The retinal projections in the goldfish: An experimental study

BRAIN RESEARCH

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THE R E T I N A L P R O J E C T I O N S I N THE GOLDFISH: A N E X P E R I M E N T A L STUDY

S. C. SHARMA

Biology Department, Washington University, St. Louis, Mo. 63130 (U.S.A.) (Accepted October 6th, 1971)

INTRODUCTION

The common goldfish, Carassius auratus, has been widely studied experimentally by anatomists, physiologists and psychologists to investigate its visual system. Most such studies have taken into consideration the primary center of the retinal termination, i.e., the optic tectum, the anatomical structure of which has been outlined in detail by Leghissa 16. The highly specific retinal projections to the tectum in goldfish have been shown anatomically by Attardi and Sperry z and electrophysiologically by Jacobson and Gaze 12. However, to date, very little information is available regarding the retinal projections to various thalamic nuclei in goldfish brain. An effort was made by Schnitzlein 18 using rapid Golgi and pyridine silver impregnation techniques to show the various thalamic nuclear masses in 4 teleosts including goldfish. The major differences in the appearance and the relations of the thalamic nuclear masses were reported. Previous studies on the visual projections in fishes have been reviewed by Schnitzlein is. The failure of older techniques to identify the precise location of the axonal terminals and the postsynaptic cells demand the reinvestigation of the earlier studies using the new and improved methods for staining the axonal degeneration. The recent advances in the neuroanatomy of the visual pathways have been made possible by modified Nauta techniques for the staining of degenerating nerve terminals 11. The reinvestigations of the visual projections in the teleosts using these modified techniques have been reported recently by Ebbesson 7 in Opsanus tau and Gymnothorax funebris and Campbell and Ebbesson 5 in Holocentrus. These studies have shown in detail the various pathways taken by,the degenerating optic nerve fibers and their sites of termination. The present study is concerned with the retinal projections in the'goldfish. METHODS

Twenty adult specimens of goldfish (Carassius auratus) were used. Unilateral eye enucleations were carried out under tricaine anesthesia (Sigma, MS-222). Animals Brain Research, 39 (1972) 213-223

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were kept at constant water temperature of 22°C and were sacrificed after 5-30 days following the surgery. The brains were removed after perfusion with 1 0 ~ formolsaline and stored in 1 0 ~ formalin. The method for embedding and sectioning was that of Ebbesson 6. Thirty micra thick sections were processed with (1) Ebbesson and Rubinson 9 modification of Nauta's technique, (2) Fink and Heimer 11 method, and (3) the Fernstrom 1° modification of the Nissl method. The brains were also impregnated with rapid Golgi and Golgi-Cox ~7 methods. The optimal surviving time, following surgery, showing degenerating axonal debris was 10 days. In Figs. 1-3, large dots and interrupted lines indicate degenerating fibers of passage and small dots show loci of terminal degeneration. RESULTS Animals, studied 5 days after eye enucleation, did not show extensive degenerating material in the optic pathways. Best results were obtained where brain was fixed 7-14 days following enucleation. The terminology used in this paper for the nuclei in thalamus is that of Schnitzlein 18 for teleosts, and for the various layers in the optic tectum is that of Leghissa 16 and Jacobson and Gaze 12. There is a complete decussation of optic nerves in the chiasma which lies ventral to the telencephalo-diencephalic junction below the dorsal hypothalamus (pre-optic area). No degeneration was observed in the ipsilateral optic centers. Both coarse and fine granules are seen in the degenerating optic tract. The optic tracts are large and cover the rostral diencephalic surface.

