Telencephalic terminals in the major retinal synaptic lamina of the goldfish optic tectum

Brain Research, 336 (1985) 363- 367 Elsevier

363

BRE 20866

Telencephalic terminals in the major retinal synaptic lamina of the goldfish optic tectum MARK J. AIRHART and RICHARD M. KRIEBEL

Department of Anatomy, Quillen-Dishner College of Medicine, East Tennessee State University, Johnson City, TN37614 and Department of Anatomy and Neurobiology, University of Vermont, College of Medicine, Burlington, VT05405 ( U. S.A .) (Accepted January 21st, 1985)

Key words: goldfish optic tectum - - degeneration --electron microscopy - - telencephalotectal terminals

Light and electron microscopic degeneration studies were used to examine the telencephalotectal pathway in goldfish. Both techniques showed that each telencephalic lobe sent bilateral projections to several tectal laminae. Degenerating synaptic terminals and fibers were observed in the major retinal projection lamina as well as in other tectal laminae. The terminals contained round to oval synaptic vesicles, asymmetric synapses and contacted relatively small postsynaptic profiles.

In teleosts, the existence of a projection from telencephalon to optic tectum, analogous to the cerebral-collicular pathway of mammals, is well established 12,16. However, there is disagreement on the distribution of the projection and on the degree to which the telencephalic input overlaps with that of the major tectal afferent, the optic nerve. Within the same order of fish, Cyprinidae, the telencephalic projection has been variously reported to be exclusively ipsilateral (Cyprinus carpio 9,10 and Carassius auratus 6) or strongly bilateral (Carassius carassius ll and Carassius auratus13). Light microscopic studies, including silver degeneration and retrograde horseradish peroxidase (HRP) tracing, have shown that the telencephalic projection is excluded from the major retinal projection lamina in Eugerres plurnerii 15, Holocentrus rufus is, Carassius auratus 6,13 and Cyprinus carpio 93°. Electron microscopic degeneration studies, however, have reported a significant and sometimes predominant projection of telencephalic fibers and terminals to the major synaptic substrate for retinal terminals in Carassius carassius la and Holocentrus rufus 8. To help clarify these controversies, we have used both light and electron microscopic degeneration

techniques to examine the telencephalic projection to the goldfish (Carassius auratus) optic tectum. We used a modified Fink and Heimer degeneration procedure 4 to determine the laminar position and distribution of telencephalotectal fibers and/or terminals. To determine conclusively if a projection to a lamina of the optic tectum consisted of fibers, terminals, or both, electron microscopic degeneration studies were employed. C o m m o n goldfish (Carassius auratus), 6.5-7.5 cm long, were obtained from Grassyfork Fisheries (Martinville, IN) and maintained in aquaria at 25 °C under normal d a y - n i g h t laboratory conditions. The telencephalon was approached through a small bone flap incised in the calvaria, and approximately two-thirds of the rostral right telencephalon was ablated. The entire telencephalic lobe was not removed because of potential damage to the optic tract that lies adjacent to the ventral posterior pole of the lobe. In control animals a bone flap was reflected as described above and the surrounding ventricle of a telencephalic lobe was torn, but the telencephalon itself was left intact. For light microscopic studies, 6 experimental and two control fish were anesthetized in 0.03% MS-222

Correspondence: M. J. Airhart, Department of Anatomy, Box, 19960A, Quillen-Dishner College of Medicine, East Tennessee State University. Johnson City, TN 37614, U.S.A. 0006-8993/85/$03.30 © 1985 Elsevier Science Publishers B.V. (Biomedical Division)

