Chapter 40: Comparative ultrastructure and opsin immunocytochemistry of the retina and pineal organ in fish

A. Ermisch. R. Landgraf and H.-J. Riihle (Eds.) Pro8ress in Brain Research, Vol. 91 Q 1992 Elsevier Science Publishers B.V.All rights reserved.

307 CHAPTER 40

Comparative ultrastructure and opsin immunocytochemistry of the retina and pineal organ in fish I. Vigh-Teichmann1s2, M.A. Ali3 and B. Vigh2 ’Neuroendocrine Laboratory of the Hungarian Academy of Sciences - Semmelweis Medical University Joint Research Organization, H-1094 Budapest, Hungary; 22nd Department of Anatomy, Semmelweis University Medical School, Budapest, Hungary; and ’Department of Biology, University of Montreal, Quebec, Canada

The pineal organ and retina were compared in developing charr and cisco, further in adult cisco, eel, creek chub, dace, zebrafish and black moli by opsin immunocytochemistry. In prehatching charr embryos, retinal rods and cones and pinealocytes displayed well-developed outer segments and formed synapses. Differentiation of the retina started centrally but was more advanced in the dorso-caudal retina than rostroventrally. The pineal organ differentiated earlier distally than proximally. In the cisco, the pineal organ and retina differentiated around hatching. In charr embryos, further in the larval and adult species studied,

opsin immunoreactivity was found in retinal rods, accessory cones and many “rod-like’’ pinealocytes, a result indicating the presence of rhodopsin and/or porphyropsin. Retinal principle cones, long and short cones and some “cone-like’’ pinealocytes were opsin-immunonegative; they are thought to represent redand/or u.v./violet-sensitive elements. The pineal organ may be involved in negative phototaxic behavior. Both the retina and pineal organ appear to be suitably differentiated to detect light in the larval and embryonic charr.

Introduction

and Vigh-Teichmann, 1988). These results are strengthened by experimental data of light absorption maxima typical of green-sensitive rhodopsin/porphyropsin (Downing et al., 1986; Vigh and Vigh-Teichmann, 1988). However, we also found (rhod)opsin-immunonegative pinealocytes in lamprey, ratfish and European minnow (VighTeichmann et al., 1983,1990; Vigh-Teichmann and Vigh, 1985), findings pointing to the general presence of different types of photoreceptors in the pineal organ, similarly to the retina. With regard to the differentiation of the fish retina the view dominates that the larvae of many teleost species with metamorphosis have pure cone retinas (Evans and Fernald, 1990). In salmonid fishes, whose larvae display negative phototaxic behavior, the rods are thought to differentiate later than cones, in the second half of the larval life (Carey and Noakes, 1981) or at the end of metamor-

By their development and morphology the retina and the pineal complex constitute light-sensitive areas of the systems of CSF-contacting neurons and circumventricular organs (Vigh-Teichmann and Vigh, 1983). The retina of fresh-water fishes is known to be composed of rods, single and paired cones, while the pineal organ contains photoreceptor pinealocytes cytologically displaying cone-like outer segments. These retinal and pineal photoreceptors can only perceive light when they are appropriately differentiated and elaborate photopigments essential for photochemical transduction. Indeed, previous immunocytochemical studies in fish revealed the presence of rhodopsin and/or cross-reacting porphyropsin in the outer segments of retinal rods and many pinealocytes (VighTeichmannet al., 1983,1990;Vighet al., 1986; Vigh

308

Fig. 1. Details of the developing pineal organ of the charr. a. Well developed pineal (P) and parapineal (asterisk) organs of embryo 2-days before hatching. D, Dorsal sac; E, epidermis; H, habenular commissure; S, subcommissural organ. (Toluidine blue, x 280). b. OS-2-immunoreactive outer segments ( 0 s) and perikarya (asterisk) of pinealocytes; E, epidermis. (Prehatching embryo, x 560.) c. Outer segment of pinealocyte. (Prehatching embryo, x 22000.) d. IgG-gold particles (black dots) mark 0 s - 2 antigenic sites of visual pigment on “rod-like” outer segment (R); IS, inner segment. (Four-week-old larva, x 16000.)

phosis (Evans and Fernald, 1990). Developmental data on the retina and pineal organ of salmonids are, however, lacking except in Atlantic salmon (Ostholm et al., 1987). Therefore, our study deals with the differentiation of the retina and pineal organ of the salmonid charr Salvelinus alpinus and cisco Coregonus albus. In addition, an attempt is made to distinguish different types of retinal and pineal photoreceptors in adult teleost species by opsin immunocytochemistry.

