Afferent and Efferent Fiber Connections of the Carp Torus Longitudinalis HIRONOBU IT0 AND REIJI KISHIDA Department of Anatomy, Osaka University Medical School, Osaka, and Department Anatomy, Yokohama City Uniuersity School of Medicine, Yokohama, Japan
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ABSTRACT The efferent and afferent pathways of the carp torus longitudinalis were studied by means of degeneration and retrograde HRP methods. Efferent projections were only seen in the most superficial layer of the ipsilateral optic tectum (stratum fibrosum marginale). Afferent pathways to the torus longitudinalis were found to originate mainly in the valvula cerebelli. Degenerating fibers course in the tractus mesencephalocerebellaris posterior within the valvula, and join the tractus mesencephalocerebellaris anterior in the tegmentum. The fibers which ascend in the tract gradually invade the optic tectum through which they are distributed t o the torus longitudinalis. The remaining fibers pass through the posterior commissure and terminate in the torus longitudinalis at the rostra1 end of the tract. Degenerating terminals were also seen in the torus longitudinalis when lesions were made in the optic tectum, tectal commissure, torus semicircularis, and in the area between the valvula and the corpus cerebelli. The possibility of projections from these areas is discussed depending upon the results of the retrograde HRP method. The teleostean tori longitudinales are a pair of longitudinal ridges which extend from the medial border of the optic tectum into the mesencephalic ventricle. The torus longitudinalis has been thought to play a role in opticostatic correlation, judging from anatomical studies of normal fibers (Ariens-Kappers et al., ’361,and from the absence or the lesser development of the structure in some deep sea fishes (Tuge and Shimamura, ’58). Fine structural investigation of the carp torus longitudinalis clarified that the small cell profiles as well as the closely packed cell pattern and peculiar synaptic configurations of so-called “synaptic glomeruli” are strikingly similar to those found in the granular layer of the valvula cerebelli (Ito, ’71). Although there have been descriptions of the relationship between the torus longitudinalis and the marginal layer of the optic tectum (Radl, ’15; Kudo, ’23; Leghissa, ’55), no experimental works have been published which deal with the fiber connections of the torus longitudinalis itself. This is the first experimental study on the fiber connections of the structure as disclosed by means of FinkJ. COMP. NEUR. (1978)181: 465-476.
Heimer and retrograde horseradish peroxidase (HRP) methods. MATERIALS AND METHODS
Cyprinus carpio was selected for the present study because of the well developed torus longitudinalis. Although the tori longitudinales of both sides lie close to each other in small specimens (below 15 cm in body length), each torus longitudinalis separates in large specimens and resembles an inverted V in the dorsal view. Therefore, lesions were easily made on the torus longitudinalis without involvement of neighboring structures in large specimens. Seventy-one specimens (20-30 cm in body length) obtained from commercial sources were used in this study. Anesthesia was initiated by immersion in an approximately 1:2,000 solution of tricaine methansulfonate (MS 222) and maintained by a more dilute solution. Operative procedures were performed during gill perfusion with aerated water. Degeneration experiments Most lesions were produced by extripation
465
466
HIRONOBU I T 0 AND REIJI KISHIDA
or electric coagulation of various structures. In some areas, hemisections and incisions were performed (table 1). Lesions of t h e valvula cerebelli and of the torus longitudinalis are illustrated in figures 1and 2. Use of a caudal approach between the tectal commissure and the corpus cerebelli, where the valvula cerebelli extends anteriorly from the corpus to protrude into the mesencephalic
ventricle, allows easy access to the dorsal surface of t h e valvula and also to the torus semicircularis without involving the optic tectum. The animals were maintained postoperatively a t 15-20°Cand sacrificed after 7 to 14 days by intracardial perfusion with 0.7% saline containing 5-10 I.U. heparin per milliliter and followed by 10% formalin. The brains were removed and stored in 10%formalin under re-
Abbreviations in tables and figures
CC, Corpus cerebelli CP, Commissura posterior CR, Crista cerebelli CT, Cornmissura tectalis EP, Epiphysis FLL, Fasciculus longitudinalis lateralis FLM, Fascieulus long7tudinalis medialis FR, Formatio reticularis LI, Lobus inferior LV, Nucleus lateralis valvulae MCA, Tractus mesencephalocerebellaris anterior MCP, Tractus mesencephalocerebellaris posterior
SAC, Stratum album centrale SFGS, Stratum fibrosum e t griseum superficiale SG, Secondary gustatory nucleus SGC, Stratum griseum centrale SM, Stratum fibrosum marginale SO, Stratum opticum SP, Spinal cord SPV, Stratum griseum periventriculare TE, Telencephalon TL, Torus longitudinalis TO, Tectum opticum TS, Torus semicircularis VC, Valvula cerebelli
Fig. 1 Lesions of t h e valvula cerebelli. The areas which were actually removed by aspiration are indicated by black, and th e areas which were electrically coagulated are indicated by dots.
467
CARP TORUS LONGITUDINALIS TABLE
1
Degeneration Animal No.
Projection area
TL
54(e), 55(e), 69(e), 83(e)
ip-SM(TO),eo-TL
Lesion
Animal No.
Degen. term. in TL
Lesion
TE
EP _. LI
95(e), 96(e), 127h) 100(r)
(-)
(-1 (-1 (+ +) bilateral ( + ) bilateral ( + 1 bilateral
TO CT TS
+ ( + + ) bilateral
vc vc-cc cc
(+)
ipsilateral
(-1 (-J
CC-CR CR SP
(-) (-)
a, aspiration; e, electric coagulation; h. bemisection; i, incision; r, removal;
contralateral;
ipailateral
TABLE 2
HRP Inject. site
Animal No.
Areas of labeled cello
TL
61, 62,77, 86, 91, 92
ip-TO(?),bi-TS(?), bi-FR(?J, ip-VC
Inject. site
Animal No.
Labeled cells in TL
TE TO
66, 87, 88, 104-107, 112-121 59, 60, 75, 76, 85, 89, 90 63,64 65
(-) (+ + ) (-) (-)
vc cc bi, bilateral; ip, ipsilateral
Fig. 2 Sites of lesions (black areal and HRP-injection (star) on the torus longitudinalis and the optic tectum.