364 (Sandoz) and killed by intracardial perfusion with 0.7% saline followed by 10% buffered formalin (pH 7.2) 4 days after surgery. Brains were e m b e d d e d in gelatin, sectioned transversely at 33/zm with a freezing m i c r o t o m e , and the sections stained by a modification of the Fink and H e i m e r m e t h o d 4. This modification consisted of a reduction in the silver nitrate concentration in the presoak step (0.4%) and the ammoniacal solution (1.4%), and extending t r e a t m e n t with potassium p e r m a n g a n a t e (0.05ck~, 25 min). These modifications resulted in a decrease in background levels of non-specific silver grains. A survival time of 4 days was used in these studies because a

higher density of degenerating profiles was o b s e r v e d in the tecta at this time in preliminary experiments which c o m p a r e d 2, 4 and 6 day postsurgicat fish. F o r electron microscopic studies, experimental an.imals were lesioned as previously described. A survival time of 3 days was chosen because a greater n u m b e r of degenerating boutons with recognizable synaptic vesicles was observed at this time in preliminary experiments comparing 2, 3 and 4 day postsurgical fish. F o u r animals were anesthetized 3 days postlesion and intracardially perfused with 5.1) ml of 0.7% saline followed by 5.0 ml of 2.5% glutaraldehyd e - 2 . 0 % p a r a f o r m a l d e h y d e - 3 . 0 % sucrose in phos-

Fig. 1. a: photomicrograph showing silver-impregnated degenerating profiles in goldfish tectum 4 days subsequent to partial tetencephalic ablation. Section is from rectum ipsilateral to lesioned telencephalon in dorsomedial region. Note the columnar organization of some of the degenerating profiles. The density of degenerating profiles is greatest in the stratum fibrosum et griseum superficiale (SFGS) but profiles are also present in the stratum griseum centrale (SGC) and stratum album centrale (SAC). Other abbreviations: stratum marginale, SM; stratum opticum, SO; stratum periventriculare, SP. x 363. b: photomicrograph showing silver-impregnated degenerating profiles from the same animal as described in a. The tectal section is contralateral to the lesion and lies lateral to the tectal section in a. Degenerating profiles are present in the SFGS, SGC and SAC but in contrast to a the density of degenerating profiles is greater in the SGC than SFGS. x 363.

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Fig. 2. An electron micrograph of the SFGS from an animal killed 3 days after partial telencephalic lobe ablation. The degenerating synaptic terminal (dt) is in the tectum ipsilateral to the lesion. It contains round to oval synaptic vesiclesand forms an asymmetric synapse that contacts a relatively small postsynaptic profile (*). × 48,500. Fig. 3. An electron micrograph of a degenerating telencephalotectal fiber (df) in the SFGS, 3 days after contralateral telencephalic le.sion. The degenerating myelinated fiber shows a dark cytoplasm, characteristic of degeneration, with relatively intact myelin. × 34,000. phate buffer (pH 6.9, 0.08 M). Brains were dehydrated in a graded series of alcohols and propylene oxide for subsequent embedding in Epon 812. Thin sections, 70-80 nm, were stained with uranyl acetate and lead citrate and examined in a Phillips 300 electron microscope. Sections examined came from the midtectal area, defined by a midpoint along the rostrocaudal axis. The only boundary between tectal laminae that could not clearly be distinguished at the ultrastructural level was between the stratum fibrosum et griseum superficiale (SFGS) and the stratum griseum centrale (SGC) 2. Degenerating fibers and terminals near this boundary were not included in this study. Following partial ablation of the right telencephalon, silver-impregnated degenerating profiles were observed throughout the rostrocaudal axis of both optic tecta. Degenerating profiles were present in the following laminae of both tecta: SFGS (the major synaptic substrate for retinal terminalsSa4), SGC and stratum album centrale (SAC) (Fig. la, b). Degenerating profiles were consistently observed in the ventrolateral margin of the stratum opticum (SO) but not

throughout it entirety, and degenerating profiles were also occasionally seen in the stratum marginale (SM). The laminar distribution of degenerating profiles were similar in both tecta. However, there were differences in degenerating profile density and organization within a given lamina, along its mediolateral axis. To conserve space, these differences will be illustrated from Fig. la and b, although the points apply equally well to material obtained from the same tectum. In the dorsal medial third of each tectum, the pattern of some degenerating profiles was columnar (i.e. running perpendicular to the pial surface) (Fig. la), while in more lateral areas the degenerating profiles were more randomly distributed (Fig. lb). The relative density of degenerated profiles between the SFGS and SGC showed some variability. For example, Fig. la shows a greater density of degenerating profiles in the SFGS than the SGC but this relationship is reversed in the more lateral tectal region (Fig. lb). Tectal sections from control animals showed only occasional, randomly placed, silver-impregnated profiles. The density of these profiles was no greater in tectal regions than elsewhere