Materials and methods Fishes studied were: landlocked charr (Salvelinus alpinus, embryos 1 - 2 days before hatching and 0, 1, 4, 7, 13 day-, 1- and 2-month-old larvae), cisco (Coregonus albus, embryos 1 - 3 days before hatching and hatched larvae), eel (Anguilla rostruta), creek chub (Semotilus atromaculatus), red-bellied dace (Chrosomus eos), zebrafish (Brachydanio rerio) and black moli (Moor moli). Sheep antibo-

309

vine rhodopsin and the monoclonal anti-visual pigment antibodies OS-2 and COS-1 demonstrating greedblue and greedred photopigments in higher vertebrates (Vigh-Teichmann et al., 1990), were used with the avidin-biotin-peroxidase (ABC) and immunogold (IgG) methods (details, see VighTeichmann et al., 1990) after 1% glutaraldehyde (GA) or 4% paraformaldehyde(PA)-0. 1070 GA fixation, Poly Bed 812 embedding and/or Naborohydride treatment.

Results The charr pineal complex consisting of the pineal and right-sided parapineal organs (Vigh-Teichmann et al., 1991) was well developed in the prehatching embryos to 2-month-old larvae (Fig. la-d), while in cisco embryos the pineal organ seemed to be less differentiated as suggested from its shortness, small size and tube-like appearance. Photoreceptor pinealocytes (Fig. l b - d), intrinsic secondary

neurons, glial cells and ribbon-containing synapses could be recognized in charr. When using the rhodopsin and monocloncal OS-2 antibodies, we found rhodopsin-, OS-24mmunoreactive outer segments (“rod-like” ones) and perikarya, inner segments of pinealocytes in the distal pineal organ of charr embryos 2 days before hatching (Fig. lb,c). There was no immunoreactivity with the COS-1 antibody in the stages studied. At the electron microscopic level, the immunogold particles marked the opsin antigenic sites on the photoreceptor membranes (Fig. 16). In charr and cisco retina, the differentiation started centrally as known from other fishes (VighTeichmann et al., 1991). However, the caudal and dorsal retinal quarters were more differentiated than the ventral and rostral ones. In the charr embryo, rods, paired and single cones could be distinguished together with synaptic pedicles (Fig. 2a,b); the rod outer segments were opsinimmunoreactive (Fig. 2 4 . These results are in ac-

- Fig. 2. Ultrastructural details of embryonic and larval charr retina. G , Melanin granules of pigment epithelium. a. OS-2-immunoreactive rod (R) and immunonegative cone (CO) of embryo 2 days before hatching ( x 2oooO). b. Synaptic pedicles (A) of embryo 2 days before hatching ( x 2oooO). c. OS-2-immunopositive rod (R) and immunonegative long single cone (CO) of 4-week-old larva ( x 22500).

310

Fig. 3 . Details of the adult (a,@ and larval (c) pineal organ. a. (Rhod)opsin-immunoreactive outer segments (arrows) of “rod-like” pinealocytes of cisco ( x 200). b. IgC-gold particles mark OS-2-immunoreactive sites on outer segment (RO)of “rod-like” pinealocyte (Creek chub, x 3oooO.) c. Opsin-immunonegative outer segment (0s) of “cone-like’’ pinealocyte. (Two-month-old charr, x 3oooO.)

cord with the presence of opsin-immunoreactive rods at the 20th embryonic day in guppy (Vigh et al., 1986), but in contrast with those in Atlantic salmon in which rods were only found after hatching (Ostholm et al., 1987) and in cisco, in which the retina was poorly differentiated 1 - 3 days before hatching. The pigment epithelium contained few melanin granules, and lamination just started dorsocaudally. Opsin-immunoreactive rods appeared around hatching. Apparently, this salmonid species differentiates just around hatching similarly to the goldfish (Negishi et al., 1990). In young cham larvae (1 - 4 days after hatching) rod perikarya, inner segments and even pedicles displayed immunoreactivity. This indicates a high degree of opsin gene expression after hatching when the growth of the retina is accelerated. Since the opsin immunoreactivity of the perikarya and inner segments of the “rod-like” pinealocytes of the charr appears somewhat earlier than in the retina, an