468
HIRONOBU IT0 AND REIJI KISHIDA
tus mesencephalocerebellaris posterior. The fibers then penetrate the nucleus lateralis valvulae (fig. 3) and enter the tractus mesencephalocerebellaris anterior which is located on the dorsomedial border of the fasciculus longitudinalis lateralis. At this level some degenerating fibers turn medially across the base of the tegmentum to join the same tract Horseradish peroxidase (HRP) of the opposite side. The tract travels rostrally A 0.2 pl of 35% solution of HRP in saline along the dorsomedial or medial side of the (0.6%)was injected with a microsyringe into fasciculus longitudinalis lateralis. After the the torus longitudinalis or into other struc- fasciculus spreads out into the torus semicirtures (table 2). The sites of injection into the cularis, the tract changes position dorsally. torus longitudinalis and t h e optic tectum are The tractus mesencephalocerebellaris anteriillustrated in figure 2. From two to five days or shows the largest diameter beneath the nuafter injection, the animals were perfused in- cleus mesencephalicus nervi trigemini, and tracardially with 0.7%saline containing 5-10 tapers to disappear a t the level of the posterior I.U. heparin per milliliter followed by a mixed commissure. At this level heavy axonal and solution of 1.6%paraformaldehyde and 10% terminal degeneration is seen in the nucleus glutaraldehyde in 0.1 M phosphate buffer (pH glomerulosus of Schnitzlein ('62) and in the 7.4).The brains were removed from the skulls, inferior lobe. Some degenerating fibers graduand postfixed in fresh fixative for 5 to 12 ally leave the tract throughout its longitudihours, and rinsed for 24 hours in 0.1 M phos- nal extent, and course dorsolaterally up to t h e phate buffer (pH 7.4) containing 5% sucrose. optic tectum. Degenerating fibers which inAfter being embedded in egg yolk (Ebbesson, vade the optic tectum of the opposite side '701, frontal serial sections were cut a t 60 could not be traced further. In the ipsilateral pm and reacted after 3,3'-diaminobenzidine optic tectum degenerating fibers course dor(Luiten, '75). After sections were individually somedially in the stratum album centrale givmounted on slides with gelatin alcohol, alter- ing off some debris around the layer (fig. 4) native slides were stained with cresyl violet and reach the torus longitudinalis where most for histological identification. of them appear to end (fig. 5). A few degenerating fibers pass through the torus longituRESULTS dinalis and run across the tectal commissure to terminate in the opposite torus longitudiI. Afferent connections nalis. At t h e level of the posterior commissure Degeneration some degenerating fibers from the rostra1 end The degeneration in the torus longitudinalis of the tractus mesencephalocerebellaris antewas produced by lesions in the optic tectum, rior enter the commissure and end bilaterally torus semicircularis, and the valvula cerebelli. in the tori longitudinales. These fibers appear Some degeneration debris was also found fol- to correspond to "die frontale Torusverbindlowing electric coagulation of the contralat- ung" described by Kudo ('24). era1 torus longitudinalis, incision of the tectal commissure, or hemisection between t h e HRP In injection of HRP into the torus longitudivalvula cerebelli and corpus cerebelli (table 1). The following is a description of cases in Fig. 3 Degenerating fibers which penetrate the nuwhich lesions were made in the valvula cere- cleus lateralis valvulae following lesion of the valvula cerbelli (fig. 1). The degeneration in the torus ebelli. Case 56, 13-day survival. X 300. longitudinalis resulting from the other lesions Fig. 4 Degenerating fibers in the stratum album centrale of the optic tectum following lesion of the valvula will be referred to in the DISCUSSION. cerebelli. Some degenerating fibers are also seen In the Essentially the same course and distribu- stratum griseum centrale and in the stratum griseum tion of degeneration were seen in all eight ani- periventriculare (arrow). Case 124, 7-day survival. X 300. mals (Nos. 56, 57, 72, 84, 97, 98, 124, and 125). Fig. 5 Degenerating fibers and terminals in the torus Lesions included both the molecular and gran- longitudinalis following lesion of the valvula cerebelli. 125, 7-day survival. X 400. ular layers in all cases. Degenerating fibers Case Fig. 6 HRP-positive cells in the molecular layer of the arise from the lesion and run ventromedially valvula cerebelli following injection of the torus longituthrough the valvula cerebelli to join the trac- dinalis. Case 77, 5-day survival. X 400.
frigeration, and then embedded in egg yolk (Ebbesson, '70) and sectioned frontally a t 60 pm on a freezing microtome. Every fifth section was processed with cresyl violet for histological identification. Other sections were processed according to a modification of the Fink-Heimer technique (Ebbesson, '70).
CARP TORUS LONGITUDINALIS
469
470
HIRONOBU IT0 AND REIJI KISHIDA
Fig. 7A Lesion of the torus longitudinalis covered with clot (arrow). X 20. Terminal degeneration produced by the lesion is shown in figure 8. B Site of HRP injection in the optic tectum (arrow). X 50. Labeled cells in the torus longitudinalis are shown in figure 10.
nalis, diffusion of HRP into the ipsilateral optic tectum was inevitable to some extent because of the close relationship of these two structures. Labeled fibers arise from injection sites of the torus longitudinalis and run laterally through the stratum album centrale of the optic tectum. If the injection site is located near the rostral end of the torus longitudinalis (fig. 2: Nos. 77 and 861, labeled fibers are found in the posterior commissure and in the rostral part of the tractus mesencephalocerebellaris anterior of both sides. The fibers which run laterally into the stratum album centrale leave the optic tectum along the tractus tectobulbaris. A few labeled neurons are
found in the stratum griseum centrale of the optic tectum. After leaving the optic tectum the fibers run ventromedially beneath the torus semicircularis to join the tractus mesencephalocerebellaris anterior. Some labeled neurons are observed in the torus semicircul a r k A t the level of the nucleus lateralis valvulae the labeled fibers turn upwards and enter the valvula cerebelli. Neurons are found to be labeled in the nucleus lateralis valvulae and in the molecular layer of the valvula cerebelli (fig. 6). In addition, several large neurons are labeled in the reticular formation of the mesencephalic tegmentum. No labeled fibers and cells are found caudal to the nucleus lat-
CARP TORUS LONGITUDINALIS
471
Fig. 8 Terminal degeneration confined to t h e stratum fibrosum marginale of the optic tectum following lesion of the torus longitudinalis. Case 69, 10-day survival. X 300. Fig. 9 Degenerating fibers which run in the stratum album centrale and penetrate dorsally through the stratum griseum centrale following lesion of the torus longitudinalis. Case 83, IOaday survival. x 200. Fig. 10 HRP-positive cells in the torus longitudinalis following a tectal injection. Case 75,5-day survival. X
400.
