Retinotectal synapses formed by ipsilaterally projecting fibers in the doubly innervated goldfish tectum

Brain Research, 325 (1985) 307-312

307

Elsevier BRE20561

Retinotectal synapses formed by ipsilaterally projecting fibers in the doubly innervated goldfish tectum MARK J. ,~IRHART* and JEANETFE J. NORDEN

Department of Anatomy, Vanderbilt Umverstty School of Medtcme, Nashvdle, TN37232 (U S.A.) (Accepted August 21st, 1984)

Key words goldfish optic tectum - - synapses - - doubly innervated tectum

When one tectum of an adult goldfish is removed, the severed retinal fibers regenerate ~psflaterally into the remaimng tectal lobe Initmlly fibers from the two eyes overlap m the tectum but EM-HRP data suggest that few mature retinal synapses are formed between the ipsflateral eye and tectum at this t~me. At longer ume periods, when some fibers appear to segregate into eye-specific termination bands, our data suggest that a sigmficant number of synapses from the ipsilateral eye are present. These findings have important ]mphcations for how eye-specific terminat]on bands are formed in doubly innervated tecta The goldfish retinotectal pathway has been used extensively as a model system to study axonal regeneration and the specificity of neuronal connections 3,7,13A5.16. This pathway projects almost exclusively to the contralateral tectum and is retinotopically organized 6,13,19,24,25. Numerous studies have shown that following lesions of the optic nerve or tract, retinal fibers regenerate to the contralateral tectum 3,7,13 and reform synaptic connections 15A6. The goldfish retinotectal pathway is also capable of considerable plasticity. For example, contralaterally projecting retinal fibers can be induced to regenerate ipsilaterally into a normally innervated tectum following removal of one tectal lobe 10,12,20,23 or following deflection and msertion of fascicles of optic fibers from one tectum to the other 14. Initially, regenerating ipsilateral and normally projecting contralateral retinal fibers overlap in their projections onto the remaining tectum12,t4,23, with regenerating fibers projecting retinotopically in a mirror-image fashion4,10,20. Over time, however, the projections from the two eyes segregate into eye-specific termination bands12A 4.23, similar to those reported for doubly innervated tecta in frogs s. It is of considerable interest whether ipsilaterally

regenerating fibers actually form synapses in the doubly innervated tectum, and if so, whether these synapses are made before the formation of eye-specific termination bands. To date all of the studies of doubly innervated tecta in goldfish have relied on light microscopic and/or electrophysiological methods to demonstrate the presence and distribution of regenerated ipsilaterally projecting fibers. In the present study, we have employed c o m b m e d EMH R P methods to examine the doubly innervated tectum ultrastructurally. We find that under the conditions used in the present study, incipient synapse formation occurs as early as 26 days following tectal lobe removal and that by 59 days, the ipsilateral eye has formed a significant complement of synapses in the remaining tectum. C o m m o n goldfish (Carasstus auratus), 5 - 7 cm long, were obtained from Grassyfork Fisheries (Martinsville, TN) and maintained in 30 °C aquaria 22,23 under normal d a y - n i g h t laboratory conditions. Animals were anesthetized in 0.03% tricaine methanesulfonate (MS-222) and the tecta exposed by reflection of a bone flap. In all animals, the right tectum was removed by aspiration and the severed optic tract and brachia were positioned agamst the

Correspondence J J Norden, Department of Anatomy, Vanderbilt University School of Medicine, Nashvdle, TN 37232, U S A * Present address Department of Anatomy, Qmllen-Dlshner College of Medicine, East Tennessee State Umversity, Johnson City, TN 37614, U.S A. 0006-8993/85/$03 30 (~) 1985 Elsevier Science Pubhshers B V

308 rostral pole of the remaining left tectum. The mduced lpsllaterally projecting fibers and terminals were labeled by mjectmg horsera&sh peroxidase (HRP, 8~1, 50% aqueous solutton) intravitreally into the left eye 4 days before sacrifice, and to maximize the labehng of as many terminals as possible, the left optic nerve was injected with HRP (4/~1, 50% solution) or severed and packed with a gelfoam pledget soaked with HRP (50% solutton) two days before sacrifice. Fish were sacrificed 26-112 days following tectal lobe removal. Animals were perfused transcardially with 0.7% saline followed by a mixture of 2.5% glutaraldehyde and 2.0% paraformaldehyde in 0.08 M phosphate buffer (pH = 6.9). Tecta were sectioned in the transverse plane using a vibratome to cut 50 and 200/tm sections. The 50 ktm sections were reacted with tetramethylbenzidine (TMB) or dmminobenzidine (DAB) and H202 and processed for light microscopy. The 200 ~m sections were reacted with DAB using the Adams 1 mo&fication and processed for electron mtcroscopy using standard EM protocols. Ultrathin (60-70 nm) and thick (1-2/~m) sections of rostral, mtdtectal and caudal areas were cut on a LKB IV Ultrotome. Ultrathin secttons were mounted on 400 mesh grids, stained w~th uranyl acetate and lead citrate, and examined in an Hitachi H-600 electron m~croscope. In 5 animals, sacrificed at 26, 29, 59, 69 and 112 days following unilateral tectal ablation, labeled regenerating fibers and/or terminals were present across the entire rostrocaudal and mediolateral extent of the remaining tectal lobe (Figs. 1 and 2). Regenerating fibers appeared to grow into the tectum over a number of routes, as shown in Fig. 2 A large number of labeled regenerating fibers were observed m the deflected optic tract and brachia. Some of these fibers appeared to enter dtrectly the medml margin of the rostral rectum and other fibers appeared to grow into the intact medial and lateral brachia of the intact optic tract. Labeled fibers were also observed in the horizontal and transverse commissures. In both TMB and DAB reacted sections, 1psilaterally projecting fibers could be seen in the stratum opticum (SO) and the stratum fibrosum et greslum superficiale (SFGS), the major fiber and synaptic layers of the tectum receiving retinal input in goldfish 19,24,25. In the tecta of animals sacrificed at 26 and 29 days following tectal lobe removal, the lpsdateral