earlier start of pineal neurochemical maturation and functioning is suggested for this species. With regard to the opsin immunoreactivity in general in the developmental stages of charr and cisco it has to be noted that the outer segments of the retinal rods were immunoreactive with the three primary antibodies used, while all cone types were immunonegative, although their photoreceptor membrane lamellae were well developed (Fig. 24. This cone immunonegativity is probably not due to a silence of the opsin genes (photoreceptor membranes consist to more than 95% of opsin), but may be caused by physicochemical properties of the cone photopigments. Therefore, in the adult teleosts we also used a milder, quicker fixation (4% PF-0.1070 GA) than for the developmental material. Under this modified technique, many outer segments of pinealocytes of charr, cisco, dace, creek chub, eel, zebrafish and black moli were rhodopsinand OS-2-immunoreactive, but COS- I-negative, as

31 1

Fig. 4. Details of the adult retina. u. OS-2-immunoreactiverods (R) and immunonegative cones (asterisks); P, pigment epithelium. (Creek chub, x 700.) b. COS-I-immunoreactiveaccessory cone (asterisk) and positive rods (R). (Black moli, X 1150.) c,d. COS-1immunoreactive rod (R) and accessory cone (AC); asterisk cilium; IS, inner segment. (Zebrafish, c, x 15000; d, x 15600.)

usual (Fig. 34b). Some pineal outer segments displaying “coarse” membrane lamellae, were immunonegative with the rhodopsin and 0 s - 2 antibodies in charr, zebrafish and eel (Fig. 3 4 . These immunonegative “cone-like” pinealocytes - lacking cross-reactivity with the antisera or having masked antigenic sites - are suggested to have red and/or u.v./violet-sensitive pigments, since in pike pineal light absorption maxima were measured not only at 530 nm but also at 380 and 620 nm (Falcon and Meissl, 1981). In the retina (except ofthe eel displaying particular antigenicities, I. Vigh-Teichmann, 1991),many rods were rhodopsin-, 0s-2- and COS-1-positive, but the short and long single cones and principal members of paired cones were still immunonegative (Fig. 4a - c). Microspectrophotometric data show that

short single cones are u.v.- or violet-sensitive, while the principle and long cones contain red-sensitive pigment (Downing et al., 1986; Lythgoe and Partridge, 1989). Interestingly, the accessory members of paired cones could be identified with the rhodopsin antiserum that cross-reacts with porphyropsin. Moreover, when Na-borohydride was omitted prior to the immunoreaction, the accessory cones immunoreacted with the COS-1 antibody (binding to green- and red-sensitive pigments in higher vertebrates), while they were immunonegative with the 0s-2 antibody (Fig. 4b,d). So far, microspectrophotometry of accessory cones revealed greensensitive rhodopsin and/or porphyropsin in accessory cones of most teleosts (Downing et al., 1986).

312

Discussion The cited experimental data and our immunocytochemical results indicate that the retinal rods and accessory cones as well as the rhodopsinand OS-2-immunoreactive “rod-like” pinealocytes contain green-sensitive rhodopsin and/or porphyropsin. The absence of COS-1 immunoreactivity of the pineal photopigment, further the borohydride sensitivity and absence of 0 s - 2 immunoreactivity of the accessory retinal cone pigment may be caused by minor differences in the amino acid sequence of the respective photopigments compared to that of the retinal rods. An analysis of the amino acid sequence of these greensensitive pigments is needed to verify this assumption. With regard to the photoreceptor development in charr, the differentiation of the pineal organ is more advanced distally in the embryonic end-vesicle, where “rod-like” pinealocytes, intrinsic neurons and synapses were found. In the prehatching embryonic retina, rods, paired and single cones were already differentiated. These results contradict the view that the rod photoreceptor cells would appear later than cones at the end of the larval life of the metamorphosing charr, but speak in favor of early rod functioning. Principally, the teleostean pineal organ serves to perceive direct skylight first of all as luminance device by its many “rod-like” pinealocytes, while the retina analyses reflected light as luminance (rod) and color (cone) detector of objects. When there is no shelter above the head of the charr larva, some pineal photoreceptors obviously perceive light and transmit this information via the secondary neurons and pineal tract to motoric brain-stem centers; the animal quickly turns off and hides from light (negative phototaxis). Since the hidden larva may still perceive reflected light with the well-developed dorsal and caudal quarters of the retina, first of all the pineal photoreceptors seem to play a role in the negative phototaxic response of the larvae. On the basis of the cytologically and neurochemically differentiated photoreceptors, both the retina and the

pineal organ are able to function as light perceiving structures in the developing salmonid. Further studies are in progress to clarify whether the immunonegative “cone-like” pinealocytes represent red- and/or u.v./violet-sensitive cells. Such cells are supposed by us to play a role in the negative or positive phototaxic responses of teleosts, and further in the detection of daily and seasonal changes of skylight quality ingeneral, similarly to the pattern in the frog pineal organ (Vigh-Teichmann and Vigh, 1990).