472
HIRONOBU IT0 AND REIJI KISHIDA
Fig. 11 Semi-diagrammatic drawing of afferent connections of the torus longitudinalis. Transverse sections pass through the (A) rostral part of t h e posterior commissure, (B) mid-level of the posterior commissure, (C) level of the nucleus mesencephalicus n. trigemini, (D)rostral end of the fasciculus longitudinalis lateralis, (E)mid-level of the nucleus lateralis valvulae, and through the (F)secondary gustatory nucleus. The course of fibers and the terminal fields are indicated by arrows and Y-shaped bifurcation respectively. Black circles show the cell of origins demonstrated by both degeneration and retrograde HRP methods. Open circles show cells demonstrated only hy the retrograde HRP method.
eralis valvulae. The afferent connections clarified by both degeneration and retrograde HRP methods are summarized in figure 11. 11. Efferent connections Degeneration In order to determine efferent projections,
lesions were made in various parts of the torus longitudinalis (fig. 2: Nos. 54, 55, 69 and 83). All lesions produced degenerating terminals only in the stratum fibrosum marginale of the ipsilateral optic tectum (figs. 7A, 8).Although the dorsomedial margin of the stratum receives degenerating fibers directly from the
CARP TORUS LONGITUDINALIS
torus longitudinalis, the lateral and ventrolatera1 parts of the stratum receive the fibers by way of the stratum album centrale. That is, most degenerating fibers from the torus longitudinalis travel in the stratum album centrale, and then exit as several fiber bundles towards the surface (fig. 9). These fiber bundles pass across t h e upper three layers (stratum griseum centrale, stratum fibrosum et griseum superficiale, and stratum opticum), and spread out in the stratum fribrosum marginale. Following lesions made in the rostral end of the torus longitudinalis (fig. 2: Nos. 54 and 69) degenerating fibers loop lateral-, then ventralwards around the optic tectum. By contrast, following lesions placed in the caudal part (fig. 2: Nos. 55 and 83)degenerating fibers run caudolateralwards in t h e stratum album centrale. HRP Although HRP labeled fibers were rarely seen in the stratum fibrosum marginale except in the vicinity of the injected site, cells in the torus longitudinalis were always labeled by injection of HRP into the optic tectum (figs. 7B, 10). Labeled cells in the torus longitudinalis tended to be localized more rostrally than the levels of the HRP-injected sites in the optic tectum. The labeled cells correspond to the “small” and “medium-sized” cells classified in the previous study (Ito, ’70). When injected sites occurred within deep layers of the optic tectum, labeled cells were found not only in the torus longitudinalis but also in the optic tectum, torus semicircularis, mesencephalic reticular formation, nucleus lateralis valvulae and in the valvula cerebelli. No cells in the torus longitudinalis were labeled by injection into the telencephalon and the cerebellum (table 2). DISCUSSION
1. Afferent connections of the torus longitudinalis Projections to the torus longitudinalis have only been reported following tectal lesions (Ebbesson and Vanegas, ’76; Sligar and Voneida, ’76). In the present study, degenerating fibers in the torus longitudinalis were produced not only by tectal lesions but also by lesions of the torus semicircularis and the valvula cerebelli. A few degenerating terminals were found after transection of the tectal commissure as well as hemisection between the valvula cerebelli and the corpus cerebelli.
473
No degeneration was observed when lesions were made more caudal than the corpus cerebelli. Some of the degenerating fibers which originated in the valvula cerebelli course beneath the torus semicircularis and within the optic tectum. Because of this, degeneration produced by lesions of both areas may be a result of damage of the fibers of passage. Some degeneration found bilaterally in the torus longitudinalis after transection of the tectal commissure might also have been produced as a result of transection of fibers destined t o the contralateral torus longitudinalis. After injection of HRP into the torus longitudinalis, cells in the optic tectum, torus semicircularis, mesencephalic reticular formation, and nucleus lateralis valvae were labeled in addition to those in the valvula cerebelli. However, since the tectal neurons send their axons to the contralateral side, by way of the tectal commissure (Ebbesson and Vanegas, ’76) HRP injected into the torus longitudinalis may be taken up by these axons and transported to the parent cell bodies. In addition, if we consider diffusion of HRP into the optic tectum, which occurred more or less in all cases, and consider similar results obtained by HRP injection into deep portions of the optic tectum, we cannot decide whether the labeled cells of the torus semicircularis, mesencephalic reticular formation and nucleus laterals valvulae project to the torus longitudinalis or to the optic tectum. Animals with a large lesion of the valvula cerebelli had a tendency to sink to the bottom of the water (fig. 1: Nos. 72, 97, 98, 124 and 125). Side-to-side equilibrium was well maintained. Within one hour after operation the functional disorder disappeared in four animals (Nos. 97, 98, 124 and 125), and they began to swim keeping their bodies in the middle level of the water. Animal No. 72 never recovered. Tuge and Shimamura (’58) reported that in some deep sea fish (Cyclothone microdon) the torus longitudinalis was completely lacking, and the valvula cerebelli was hardly detectable in spite of the well developed corpus cerebelli. It may be inferred, therefore, that the valvula cerebelli is associated with some function different from that of the corpus cerebelli, and having close connection with the torus longitudinalis.