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Fig. 1. Transverse section, drawn from Fink and Heimer preparation, rostral to the telencephalodiencephalic junction of the brain at the level of optic chiasma. Degenerating debris of the optic axons is drawn. Abbreviations: A, marginal outer layer of the tectum; APr, area pretectalis; APL, area preopticus lateralis; AVLT, area ventralis lateralis of the thalamus; B, piano delle fibre afferenti of Leghissa; C, piano plessiforme externo of Leghissa; Cer, cerebrum; D, piano plessiforme delle fibre retiniche of Leghissa; E, internal plexiform layer; F, stratum fibrosum periventriculare; fMTrO, fasciculus medialis tractus opticus; G, stratum griseum periventriculare; GN, geniculate nucleus; NC, nucleus corticus; NDL, nucleus dorsolateralis pars lateralis; NDM, nucleus dorsolateralis pars medialis; NPM, nucleus preopticus pars magnocellularis; NPP, nucleus preopticus pars parvocellularis; NR, nucleus rotundus; OT, optic tectum; TL, torus longitudinalis; TrO, tractus opticus. Brain Research, 39 (1972) 213-223

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RETINAL PROJECTIONS IN GOLDFISH

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Fig. 2. Transverse section, drawn from Fink and Heimer preparation, at the rostral level of the optic tectum. For abbreviations see Fig. 1. The optic tracts, at the caudal end of the chiasma, come into relation with the hypothalamus. A dorsomedial tract of the optic nerve leaves it at the level of chiasma towards area preopticus lateralis. A few branches of this tract terminate on the large cells of the nucleus preopticus, pars magnocellularis (Fig. 1). This branch of the optic tract is called the fasciculus medialis tractus opticus (Figs. 1 and 2). A few fibers from this tract bend towards the main optic tract and join it. A small number of fibers are seen terminating on the dorsal side of the area preopticus lateralis, medial to the optic tract. The optic terminations in the nucleus preopticus pars parvocellularis were never seen. The diencephalic areas in goldfish are fused caudally across the ventricle at the junction of the ventral thalamus and hypothalamus. The thalamus has a definite nucleus dorsomedialis, a nucleus dorsolateralis, and a nucleus rotundus. Slightly caudal to the preoptic area, the optic tract divides into two major subdivisions, a medial and a lateral tract of the optic nerve. The medial tract fibers terminate into the rostral and dorsal part of the optic rectum, whereas, the lateral tract fibers innervate the ventral and lateral parts of the tectum. Between the two divisions of the optic tract lies a spherical nucleus called nucleus rotundus 18. Rather large sized, densely packed cells form the outer boundary of this nucleus. A few degenerating axons were seen entering this nucleus and terminating in the center of the nucleus, which contains loosely arranged cells (Fig. 4). Medial to the nucleus rotundus and the optic Brain Research, 39 (1972) 213-223

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Fig. 3. Transverse section, drawn from Fink and Heimer preparation, slightly caudal to Fig. 2 showing different layers of optic fiber degeneration in the tecturn. Abbreviations see Fig. 1. tracts lies the nucleus dorsolateralis thalami. Both lateral and medial part of this nucleus receive optic fibers (Fig. 3). A trajectory of the lateral tract of the optic nerve, just ventral to the nucleus rotundus extends medially and bends towards area ventralis lateralis of ventral thalamus and terminates in the vicinity of large sized cells of that area (Fig. 3). Lateral geniculate nucleus in goldfish is composed of small sized ceils packed into an elongated sheet. This nucleus lies laterally to the nucleus rotundus and is placed in between two major tracts of the optic nerve. The fibers from the optic tract or its branches terminate on these cells. This nucleus is connected by small fascicles to the optic tectum (Figs. 2 and 5). A few optic fibers terminate in area pretectalis, which lies medial to the medial optic tract and slightly dorsal to the nucleus rotundus (Fig. 2). This area extends caudally joining the tegmentum. A few large cells are grouped together at the ventromedial part of the tectum, receiving a few optic fibers in the area termed area corticalis which is not very well marked in goldfish (Fig. 3). Most of the degenerating optic axons are distributed in the optic tectum. The medial optic tract fibers are grouped into fascicles which expand rostrocaudally Brain Research, 39 (1972) 213-223

Fig. 4. Nucleus rotundus and geniculate nucleus of goldfish. Fink and Heimer preparation reveals degenerating retinal axons in the nucleus rotundus. Geniculate nucleus is the long band of dark granules on the right hand side of the nucleus rotundus (arrow). x 700.