1

Ftg. l A 50 ,um transverse section through the mldtectal regton of the optic tectum of an animal sacrificed 29 days following removal of one tectal lobe T h e regenerating lpsdateral fibers are

labeled with HRP HRP reaction product is present uniformly throughout the stratum opt~cum(SO) and the stratum fibrosum et greslum superficlale (SFGS) of the remaining tectum TMB reacted: neutral red counterstam, x40 projection to the SFGS was densely, homogeneously, and uniformly labeled across the tectum (Fig. 1). In animals survtvmg 2-4 months, 'patches' or alternatmg areas of htgh and low denstty labeling were seen m parts of the SFGS (Fig. 2) These patches were variable in size and number between animals but similar in appearance to eye-specific termination bands previously described m goldfish 12,14,23 Ultrastructural examination of the SO and the SFGS revealed labeled unmyelmated axons m all animals. In animals sacrificed at 59-112 days. labeled myelinated axons were seen occasionally Labeled synapses (Fig. 3a) could be adentified in the SFGS at all times examined although the number of labeled synapses was dependent on the time since tectal ablation. Quantitative EM data on the number of labeled synapses in the SFGS of animals sacrtficed at 26 and 29 days indicate that labeled mature synapses are rare in these animals (2 labeled synapses; 800 synapses examined). While a comparable quantitative analysis of the SFGS is not complete m animals sacn-

2

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....

F~g 2. a: a 50/tm transverse section through the rostral tectum of an animal sacrificed 59 days following unilateral tectal ablation. Regenerating ipsdaterally projecting fibers are labeled w~th HRP and can be seen in a number of structures including the deflected optic tract (DOTr), dorsal medial (DB) and ventral lateral (VB) brachla, transverse commissure (TC), horizontal commissure (HC), and the SO and the SFGS of the remaining tectum The large arrow sets off an area of lower fiber and/or terrmnal density m the SFGS TMB reacted; neutral red counterstam; ×40. b a 50/~m transverse section from the mtdtectal region of the optic tectum of an animal sacrificed 59 days after removal of one tectal lobe Large arrows denote areas of lower fiber and/or terminal densities within the SFGS which may represent eye-specific termination bands. TMB reacted; neutral red counterstam; × 40 Fig. 3 a: labeled synapses (*) in the SFGS of an animal sacrificed 59 days following umlateral tectal ablation. These synapses have been formed between regenerating ~psdateral fibers and postsynapt~c neurons m the remaining tectal lobe The presynaptlc terminal contains predominantly round synaptlc vesicles (SV) and a large mltochondrion (MIT) and ~s making asymmetric contacts onto small postsynaptlc profiles x47,814 b' an unlabeled immature synaptic contact (*) in the SFGS of the animal sacrificed 29 days following removal of one tectal lobe Paramembranous thlckemngs are present, but synapt~c vesicles (SV) are not clustered at the presynapt~c membrane ×55,600

310 riced between 59-112 days, labeled synapses are frequently encountered. While all synapses made by lpsilaterally projecting fibers might not be labeled with HRP in a given animal, these data suggest that the number of mature synapses made by the lpsilateral eye is likely to be higher in animals sacrificed at the longer time intervals. This conclusion is also supported by our preliminary quantitative analysis of synaptic densities 17which indicates that the total number of synapses (labeled and unlabeled) is sigmficantly higher in areas of the SFGS m the animal sacrificed at 59 days compared to synaptic counts in comparable areas of the tectum in the ammals sacrificed at 26 or 29 days. In all animals, labeled presynaptic terminals contained round to oval synaptic vesicles and often made multiple synapses onto postsynaptlc profiles (Fig. 3a). Labeled synapses were asymmetric and postsynaptic profiles were primarily small dendritic elements. All of these parameters are characteristic of normal retinotectal terminals and synapses in goldfish 2. In addition to labeled synapses, unlabeled immature synaptic contacts could be identified in the SFGS of all animals examined. As indicated in Fig. 3b, such incipient synapses could be recognized by the presence of pre- and postsynaptic specializations. Unlike mature synapses, however, immature contacts did not show a clustering of synaptic vesicles at the presynaptic membrane. The greatest number of ~mmature contacts was seen in the tecta of ammals sacrificed at the earher time periods, but some unlabeled immature synapses could still be identified m animals 112 days following tectal ablation. In agreement with previous studies, our results indicate that following ablation of one tectal lobe in goldfish, severed retinal fibers can be induced to regenerate ipsilaterally over a number of pathways to innervate the remaimng tectum 5 9,11,12,2o In the present study, all of the pathways taken by regenerating fibers have been shown to contain at least a small complement of retinal fibers in normal goldfish24 and this complement is known to increase during nerve regenerationZl. In goldfish there may be a small ipsilateral projection to a deep tectal layer, the stratum albums centrale, w~th some experimental ammals showing a few ipsdateral fibers m the rostral SFQS 24. It Is unlikely that our findings are contaminated by these putative ipsilateral fibers in the SFGS, howev-