II. Efferent connections of the torus longitudinalis Fine structure of the teleostean optic tec-
474
HIRONOBU I T 0 AND REIJI KISHIDA
tum has been studied by several authors (Ito, '70; Laufer and Vanegas, '74; Marotte and Mark, '75; Ito et al., '78a, in manuscript), and special attention has been paid to the stratum fibrosum marginale because of the striking similarity with the molecular layer of the cerebellum. However, only one paper to date has reported projections to the stratum fibrosum marginale (Marotte and Mark, '75). They identified degenerating terminals electron microscopically in the stratum as a result of cutting the tectal commissure. We never found any degeneration in the stratum after transection of the tectal commissure. Degeneration in the stratum was only produced by lesions of the ipsilateral torus longitudinalis or of the ipsilateral optic tectum itself. This result was supported by the present HRP experiment, t h a t is, cells in the torus longitudinalis were labeled following HRP injection into the ipsilateral optic tectum. No attempt was made in Marotte and Mark's study to make lesions in the torus longitudinalis. One possible explanation for the difference may lie in the different methods employed. The torus longitudinalis may also send only a few fibers t o t h e contralateral stratum fibrosum marginale. Many people have attempted to characterize inputs to the optic tectum in relation to the layers. Retinal projections are seen in the stratum opticum and stratum fibrosum et griseum superficiale (Ebbesson, '68; Sharma, '72; Vanegas and Ebbesson, '73; Laufer and Vanegas, '74; Gulley et al., '75; Voneida and Sligar, '76), telencephalic projections are in the stratum griseum centrale (Vanegas and Ebbesson, '76; Ito and Kishida, '77), contralatera1 tectal projections are in a deep area of the stratum griseum centrale (Ebbesson and Vanegas, '76; Ito e t al., '78b, in manuscript), and diencephalic projections a r e distributed throughout t h e optic tectum except t h e stratum fibrosum marginale (It0 et al., '78c, in manuscript). The results of the present study, namely that the torus longitudinalis projects exclusively to the stratum fibrosum marginale, help fill one of the blank spaces of inputs t o t h e optic tectum. ACKNOWLEDGMENTS
We wish to thank Doctor Frank Atencio for his suggestions concerning this manuscript. Thanks are also due to Miss Masami Yoshimot0 for her excellent help in preparing the
histological materials. This research was supported by a grant for scientific research (No. 212113) from the Ministry of Education of Japan. LITERATURE CITED Ariens-Kappers, C. U., G. C. Huber and E. C. Crosby 1936 The Comparative Anatomy of the Nervous System of Vertebrates, Including Man. McMillan, New York (reprinted by Hafner, New York, 1960). Ebbesson, S. 0. E. 1968 Retinal projections in two teleost fishes. An experimental study with silver impregnation methods. Brain, Behav. and Evol., 1: 134-154. - 1970 Selective silver impregnation of degenerating axons and their synaptic endings in non-mammalian species. In: Contemporary Research Methods in Neuroanatomy. W. J. H. Nauta and S. 0. E. Ebbesson, eds. Springer-Verlag, New York, pp. 132-161. Ebbesson, S. 0. E., and H. Vanegas 1976 Projections of the optic tectum in two teleost species. J. Comp. Neur., 165: 161- 180. Gulley, R. L., M. Cochran and S. 0. E. Ebbesson 1975 The visual connections of the adult flatfish,Achirus lineatus. J. Comp. Neur., 162: 309-320. Ito, H. 1970 Fine structures of the carp tectum opticum. J. Hirnforsch., 12: 325-354. 1971 Fine structure of the carp torus longitudinalis. J. Morph., 135: 153-164. Ito, ZT., S. 0. E. Ebbesson and A. B. Butler (1978a, in manuscript) Synaptic organization of the optic tectum in a teleost, Hofocentrus rufus. (1978b, in manuscript) The ultrastructure of degenerating tectal afferents from the retina, the telencephalon and the contralateral tectum in a teleost ( H o b centrus rufus). Ito, H., and R. Kishida 1977 Tectal afferent neurons identified by the retrograde HRP method in the carp telencephalon. Brain Res., 130: 142-145. Ito, H., R. Kishida and Y. Morita 11978c, in manuscript) Diencephaiic projections to the telencephalon and the optic tectum in the carp, Cyprinus caprio and Carassius carassius. Kudo, K. 1923 Uber den Torus longitudinalis. I. Mitteilung rneiner Beitrage zur Anatomie des Zwischen- und Mittelhirns der Knochenfische. Anat. Anz., 56: 359-367. 1924 Beitrage zur Anatomie des Zwischen- und Mittelhirns der Knochenfische. 111. Eine frontale Verbindung des Torus longitudinalis. Anat. Anz., 57: 271-275. Laufer, M., and H. Vanegas 1974 The optic tectumof a perciform teleost. 11. Fine structure. J. Camp. Neur., 254: 61-96. Leghissa, S. 1955 La s truttura microscopica e la citoarchittetonica del tetto ottico dei pesci teleostei. 2. Anat. Engwicklungsgesch., 128: 427-463. Luiten, P. G. M. 1975 The horseradish peroxidase technique applied to the teleostean nervous system. Brain Res., 89: 181-186. Marotte, L. R., and R. F. Mark 1975 Ultrastructural localization of synaptic input to the optic lobe of carp (Carcrssius carassius). Exp. Neur., 49: 772-789. Radl, E. 1915 Zur Morphologie der Sehzentren der Knochenfische. Morph. Jb., Leipzig, 49: 509-535. Schnitzlein, H. N. 1962 The habenula and the dorsal thalamus of some teleosts. J. Camp. Neur., 118: 225-267.
-
CARP TORUS LONGITUDINALIS Sligar, C. M., and T. J. Voneida 1976 Tectal efferent8 in the blind cave fishAstyanuz hubbsi. J. a m p . Neur., 165: 107-124. Sharma, S. C. 1972 The retinal projections in the goldfish: a n experimental study. Brain Res., 39: 213-223. Tuge, H.,and H. Shimamura 1958 Study on the brains of t h e deep sea fishes, Cyclothon microdon, Gonostoma ritiogr and Coryphaenoides acrolepis. Ann. Rept. Jap. Sea Reg. Fish. Res. Lab., 4: 197-220.