Fig. 5. A single optic fiber terminal as seen in rapid Golgi preparation. Arrow indicates the optic fiber in outermost area of the tectum (A). The bell shaped terminal is in the area C. Bar represents 30 ttm.

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Fig. 6. Photomicrograph of a transverse section of the caudal optic tectum, showing the degenerating axonal debris in B, C, and D layers. Modified Nauta technique, x 750.

b e n d i n g laterally. The degenerating fascicles, when seen in the transverse sections, are groups o f large granules; each g r o u p o f fascicles is separate from one a n o t h e r and are obliquely oriented in transverse sections. These fascicles are more a p p a r e n t on the mediodorsal surface of the tectum. Most o f those fascicles are seen in area B Brain Research, 39 (1972) 213-223

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Fig. 7. Transverse section of the rostral optic tectum. Degenerating debris is distributed in layers B, C, D, and F. Modified Nauta technique. Layers A and G are not shown. × 750.

and C of the tectum which are outer layers of stratum griseum et fibrosum superficiale of Leghissa. These fiber bundles are longitudinally oriented. A very few fibers in the outermost layers, i.e., stratum opticum, have been seen with Fink and Heimer method 11. When seen with Golgi rapid preparation, the outermost layer contains a few fibers which travel a distance in this layer and bend ventrally terminating in layer B and C (Fig. 5). The majority of optic fibers terminate in layers B, C, and D (stratum griseum et fibrosum superficiale). The fibers from the lateral optic tract enter the tectum in layers B, C, and D and expand ventrally and caudally terminating all along its way in these layers (Fig. 3). A remarkable difference in the area of optic nerve termination in the rostral and caudal part of the tectum has been observed. In the caudal part of the tectum, the degenerating pattern of the optic nerve is restricted to B, C, and D layers only (Fig. 6). However, in the rostral part of the rectum a branch of the lateral optic tract Brain Research, 39 (1972) 213-223

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enters the layer F (stratum fibrosum periventriculare). These fibers, although very few in number, expand lateromedially and terminate in layer F, probably near the large pyramidal cells. A few scattered degenerating granules were also seen on the rostrolateral aspect of layer E (Fig. 7). There is a lack o f organized layering of degenerating granules in any zone of the optic tectum. A large number of coarse fibers are found in layer D. A few fine irregular degenerating granules are seen in all the degenerating layers. DISCUSSION

Area preopticus lateralis and the nucleus preopticus, pars magnocellularis, both part of the hypothalamus, receive optic nerve fibers from fasciculuz medialis tractus optici which arise at the level of optic chiasma. This tract has recently been described by Ebbesson 7 and Campbell and Ebbesson ~ in 3 different teleosts. A few fibers of this tract rejoin the optic nerve. The present findings confirm the observations of Bellonci 3, Jansen 13 and Campbell and Ebbesson 5. Ebbesson 7 described that a branch of this tract innervates the nucleus preopticus pars parvocellularis in toadfish and eel. Such a termination has not been observed in the present study. The hypothalamic projection of the optic nerve has been observed in elasmobranchs s, teleosts 5 and amphibianslL The significance of these projections has not yet been determined. The termination of optic fibers in the area ventralis lateralis of the thalamus was seen using Fink and Heimer method. However, no such termination was reported by Schnitzlein 18 who also used the same animal. The presence of such termination has been reported recently by Campbell and Ebbesson 5 in Holocentrus. The optic fiber innervation to the nucleus rotundus has been reported in the present findings; however, Schnitzlein is was unable to see such termination in goldfish. Lubson (according to Brickner 4) reported the entrance of medial optic tract fibers into the nucleus rotundus, and his findings are corroborated in this investigation. Both pars medialis and pars lateralis of the nucleus dorsolateralis receive optic fibers. Schnitzlein 18 reported the optic fiber innervation to pars lateralis but not to pars medialis in goldfish. The nucleus geniculatus lateralis has been reported by various authors in teleosts, and its location appears to vary in different species 18. In goldfish, this nucleus is a sheet of deeply staining small cells without apparent lamination which lies lateral to the nucleus rotundus. Terminations of optic nerve fibers to this nucleus in the teleostean brain have been previously reported by several authors. Bellonci 3 suggested that only the collaterals of optic axons terminate in this nucleus. In the present study it was not possible to clarify this point. The dorsomedial tract of the optic nerve 13 terminates in the area pretectalis 18. Area pretectalis was a recipient of optic nerve fibers in the present study. This area is similar to the nucleus pretectalis dorsomedialis of Ebbesson 7 and area pretectalis in elasmobranchs 6. This area is included in the nucleus tegmenti motorius dorsalis Brain Research, 39 (1972) 213-223