3~0 in the control brains. The telencephalic projection was examined at the ultrastructural level to localize terminal fields in the tecta. Degenerating synaptic terminals were identified by electron-dense cytoplasm typical of the 'dark reaction' described by Heimer and Peters 7. Three days after unilateral telencephalic ablation, degenerating terminals were observed in the SM. SFGS, SGC and SAC of both tecta, with degenerating terminals most frequently observed in the SFGS and SGC. Telencephalic terminals in the early stages of degeneration contained spherical synaptic vesicles and made asymmetric synaptic contacts (Fig. 2). These degenerating terminals were observed contacting relatively small postsynaptic profiles (Fig. 2). Degenerating myelinated fibers were observed in the SO, SAC and coursing through the neuropil of the SFGS and SGC (Fig. 3). No degenerating unmyelinated fibers were observed. Both light and electron microscopic data support the conclusions that in goldfish one telencephalic lobe projects to both tecta and that there is a projection to both the SFGS and SGC in each tectum. The laminar distribution of telencephalotectal fibers and terminals, observed initially light microscopically, was confirmed by the electron microscopic degeneration study. Furthermore, the ultrastructural investigation showed that telencephalic terminals synapsed in both the SFGS and SGC and that the tentative projection to the SM suggested by the light microscopic study was real. For both studies, it was critical that the partial telencephalic lobe ablation did not damage the ipsilateral optic tract and consequently induce degeneration of retinal fibers and terminals in the SO and SFGS. This is why only the rostral two-thirds of the telencephalic lobe was removed. The following observations indicated the absence of damage to the ipsilateral optic tract: (1) histological examination of light microscopic sections revealed no damage to the ipsilateral optic tract or brachia; and (2) the pattern of degeneration in the contralateral tectum, where the optic tract could not have been damaged, was similar to that in the ipsilateral tectum. In addition, the telencephalic lobe contralateral to the lesion was also examined for potential damage; no obvious indicators such as gliosis or blood clots were observed. Although the present investigation demonstrates a

close correlation between the light and electron m~croscopic studies, a similar investigation tracing telencephalotectal fibers and terminals in Holocentrus rufus showed conflicting results between these ~echniques s,15. In the light microscopic study~ fish killed 7-35 days after the lesion showed degenerating profiles only in the SO and lower tectal laminae (SGC and SAC). In the correlative electron microscopic study in which fish were killed 3 days postlesion, degenerating terminals were found in the SFGS, while in the SGC both degenerating fibers and terminals were observed. We suggest that the light microscopic study reflected fiber degeneration since telencephalic terminals at these late postlesion times probably had been phagocytosed. A similar explanation could account for the differences between our results and the light microscopic degeneration results of Oka and Ueda 13. They traced telencephalic efferents in goldfish using a silver degeneration technique and found a bilateral tectal projection but no degenerating profiles in the SFGS. Although they used goldfish of approximately the same size as in the present investigation, the survival times were longer, i.e~ 6-12 days. These relatively long survival times may have resulted in the nearly complete phagocytosis of degenerating terminals and fibers in the SFGS. The persistence of degenerating profiles in the lower tectal laminae (SGC and SAC) may reflect a high ratio of telencephalic fibers to terminals and the longer time it takes for fibers to completely degenerate and lose their argyrophilia. A second possible explanation is that our modification of the silver degeneration technique is more sensitive than the technique used by Oka and Ueda (Ebbesson's modification) of the Fink-Heimer method). Our results also differ from previous retrograde HRP studies that traced telencephatotectal efferents in C. auratus as well as other fish in the order Cyprinidae. In these investigations a small amount of H R P was injected into a tectal lobe and only those injections that included the lower laminae (SGC and SAC) resulted in labeled cells in the ipsilateral telencephalonO, 9,10. Such discrepancies between retrograde HRP and anterograde degeneration studies may be due to differences in the mechanisms of labeling and the distribution of telencephalotectal fibers and terminals. For example, if HRP uptake were greater in damaged axons than terminals and asso-

367 ciated collaterals, and the diameter and density of

cephalic terminals have been observed in the SFGS.

telencephalic fibers was greater in lower tectal laminae, then H R P injection into lower tectal strata would be much more effective in retrogradely filling

Therefore, the generalization stated in a recent review12 that telencephalotectal fibers and terminals in

telencephalic cells. The present results are similar to an earlier electron microscopic study tracing the telencephalotectal projection in a closely related species, C. carassius ~1.

teleosts are restricted to the lower tectal laminae (SGC and SAC) and do not overlap with the major retinal synaptic lamina (SFGS) does not pertain at least to the following fish: C. auratus, C. carassius and H. rufus.