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Vanegas, H., and S. 0. E. Ebbeeson 1973 Retinal projections i n the perch-like teleost Eugerres plumieri. J. Comp. Neur., 151: 331-358. 1976 Telencephalic projections in two teleost species. J. a m p . Neur., 165: 181-196. Voneida, T. J., and C. M. Sligar 1976 A comparative neuroanatomic study of retinal projections in two fishes: Astyanuz hubbsi (the blind cave fish), and Astyanux mexicanus. J. a m p . Neur., 165: 89-106.
of
ABSTRACT The efferent and afferent pathways of the carp torus longitudinalis were studied by means of degeneration and retrograde HRP methods. Efferent projections were only seen in the most superficial layer of the ipsilateral optic tectum (stratum fibrosum marginale). Afferent pathways to the torus longitudinalis were found to originate mainly in the valvula cerebelli. Degenerating fibers course in the tractus mesencephalocerebellaris posterior within the valvula, and join the tractus mesencephalocerebellaris anterior in the tegmentum. The fibers which ascend in the tract gradually invade the optic tectum through which they are distributed t o the torus longitudinalis. The remaining fibers pass through the posterior commissure and terminate in the torus longitudinalis at the rostra1 end of the tract. Degenerating terminals were also seen in the torus longitudinalis when lesions were made in the optic tectum, tectal commissure, torus semicircularis, and in the area between the valvula and the corpus cerebelli. The possibility of projections from these areas is discussed depending upon the results of the retrograde HRP method. The teleostean tori longitudinales are a pair of longitudinal ridges which extend from the medial border of the optic tectum into the mesencephalic ventricle. The torus longitudinalis has been thought to play a role in opticostatic correlation, judging from anatomical studies of normal fibers (Ariens-Kappers et al., ’361,and from the absence or the lesser development of the structure in some deep sea fishes (Tuge and Shimamura, ’58). Fine structural investigation of the carp torus longitudinalis clarified that the small cell profiles as well as the closely packed cell pattern and peculiar synaptic configurations of so-called “synaptic glomeruli” are strikingly similar to those found in the granular layer of the valvula cerebelli (Ito, ’71). Although there have been descriptions of the relationship between the torus longitudinalis and the marginal layer of the optic tectum (Radl, ’15; Kudo, ’23; Leghissa, ’55), no experimental works have been published which deal with the fiber connections of the torus longitudinalis itself. This is the first experimental study on the fiber connections of the structure as disclosed by means of FinkJ. COMP. NEUR. (1978)181: 465-476.
Heimer and retrograde horseradish peroxidase (HRP) methods. MATERIALS AND METHODS
Cyprinus carpio was selected for the present study because of the well developed torus longitudinalis. Although the tori longitudinales of both sides lie close to each other in small specimens (below 15 cm in body length), each torus longitudinalis separates in large specimens and resembles an inverted V in the dorsal view. Therefore, lesions were easily made on the torus longitudinalis without involvement of neighboring structures in large specimens. Seventy-one specimens (20-30 cm in body length) obtained from commercial sources were used in this study. Anesthesia was initiated by immersion in an approximately 1:2,000 solution of tricaine methansulfonate (MS 222) and maintained by a more dilute solution. Operative procedures were performed during gill perfusion with aerated water. Degeneration experiments Most lesions were produced by extripation
465
466
HIRONOBU I T 0 AND REIJI KISHIDA
or electric coagulation of various structures. In some areas, hemisections and incisions were performed (table 1). Lesions of t h e valvula cerebelli and of the torus longitudinalis are illustrated in figures 1and 2. Use of a caudal approach between the tectal commissure and the corpus cerebelli, where the valvula cerebelli extends anteriorly from the corpus to protrude into the mesencephalic
ventricle, allows easy access to the dorsal surface of t h e valvula and also to the torus semicircularis without involving the optic tectum. The animals were maintained postoperatively a t 15-20°Cand sacrificed after 7 to 14 days by intracardial perfusion with 0.7% saline containing 5-10 I.U. heparin per milliliter and followed by 10% formalin. The brains were removed and stored in 10%formalin under re-
Abbreviations in tables and figures
CC, Corpus cerebelli CP, Commissura posterior CR, Crista cerebelli CT, Cornmissura tectalis EP, Epiphysis FLL, Fasciculus longitudinalis lateralis FLM, Fascieulus long7tudinalis medialis FR, Formatio reticularis LI, Lobus inferior LV, Nucleus lateralis valvulae MCA, Tractus mesencephalocerebellaris anterior MCP, Tractus mesencephalocerebellaris posterior
SAC, Stratum album centrale SFGS, Stratum fibrosum e t griseum superficiale SG, Secondary gustatory nucleus SGC, Stratum griseum centrale SM, Stratum fibrosum marginale SO, Stratum opticum SP, Spinal cord SPV, Stratum griseum periventriculare TE, Telencephalon TL, Torus longitudinalis TO, Tectum opticum TS, Torus semicircularis VC, Valvula cerebelli
Fig. 1 Lesions of t h e valvula cerebelli. The areas which were actually removed by aspiration are indicated by black, and th e areas which were electrically coagulated are indicated by dots.
467
CARP TORUS LONGITUDINALIS TABLE
1
Degeneration Animal No.
Projection area
TL
54(e), 55(e), 69(e), 83(e)
ip-SM(TO),eo-TL
Lesion
Animal No.
Degen. term. in TL
Lesion
TE
EP _. LI
95(e), 96(e), 127h) 100(r)
(-)
(-1 (-1 (+ +) bilateral ( + ) bilateral ( + 1 bilateral
TO CT TS
+ ( + + ) bilateral
vc vc-cc cc
(+)
ipsilateral
(-1 (-J
CC-CR CR SP
(-) (-)
a, aspiration; e, electric coagulation; h. bemisection; i, incision; r, removal;
contralateral;
ipailateral
TABLE 2
HRP Inject. site
Animal No.
Areas of labeled cello
TL
61, 62,77, 86, 91, 92
ip-TO(?),bi-TS(?), bi-FR(?J, ip-VC
Inject. site
Animal No.
Labeled cells in TL
TE TO
66, 87, 88, 104-107, 112-121 59, 60, 75, 76, 85, 89, 90 63,64 65
(-) (+ + ) (-) (-)
vc cc bi, bilateral; ip, ipsilateral
Fig. 2 Sites of lesions (black areal and HRP-injection (star) on the torus longitudinalis and the optic tectum.