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of Brickner 4. The division of this nucleus into dorsal and ventral parts, as described by Ebbesson 7 in moray eel and toadfish, was not observed in goldfish. There is a continuous uniform degeneration in this area. The visual projection to the nucleus corticalis as described by Schnitzlein 18 and Ebbesson 7 is the same as seen in the present study. The presence of an accessory optic tract, as described by Campbell and Ebbesson 5 in Holocentrus, was not observed in the present study. This discrepancy may be due to species difference. The role played by various projections of the optic nerve to thalamic and hypothalamic areas is questionable. Do the projections to the hypothalamus control light-triggered seasonal endocrine changes? This remains an open question. Similarly, do the thalamic nuclei which receive optic innervations act like relay centers for various visual modalities? Experiments involving selective ablation of these centers might help elucidate these points. Retino-tectalprojections. The orderly projection of the retinal fibers to the optic tectum in teleosts has been described by degenerating and regenerating techniques. Some of the important studies in this category are those of Akert 1, Leghissa 16, Attardi and Sperry 2. Similarly, point to point electrophysiological representation of the visual field onto the tectum in teleosts has been shown by Jacobson and Gaze 1~ and Schwassman and Kruger 19. The present study reveals in detail the terminations of the degenerating optic fibers in various layers of the tectum. Most degenerating optic fibers enter the tectum in fascicles through layers B, C, and D of the stratum griseum et fibrosum superficiale 7. However, a few fibers stained with Fink and Heimer method 11 and with rapid Golgi method reveal the presence of very few fibers entering the tectum via the most superficial layer 14. Jacobson and Gaze 12 studied visually evoked unit responses from the goldfish optic rectum. They reported a lack of activity in the outermost layer (up to 50-100 # m from the surface of the tectum) to any visual stimulation. Termination of optic nerve fibers in area A was never seen either in Golgi or Fink and Heimer preparations. The source of visual responses recorded in their study has been attributed to presynaptic terminations of the arborizing optic nerve fibers. Their inability to find any orderly sequence of different types of units in areas B and C is corroborated by the fact that retinal fiber degeneration is not organized in any layering in the tectum. However, the distinctiveness of layer D in having mostly sustained responses is difficult to account for since no distinct difference was observed in areas B, C, and D. There were no visual responses in layer E which was approximately 200 /*m thick 12. in the present study very sparsely scattered optic fibers were observed, restricted only to the rostrolateral aspect of the tectum. Jacobson and Gaze lz recorded visual responses in layer F (stratum fibrosum periventriculare of Leghissa) and reported that 'the responses were only recorded from the rostral half of the tectum and that the more rostral the electrode track the more easily were the responses recorded'. Layer F in the caudal part of the tectum does not have any optic fiber terminations. However, degenerating optic axonal debris, restricted to the rostral part of the tectum, was seen in layer F in the present

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study. Similar fibers in layer F have been reported recently in Holocentrus 5. The specific role of the optic fibers in layer F is not clear at present. SUMMARY

The retinal projections of a teleost fish (goldfish, Carassius auratus) are studied using the Nauta-Gygax and Fink-Heimer methods following unilateral eye enucleation. Totally crossed projections were revealed to (1) a small magnocellular nucleus of the hypothalamus; (2) nucleus rotundus; (3) medial and lateral part of the nucleus dorsolateralis thalami; (4) area ventralis lateralis of thalamus; (5) lateral geniculate nucleus; (6) area pretectalis; (7) area corticalis; and (8) the optic tectum. Projections to layer F were restricted to the rostral tectum only. No accessory optic tract was found. ACKNOWLEDGEMENT

This work was supported by Grant NB 0571 of the U.S. Public Health Service to Dr. V. Hamburger.