In this fish a single telencephalic lobe projected bilaterally to the optic tecta and telencephalic terminals were observed in the following laminae: SM, SFGS and SGC. In fact, in all teleosts in which the telencephalotectai projection has been examined electron microscopically ( C. auratusl, present study, C. carassius 11 and 11. rufus8), a significant n u m b e r of telen-

1 Airhart, M. J., Telencephalotectal projections in the goldfish, C. auratus: a light and electron microscopic study, Anat. Rec., 193 (1979) 468. 2 Airhart, M. J. and Kriebel, R. M., Retinal terminals in the goldfish optic tectum: identification and characterization, J. comp. Neurol., 226 (1984) 377-390. 3 Ebbesson, S. O. E., Selective silver impregnation of degenerating axons and synaptic endings in non-mammalianspecies. In W. J. Nauta and S. O. E. Ebbesson (Eds.), Contemporary Research Methods in Neuroanatomy, Springer-Verlag, New York, 1970, pp. 132-161. 4 Fink, R. P. and Heimer, L., Two methods for selective silver impregnation for degenerating axons and the synaptic endings in the central nervous system, Brain Research, 4 (1967) 369-374. 5 Grafstein, B., Transport of protein by goldfish optic nerve fibers, Science, 157 (1967) 196-198. 6 Grover, B. G. and Sharma, S. C., Organization of extrinsic tectal connections in goldfish ( Carassius auratus), J. comp. Neurol., 196 (1981) 471-488. 7 Heimer, L. and Peters, A., An electron microscopic study of a silver stain for degenerating boutons, Brain Research, 8 (1968) 337-346. 8 Ito, H., Butler, A. B. and Ebbesson, S. O, E., An ultrastructural study of the normal synaptic organization of the optic tectnm and the degenerating tectal afferents from ret-

The authors wish to thank Drs. R. Baisden and M. H o u g l a n d for their critical review of the manuscript, We thank Cindy Canter for typing the manuscript. This work was supported by an E T S U Institutional G r a n t to M.J.A. and PHS 5429-16-19 to R.M.K.

ina, telencephalon, and contralateral tectum in a teleost, Holocentrus rufus, J. comp. Neurol., 191 (1980) 639-659. 9 Ito, H. and Kishida, R., Tectal afferent neurons identified by the retrograde HRP method in the carp telencephalon, Brain Research, 130 (1977) 142-145. 10 Luiten, P. G. M., Afferent and efferent connections of the optic tectum in the carp (Cyprinus carpio L.), Brain Research, 220 (1981) 51-65. 11 Marotte, L. R, and Mark, R. F., Ultrastructural localization of synaptic input to the optic lobe of carp (Carassius carassius), Exp. Neurol., 49 (1975) 772-789. 12 Meek, J., Functional anatomy of the tectum mesencephali of the goldfish. An explorative analysis of the functional implications of the laminar structural organization of the tecturn, Brain Res. Rev., 6 (1983) 247-297. 13 Oka, Y. and Ueda, K., Telencephalic projections in the goldfish (Carassius auratus): an anterograde degeneration study, J. Fac. Sci. Univ. Tokyo, Ser. IV, 15 (1981) l-8. 14 Sharma, S. C., The retinal projections in goldfish: an experimental study, Brain Research, 39 (1972) 213-223. 15 Vanegas, H. and Ebbesson, S. O. E., Telencephalic projections in two teleost species, J. comp. Neurol., 165 (1976) 181-196. 16 Vanegas, H. and Ito, H., Morphological aspects of the teleostean visual system: a review. Brain Res. Rev., 6 (1983) 117-137.