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HIRONOBU IT0 AND REIJI KISHIDA
tus mesencephalocerebellaris posterior. The fibers then penetrate the nucleus lateralis valvulae (fig. 3) and enter the tractus mesencephalocerebellaris anterior which is located on the dorsomedial border of the fasciculus longitudinalis lateralis. At this level some degenerating fibers turn medially across the base of the tegmentum to join the same tract Horseradish peroxidase (HRP) of the opposite side. The tract travels rostrally A 0.2 pl of 35% solution of HRP in saline along the dorsomedial or medial side of the (0.6%)was injected with a microsyringe into fasciculus longitudinalis lateralis. After the the torus longitudinalis or into other struc- fasciculus spreads out into the torus semicirtures (table 2). The sites of injection into the cularis, the tract changes position dorsally. torus longitudinalis and t h e optic tectum are The tractus mesencephalocerebellaris anteriillustrated in figure 2. From two to five days or shows the largest diameter beneath the nuafter injection, the animals were perfused in- cleus mesencephalicus nervi trigemini, and tracardially with 0.7%saline containing 5-10 tapers to disappear a t the level of the posterior I.U. heparin per milliliter followed by a mixed commissure. At this level heavy axonal and solution of 1.6%paraformaldehyde and 10% terminal degeneration is seen in the nucleus glutaraldehyde in 0.1 M phosphate buffer (pH glomerulosus of Schnitzlein ('62) and in the 7.4).The brains were removed from the skulls, inferior lobe. Some degenerating fibers graduand postfixed in fresh fixative for 5 to 12 ally leave the tract throughout its longitudihours, and rinsed for 24 hours in 0.1 M phos- nal extent, and course dorsolaterally up to t h e phate buffer (pH 7.4) containing 5% sucrose. optic tectum. Degenerating fibers which inAfter being embedded in egg yolk (Ebbesson, vade the optic tectum of the opposite side '701, frontal serial sections were cut a t 60 could not be traced further. In the ipsilateral pm and reacted after 3,3'-diaminobenzidine optic tectum degenerating fibers course dor(Luiten, '75). After sections were individually somedially in the stratum album centrale givmounted on slides with gelatin alcohol, alter- ing off some debris around the layer (fig. 4) native slides were stained with cresyl violet and reach the torus longitudinalis where most for histological identification. of them appear to end (fig. 5). A few degenerating fibers pass through the torus longituRESULTS dinalis and run across the tectal commissure to terminate in the opposite torus longitudiI. Afferent connections nalis. At t h e level of the posterior commissure Degeneration some degenerating fibers from the rostra1 end The degeneration in the torus longitudinalis of the tractus mesencephalocerebellaris antewas produced by lesions in the optic tectum, rior enter the commissure and end bilaterally torus semicircularis, and the valvula cerebelli. in the tori longitudinales. These fibers appear Some degeneration debris was also found fol- to correspond to "die frontale Torusverbindlowing electric coagulation of the contralat- ung" described by Kudo ('24). era1 torus longitudinalis, incision of the tectal commissure, or hemisection between t h e HRP In injection of HRP into the torus longitudivalvula cerebelli and corpus cerebelli (table 1). The following is a description of cases in Fig. 3 Degenerating fibers which penetrate the nuwhich lesions were made in the valvula cere- cleus lateralis valvulae following lesion of the valvula cerbelli (fig. 1). The degeneration in the torus ebelli. Case 56, 13-day survival. X 300. longitudinalis resulting from the other lesions Fig. 4 Degenerating fibers in the stratum album centrale of the optic tectum following lesion of the valvula will be referred to in the DISCUSSION. cerebelli. Some degenerating fibers are also seen In the Essentially the same course and distribu- stratum griseum centrale and in the stratum griseum tion of degeneration were seen in all eight ani- periventriculare (arrow). Case 124, 7-day survival. X 300. mals (Nos. 56, 57, 72, 84, 97, 98, 124, and 125). Fig. 5 Degenerating fibers and terminals in the torus Lesions included both the molecular and gran- longitudinalis following lesion of the valvula cerebelli. 125, 7-day survival. X 400. ular layers in all cases. Degenerating fibers Case Fig. 6 HRP-positive cells in the molecular layer of the arise from the lesion and run ventromedially valvula cerebelli following injection of the torus longituthrough the valvula cerebelli to join the trac- dinalis. Case 77, 5-day survival. X 400.
frigeration, and then embedded in egg yolk (Ebbesson, '70) and sectioned frontally a t 60 pm on a freezing microtome. Every fifth section was processed with cresyl violet for histological identification. Other sections were processed according to a modification of the Fink-Heimer technique (Ebbesson, '70).
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HIRONOBU IT0 AND REIJI KISHIDA
Fig. 7A Lesion of the torus longitudinalis covered with clot (arrow). X 20. Terminal degeneration produced by the lesion is shown in figure 8. B Site of HRP injection in the optic tectum (arrow). X 50. Labeled cells in the torus longitudinalis are shown in figure 10.
nalis, diffusion of HRP into the ipsilateral optic tectum was inevitable to some extent because of the close relationship of these two structures. Labeled fibers arise from injection sites of the torus longitudinalis and run laterally through the stratum album centrale of the optic tectum. If the injection site is located near the rostral end of the torus longitudinalis (fig. 2: Nos. 77 and 861, labeled fibers are found in the posterior commissure and in the rostral part of the tractus mesencephalocerebellaris anterior of both sides. The fibers which run laterally into the stratum album centrale leave the optic tectum along the tractus tectobulbaris. A few labeled neurons are
found in the stratum griseum centrale of the optic tectum. After leaving the optic tectum the fibers run ventromedially beneath the torus semicircularis to join the tractus mesencephalocerebellaris anterior. Some labeled neurons are observed in the torus semicircul a r k A t the level of the nucleus lateralis valvulae the labeled fibers turn upwards and enter the valvula cerebelli. Neurons are found to be labeled in the nucleus lateralis valvulae and in the molecular layer of the valvula cerebelli (fig. 6). In addition, several large neurons are labeled in the reticular formation of the mesencephalic tegmentum. No labeled fibers and cells are found caudal to the nucleus lat-
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471
Fig. 8 Terminal degeneration confined to t h e stratum fibrosum marginale of the optic tectum following lesion of the torus longitudinalis. Case 69, 10-day survival. X 300. Fig. 9 Degenerating fibers which run in the stratum album centrale and penetrate dorsally through the stratum griseum centrale following lesion of the torus longitudinalis. Case 83, IOaday survival. x 200. Fig. 10 HRP-positive cells in the torus longitudinalis following a tectal injection. Case 75,5-day survival. X
400.