er, since the normal prolection, when present, is extremely sparse24, while the induced ipsilateral projection is robust and completely Innervates the SFGS. Our EM examination of the SFGS, however, suggests that few morphologically mature synapses have been formed between ipsllateral presynaptlc fibers and postsynaptic tectal neurons at times when hght microscopy and electrophysiological mapping of presynaptic terminals indicate a complete innervatlon of the remaining tectum by ipsilaterally projecting fibers. The inability to identify labeled synapses is not likely to be due to an insufficient density of innervation or to an insufficient transport of H R P by lpsilaterally projecting fibers since labeled fibers and preterminals could be identified both hght and electron microscopically throughout the entire tectum Furthermore, the apparent lack of labeled mature synapses at these early times was not due to a gradient in synaptogenesis which occurs during remnervation 26 since both rostral and midtectal regions were exam1ned. The fact that mature retinotectal synapses formed by the ipsilateral eye are labeled by 59 days post-lesion, suggests that a number of the unlabeled immature synapses identified at 26 and 29 days are incipient synapses being formed by the lpsalateral eye. Similar immature contacts have been reported during original synaptogenesis in the optic tectum of the trout TM and during reinnervation of the contralateral tectum following optic tract section in the goldfish 15, The absence of label in the presynaptic elements of immature contacts may reflect some difficulty in the transport of H R P to the tips of regenerating axons 16 Because contralateral and ipsilateral fibers are just beginning to segregate between 22 and 30 days under the experimental conditions used in the present study 23, however, we cannot rule out the possibility that some of the ~mmature contacts ldennfled at early times following regeneration, or the immature contacts identified at later times, m~ght represent synaptogenesis by collateral sprouts of contralaterally projecting fibers Numerous labeled mature retmotectal synapses could be ~dentlfied in the remaining tectum of all animals surviving 2 months or longer. All of these labeled synapses showed the morphology and type of synapt~c contact characterlsnc of retmotectal synapses in goldfish, Including round to oval synaptic vesicles

311 and type of synaptic contact 2. W h i l e it is not known if any visual function can be m e d i a t e d by the ipsilateral

may arise either by the preferential stabilization of

retinotectal p a t h w a y during the early p e r i o d of overlap of ipsilateral and c o n t r a l a t e r a l retinal fibers, vision m e d i a t e d by an ipsilateral p a t h w a y has been d e m o n s t r a t e d in animals m a i n t a i n e d at 23 °C and sacrificed 125-148 days following tectal ablatton 27,28. O u r results suggest that the return of vision m a y be due to the establishment of a sufficient density of regenerating ipsilateral retinotectal synapses at these times. These findings provide a necessary b a c k g r o u n d agamst which to assess the mechanism by which eyespecific termination bands develop. It is possible that ipstlaterally projecting fibers cannot form synapses unless and until some of the normal contralaterally projecting fibers rehnquish their synaptic connections. This might explain why ipsilateral fibers appear not to have established m a n y m a t u r e synapses at the earliest time periods we examined. A l t e r n a t i vely, there m a y be no initial segregation at the synaptic level and the segregatton which develops later

or by the loss of synapses from the contralateral eye. The formation of eye-specific termination bands would involve, in the f o r m e r case, a r e o r d e r i n g of contralaterally projecting fibers and synapses, and in the latter case, the reordering of both ipsilateral and contralateral fibers and synapses. Certainly our finding of i m m a t u r e contacts up to 112 days following unilateral tectal ablation argues for a continuing dynamic synaptogenesis in the doubly m n e r v a t e d tectum. Thus, as the goldfish retmotectal p r o j e c t i o n has proven useful in the study of the specificity of synaptic connections, the doubly innervated goldfish tectum promises to be a valuable m o d e l for the study of competitive interactions at the synaptic level.

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The authors would like to thank Prof. J. A. F r e e man for helpful c o m m e n t s on the manuscript. Supp o r t e d by N I H Post-Doctoral Fellowship E Y 05567 and an E T S U Institutional G r a n t to M . J . A . and N E I G r a n t E Y 03718 to J.J.N.

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