REFERENCES 1 AKERT, K., Experimenteller Beitrag betr. die Zentrale Netzhaut-Repr/isentation in Tectum Opticum, Schweiz. Arch. Neurol. Psychiat., 64 (1949) 1-16. 2 ATTARDI, O. G., AND SPERRY, R. W., Preferential selection of central pathways by regenerating optic fibers, Exp. Neurol., 7 (1963) 46-64. 3 BELLONCI,J., I~)ber die Zentrale Endigung des Nervus opticus bei den Vertebraten, Z, wiss. Zool., 47 (1888) 146. 4 BRICKNER, R. M., A description and interpretation of certain parts of the teleostean midbrain and thalamus, J. comp. Neurol., 47 (1929) 225-282. 5 CAMPBELL, C. B. G., AND EaaESSON, S. O. E., The optic system of a teleost: Holocentrus Reexamined, Brain Behav. Evol., 2 (1969) 415-430. 6 ErmESSON, S. O. E., Ascending axon degeneration following hemisection of the spinal cord in the Tegu lizard (Tupinambis nigropunetatus), Brain Research, 5 (1967) 178-206. 7 EnBESSON, S. O. E., Retinal projections in two teleost fishes (Opsanus tau and Gymnothorax funebris). An experimental study with silver impregnation methods, Brain Behav. Evol., 1 (1968) 134-154.

8 EaaESSON, S. O. E., AND RAMSEY, J. E., The optic tracts of two species of sharks (Gialeocerdo cuvier and Ginglymostoma cirratum), Brain Research, 8 (1968) 36-53. 9 EBBESSON,S. O. E., AND RUBINSON, K., A simplified Nauta procedure, Physiol. Behav., 4 (1969) 281-282. 10 FERNSTROM, R. C., A durable Nissl stain for frozen and paraffin sections, Stain Technol., 33 (1958) 175-176. 11 FINK, R. P., AND HEIMER, L., Two methods for selective silver impregnation of degenerating axons and their synaptic endings in the central nervous system, Brain Research, 4 (1967) 369-374. 12 JACOBSON, M., AND GAZE, R. M., Types of visual responses from single units in the optic tectum and optic nerve of the goldfish, Quart. J. exp. Physiol., 49 (1964) 199-209. 13 JANSEN, J., A note on the optic tract in teleosts, Proc. kon. ned. Akad. Wet., 32 (1929) 1104-1117. 14 KAPPERS, A. C. U., HUBER, G. C., AND CROSBY, E. C., The Comparative Anatomy of the Nervous System of Vertebrates Including Man, Hafner, New York, 1960. 15 KNAPP, H., SCALIA, F., AND RISS, W., The optic tracts of Rana pipiens, Acta neurol, scand., 41 (1965) 325 355.

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16 LEGHISSA,S., La Strutture microscopia e la citoarchitettonica del tetto ottico dei pesci teleostei, Z. Anat. EntwickL-Gesch., 118 (1955) 427-463. 17 RAMON-MOLINER, E., A tungsten modification of the Golgi-Cox method, Stain Technol., 33 (1958) 19-20. 18 SCHNITZLEIN,Ho N., The habenula and dorsal thalamus of some teleosts, J. comp. Neurol., 118 (1962) 225-267. 19 SCHWASSMAN,H. O., AND KRUGER, L., Anatomy of the visual centers in teleosts. In D. INGLE (Ed.), Central Nervous System and Fish Behavior, Univ. of Chicago Press, Chicago, 1968, pp. 3-17.

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