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HIRONOBU IT0 AND REIJI KISHIDA
Fig. 11 Semi-diagrammatic drawing of afferent connections of the torus longitudinalis. Transverse sections pass through the (A) rostral part of t h e posterior commissure, (B) mid-level of the posterior commissure, (C) level of the nucleus mesencephalicus n. trigemini, (D)rostral end of the fasciculus longitudinalis lateralis, (E)mid-level of the nucleus lateralis valvulae, and through the (F)secondary gustatory nucleus. The course of fibers and the terminal fields are indicated by arrows and Y-shaped bifurcation respectively. Black circles show the cell of origins demonstrated by both degeneration and retrograde HRP methods. Open circles show cells demonstrated only hy the retrograde HRP method.
eralis valvulae. The afferent connections clarified by both degeneration and retrograde HRP methods are summarized in figure 11. 11. Efferent connections Degeneration In order to determine efferent projections,
lesions were made in various parts of the torus longitudinalis (fig. 2: Nos. 54, 55, 69 and 83). All lesions produced degenerating terminals only in the stratum fibrosum marginale of the ipsilateral optic tectum (figs. 7A, 8).Although the dorsomedial margin of the stratum receives degenerating fibers directly from the
CARP TORUS LONGITUDINALIS
torus longitudinalis, the lateral and ventrolatera1 parts of the stratum receive the fibers by way of the stratum album centrale. That is, most degenerating fibers from the torus longitudinalis travel in the stratum album centrale, and then exit as several fiber bundles towards the surface (fig. 9). These fiber bundles pass across t h e upper three layers (stratum griseum centrale, stratum fibrosum et griseum superficiale, and stratum opticum), and spread out in the stratum fribrosum marginale. Following lesions made in the rostral end of the torus longitudinalis (fig. 2: Nos. 54 and 69) degenerating fibers loop lateral-, then ventralwards around the optic tectum. By contrast, following lesions placed in the caudal part (fig. 2: Nos. 55 and 83)degenerating fibers run caudolateralwards in t h e stratum album centrale. HRP Although HRP labeled fibers were rarely seen in the stratum fibrosum marginale except in the vicinity of the injected site, cells in the torus longitudinalis were always labeled by injection of HRP into the optic tectum (figs. 7B, 10). Labeled cells in the torus longitudinalis tended to be localized more rostrally than the levels of the HRP-injected sites in the optic tectum. The labeled cells correspond to the “small” and “medium-sized” cells classified in the previous study (Ito, ’70). When injected sites occurred within deep layers of the optic tectum, labeled cells were found not only in the torus longitudinalis but also in the optic tectum, torus semicircularis, mesencephalic reticular formation, nucleus lateralis valvulae and in the valvula cerebelli. No cells in the torus longitudinalis were labeled by injection into the telencephalon and the cerebellum (table 2). DISCUSSION
1. Afferent connections of the torus longitudinalis Projections to the torus longitudinalis have only been reported following tectal lesions (Ebbesson and Vanegas, ’76; Sligar and Voneida, ’76). In the present study, degenerating fibers in the torus longitudinalis were produced not only by tectal lesions but also by lesions of the torus semicircularis and the valvula cerebelli. A few degenerating terminals were found after transection of the tectal commissure as well as hemisection between the valvula cerebelli and the corpus cerebelli.
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No degeneration was observed when lesions were made more caudal than the corpus cerebelli. Some of the degenerating fibers which originated in the valvula cerebelli course beneath the torus semicircularis and within the optic tectum. Because of this, degeneration produced by lesions of both areas may be a result of damage of the fibers of passage. Some degeneration found bilaterally in the torus longitudinalis after transection of the tectal commissure might also have been produced as a result of transection of fibers destined t o the contralateral torus longitudinalis. After injection of HRP into the torus longitudinalis, cells in the optic tectum, torus semicircularis, mesencephalic reticular formation, and nucleus lateralis valvae were labeled in addition to those in the valvula cerebelli. However, since the tectal neurons send their axons to the contralateral side, by way of the tectal commissure (Ebbesson and Vanegas, ’76) HRP injected into the torus longitudinalis may be taken up by these axons and transported to the parent cell bodies. In addition, if we consider diffusion of HRP into the optic tectum, which occurred more or less in all cases, and consider similar results obtained by HRP injection into deep portions of the optic tectum, we cannot decide whether the labeled cells of the torus semicircularis, mesencephalic reticular formation and nucleus laterals valvulae project to the torus longitudinalis or to the optic tectum. Animals with a large lesion of the valvula cerebelli had a tendency to sink to the bottom of the water (fig. 1: Nos. 72, 97, 98, 124 and 125). Side-to-side equilibrium was well maintained. Within one hour after operation the functional disorder disappeared in four animals (Nos. 97, 98, 124 and 125), and they began to swim keeping their bodies in the middle level of the water. Animal No. 72 never recovered. Tuge and Shimamura (’58) reported that in some deep sea fish (Cyclothone microdon) the torus longitudinalis was completely lacking, and the valvula cerebelli was hardly detectable in spite of the well developed corpus cerebelli. It may be inferred, therefore, that the valvula cerebelli is associated with some function different from that of the corpus cerebelli, and having close connection with the torus longitudinalis.
