. microtubule-rich processes in the inner plexiform layer have been interpreted as neurites of amacrine cells (Witkovsky and Dowling, '69). Previous studies showed that FMRFamide-ir centrifugal fibers made synaptic contacts with GABAergic and glycinergic amacrine cells (Ball and St. Uenis, '87) as well as dopaminergic interplexiform cells (Stell et al., '87; Zucker and Dowling, '87). We have clarified here that output synapses were found mainly between the centrifugal fibers and neurites in the inner plexiform layer, and only infrequently between the centrifugal fibers and somata in the inner nuclear layer. This does not mean that previous reports describing mainly axosomatic contacts of' centrifugal fibers were in error, or in conAict with the present account. But the previous studies focused upon contacts with pre-labeled cells, whose somata provided a readily detectable and convenient reference structure for learning something about centrifugal fiber synapses in the retina. Since the target somata, especially those of the dopaminergic interplexiform cells, may be widely scattered in the retina (Marc, ' 8 2 ) , it should not be surprising if the axosomatic contacts represent only a small fraction of the synaptic output of the centrifugal fibers. We found that layer 1 of the inner plexiform layer is densely covered by the output synapses of the centrifugal fibers. Thus, the retinal processes running in layer 1 are readily available as targets for the centrifugal fibers (Marc, '82). The processes running in layers 1to 3 of the inner plexiform layer are involved preferentially in the OFF-pathway, in which retinal neurons respond to light with hyperpolarization and/or a decrease in nerve impulse activity (Famiglietti et al., '77). The centrifugal fibers seem to co-stratify with retinal neurites involved in such OFF-pathways. I t is not surprising, therefore, that recently we found synaptic contacts between FMRFamide-ir axons and HRP filled OFF-type amacrine cells in the goldfish retina (Ohtsuka et al., '89). In the present study, we have found that about 65 centrifugal fibers originating in the terminal nerve ganglion innervate each eye and terminate by an estimated 840,000 output synapses upon processes of retinal neurons in IPL layer 1. Although the functional role of the centrifugal fibers is not known yet, this presumed large number of output synapses indicates clearly that terminal nerve information is transmitted directly to the retina and probably modulates retinal cell activities. We need further physiological study to clarify the functional role of this centrifugal control system.

ACKNOWLEDGMENTS We thank Dr. H. Uchiyama for his assistance in immunocytochemistry during the early stages of this study. This work was supported by grants from the Mochida Memorial Foundation for Medical and Pharmaceutical Research (T.O.), the Alberta Heritage Foundation for Medical Research (T.O., W.K.S.), and the Natural Sciences and Engineering Research Council and the Medical Research Council o l Canada (W.K.S.).

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