Acknowledgements This study was supported by the Hungarian OTKA Grant nr. 1109.

References Carey, W.E. and Noakes, D.L.G. (1981) Development of photobehavioral responses in young rainbow trout, Salmo gairdneri Richardson. J. Fish Biol., 19: 285 -296. Downing, J.E.G., Djamgoz, M.B.A. and Bowmaker, J.K. (1986) Photoreceptors of a cyprinid fish, the roach: morphological and spectral characteristics. J . Comp. Physiol., A, 159: 859-868.

Evans, B.I. and Fernald, R.D. (1990) Metamorphosisand fish vision. J. Neurobiol.. 21: 1037- 1052. Falcon, J. and Meissl, H. (1981) The photosensory function of the pineal organ of the pike (Esox lucius L.). Correlation between structure and function. J. Comp. Physiol., 144: 127137.

Lythgoe, J.N. and Partridge, J.C. (1989) Visual pigments and the acquisition of visual information. J. Exp. Biol., 146: 1 - 20. Negishi, K., Stell, W . K . and Takasaki, Y. (1990) Early histogenesis of the teleostean retina: studies using a novel immunochemical marker, proliferating cell nuclear antigen (PCNA/cyclin). Dev. Brain Res., 5 5 : 121 - 126. Ostholm, T., Brannas, E. and Van Veen, T. (1987) The pineal organ is the first differentiated light receptor in the embryonic salmon, Salmo salar L. Cell Tissue Res., 249: 641 -646. Vigh, B. and Vigh-Teichmann, I . (1988) Comparative neurohistology and immunocytochemistry of the pineal complex with special reference to CSF-contacting neuronal structures. Pineal Res. Rev.. 6: 1 -65. Vigh, B . , Vigh-Teichmann, I., Reinhard, I., Szd, A. and Van Veen, T. (1986) Opsin immunoreaction in the developing and adult pineal organ. In: D. Gupta and R.J. Reiter (Eds.), The Pineal Gland during Development, Croom-Helm, London, Sydney, pp. 31 -42. Vigh-Teichmann, I. (1991) Immunocytochemktry of Pineal

313

Photoneuroendocrine Organs, Thesis, Hungarian Academy of Sciences, Budapest, 135 pp. Vigh-Teichmann, I. and Vigh, B. (1983) The system of cerebrospinal fluid contacting neurons. Arch. Histol. Jpn., 46: 427 - 468. Vigh-Teichmann, I. and Vigh, B. (1985)CSF-contacting neurons and pinealocytes. In: B. Mess, Cs. Ruzsas, L. Tima and P. Pevet (Eds.), Current State of Pineal Research, Akademiai Kiado, Budapest, pp. 71 - 88. Vigh-Teichmann, I. and Vigh, B. (1990) Opsin immunocytochemical characterization of different types of photoreceptors in the frog pineal organ. J. Pineal Res., 8 : 323 - 333. Vigh-Teichmann, I., Korf, H.-W., Nurnberger, F., Oksche, A.,

Vigh, B. and Olsson, R . (1983) Opsin-immunoreactive outer segments in the pineal and parapineal organs of the lamprey (Lampetrafluviatilis), the eel (Anguilla anguilla) and the rainbow trout (Salmogairdneri). Cell Tissue Rex, 230: 289 - 307. Vigh-Teichmann, I., SzeI, A., Rohlich, P. and Vigh, B. (1990)A comparison of the ultrastructure and opsin immunocytochemistry of the pineal organ and retina of the deep-sea fish Chimaera monstrosa. Exp. Biol.. 48: 361 - 311. Vigh-Teichmann, I., Ali, M.A., %el, A. and Vigh, B. (1991) Ultrastructure and opsin immunocytochemistry of the pineal complex of the larval Arctic charr Salvelinus alpinus: a comparison with the retina. J . Pineal Res., 10: 196- 209.