II. Efferent connections of the torus longitudinalis Fine structure of the teleostean optic tec-
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HIRONOBU I T 0 AND REIJI KISHIDA
tum has been studied by several authors (Ito, '70; Laufer and Vanegas, '74; Marotte and Mark, '75; Ito et al., '78a, in manuscript), and special attention has been paid to the stratum fibrosum marginale because of the striking similarity with the molecular layer of the cerebellum. However, only one paper to date has reported projections to the stratum fibrosum marginale (Marotte and Mark, '75). They identified degenerating terminals electron microscopically in the stratum as a result of cutting the tectal commissure. We never found any degeneration in the stratum after transection of the tectal commissure. Degeneration in the stratum was only produced by lesions of the ipsilateral torus longitudinalis or of the ipsilateral optic tectum itself. This result was supported by the present HRP experiment, t h a t is, cells in the torus longitudinalis were labeled following HRP injection into the ipsilateral optic tectum. No attempt was made in Marotte and Mark's study to make lesions in the torus longitudinalis. One possible explanation for the difference may lie in the different methods employed. The torus longitudinalis may also send only a few fibers t o t h e contralateral stratum fibrosum marginale. Many people have attempted to characterize inputs to the optic tectum in relation to the layers. Retinal projections are seen in the stratum opticum and stratum fibrosum et griseum superficiale (Ebbesson, '68; Sharma, '72; Vanegas and Ebbesson, '73; Laufer and Vanegas, '74; Gulley et al., '75; Voneida and Sligar, '76), telencephalic projections are in the stratum griseum centrale (Vanegas and Ebbesson, '76; Ito and Kishida, '77), contralatera1 tectal projections are in a deep area of the stratum griseum centrale (Ebbesson and Vanegas, '76; Ito e t al., '78b, in manuscript), and diencephalic projections a r e distributed throughout t h e optic tectum except t h e stratum fibrosum marginale (It0 et al., '78c, in manuscript). The results of the present study, namely that the torus longitudinalis projects exclusively to the stratum fibrosum marginale, help fill one of the blank spaces of inputs t o t h e optic tectum. ACKNOWLEDGMENTS
We wish to thank Doctor Frank Atencio for his suggestions concerning this manuscript. Thanks are also due to Miss Masami Yoshimot0 for her excellent help in preparing the
histological materials. This research was supported by a grant for scientific research (No. 212113) from the Ministry of Education of Japan. LITERATURE CITED Ariens-Kappers, C. U., G. C. Huber and E. C. Crosby 1936 The Comparative Anatomy of the Nervous System of Vertebrates, Including Man. McMillan, New York (reprinted by Hafner, New York, 1960). Ebbesson, S. 0. E. 1968 Retinal projections in two teleost fishes. An experimental study with silver impregnation methods. Brain, Behav. and Evol., 1: 134-154. - 1970 Selective silver impregnation of degenerating axons and their synaptic endings in non-mammalian species. In: Contemporary Research Methods in Neuroanatomy. W. J. H. Nauta and S. 0. E. Ebbesson, eds. Springer-Verlag, New York, pp. 132-161. Ebbesson, S. 0. E., and H. Vanegas 1976 Projections of the optic tectum in two teleost species. J. Comp. Neur., 165: 161- 180. Gulley, R. L., M. Cochran and S. 0. E. Ebbesson 1975 The visual connections of the adult flatfish,Achirus lineatus. J. Comp. Neur., 162: 309-320. Ito, H. 1970 Fine structures of the carp tectum opticum. J. Hirnforsch., 12: 325-354. 1971 Fine structure of the carp torus longitudinalis. J. Morph., 135: 153-164. Ito, ZT., S. 0. E. Ebbesson and A. B. Butler (1978a, in manuscript) Synaptic organization of the optic tectum in a teleost, Hofocentrus rufus. (1978b, in manuscript) The ultrastructure of degenerating tectal afferents from the retina, the telencephalon and the contralateral tectum in a teleost ( H o b centrus rufus). Ito, H., and R. Kishida 1977 Tectal afferent neurons identified by the retrograde HRP method in the carp telencephalon. Brain Res., 130: 142-145. Ito, H., R. Kishida and Y. Morita 11978c, in manuscript) Diencephaiic projections to the telencephalon and the optic tectum in the carp, Cyprinus caprio and Carassius carassius. Kudo, K. 1923 Uber den Torus longitudinalis. I. Mitteilung rneiner Beitrage zur Anatomie des Zwischen- und Mittelhirns der Knochenfische. Anat. Anz., 56: 359-367. 1924 Beitrage zur Anatomie des Zwischen- und Mittelhirns der Knochenfische. 111. Eine frontale Verbindung des Torus longitudinalis. Anat. Anz., 57: 271-275. Laufer, M., and H. Vanegas 1974 The optic tectumof a perciform teleost. 11. Fine structure. J. Camp. Neur., 254: 61-96. Leghissa, S. 1955 La s truttura microscopica e la citoarchittetonica del tetto ottico dei pesci teleostei. 2. Anat. Engwicklungsgesch., 128: 427-463. Luiten, P. G. M. 1975 The horseradish peroxidase technique applied to the teleostean nervous system. Brain Res., 89: 181-186. Marotte, L. R., and R. F. Mark 1975 Ultrastructural localization of synaptic input to the optic lobe of carp (Carcrssius carassius). Exp. Neur., 49: 772-789. Radl, E. 1915 Zur Morphologie der Sehzentren der Knochenfische. Morph. Jb., Leipzig, 49: 509-535. Schnitzlein, H. N. 1962 The habenula and the dorsal thalamus of some teleosts. J. Camp. Neur., 118: 225-267.
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CARP TORUS LONGITUDINALIS Sligar, C. M., and T. J. Voneida 1976 Tectal efferent8 in the blind cave fishAstyanuz hubbsi. J. a m p . Neur., 165: 107-124. Sharma, S. C. 1972 The retinal projections in the goldfish: a n experimental study. Brain Res., 39: 213-223. Tuge, H.,and H. Shimamura 1958 Study on the brains of t h e deep sea fishes, Cyclothon microdon, Gonostoma ritiogr and Coryphaenoides acrolepis. Ann. Rept. Jap. Sea Reg. Fish. Res. Lab., 4: 197-220.
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Vanegas, H., and S. 0. E. Ebbeeson 1973 Retinal projections i n the perch-like teleost Eugerres plumieri. J. Comp. Neur., 151: 331-358. 1976 Telencephalic projections in two teleost species. J. a m p . Neur., 165: 181-196. Voneida, T. J., and C. M. Sligar 1976 A comparative neuroanatomic study of retinal projections in two fishes: Astyanuz hubbsi (the blind cave fish), and Astyanux mexicanus. J. a m p . Neur., 165: 89-106.
