Localization of α-bungarotoxin binding sites to the goldfish retinotectal projection

Brain Research, 187 (1980) 113-127 © Elsevier/North-HollandBiomedicalPress

113

LOCALIZATION OF a-BUNGAROTOXIN BINDING SITES TO THE GOLDFISH RETINOTECTAL PROJECTION

ROBERT E. OSWALD,JOHN T. SCHMIDT,JEANETTEJ. NORDEN and JOHN A. FREEMAN* Departments of Biochemistry and Anatomy, Vanderbilt University School of Medicine, Nashville, Tenn. 37232 (U.S.A.)

(Accepted July 26th, 1979)

SUMMARY The optic tectum of the goldfish Carassius auratus is a rich source of a-bungarotoxin (a-Btx) binding protein. In order to determine whether some fraction of these receptors is present at retinotectal synapses, we have compared the histological distribution of receptors revealed by the use of [t~sI]a-Btx radioautography to the distribution of optic nerve terminals revealed by the use of cobalt and horseradish peroxidase (HRP) techniques. The majority of a-Btx binding is concentrated in those tectal layers containing primary retinotectal synapses. The same layers contain high concentrations of acetylcholinesterase (ACHE), revealed histochemically. Following enucleation of one eye, there is a loss of a-Btx binding in the contralateral tectum, observed both by radioautography and by a quantitative binding assay of a-Btx binding. Approximately 40 ~ of the a-Btx binding sites are lost within two weeks following enucleation. By contrast, no significant change in AChE activity could be demonstrated up to 6 months following enucleation. These results are discussed in light of recent studies which show that the a-Btx binding protein and the nicotinic acetylcholine receptor are probably identical in goldfish tectum. We conclude that the 3 main classes of retinal ganglion cells projecting to the goldfish tectum are nicotinic cholinergic and that little or no postdenervation hypersensitivity due to receptor proliferation occurs in tectal neurons following denervation of the retinal input.

INTRODUCTION The retinotectal system of the goldfish has been used extensively as a model system for the study of synaptogenesis. It provides three principal advantages. First, * Author for all correspondence.

114 the optic nerve in goldfish (but not in reptiles, birds, or mammals) will regenerate following injury. Second, fibers from different classes of retinal ganglion cells are located in well-defined tectal layersZg: regenerating fibers re-establish this laminar pattern 44 and make functional synapses, restoring vision. Third, relative to the complexity of many other CNS structures, the goldfish optic projection is fairly simple. There appear to be only three main classes of optic fibers: two which terminate in the stratum opticum and superficial gray and white, and one which terminates in the central gray and deep white layers3S,39. Despite numerous studies of the time-course and morphological sequence of events that occur during changes in optic innervationl,21, 43, relatively little is known about the molecular events associated either with degeneration or regeneration. One important class of synaptic macromolecules of interest includes neurotransmitter receptors, which have been postulated to play a regulatory role in synapse stabilization4,6. Several studies have provided evidence for the presence, in high concentration, of nicotinic acetylcholine receptors (nAChR) in goldfish tectum. In a preliminary physiological study42, responses of tectal neurons following optic nerve stimulation were found to be abolished by a variety of nicotinic but not muscarinic antagonists. Other studies have provided evidence for the presence of receptors that specifically bind the snake neurotoxin a-Btx, a specific ligand for the nAChR at neuromuscular synapses. For example, Foders and Salvaterra 5 reported that the tectum binds more [125I]a-Btx than any other brain region in goldfish. Schechter et al.36 have shown using a centrifugation assay of tectal homogenates that the concentration of a-Btx binding sites decreases following optic nerve crush and increases during subsequent optic nerve regeneration. The nAChR of goldfish brain has been characterized biochemically by Oswald and Freeman 2s. Like the nAChR of Electrophorus electroplax a3, the aBtx-nAChR complex in goldfish brain is acidic with a p! of approximately 5 and molecular weight of approximately 340,000. A further set of studies has shown that a-Btx is effective in abolishing synaptic activation of tectal neurons following optic nerve stimulation1°,4z and that intracellular responses to iontophoretically applied acetylcholine are blocked by a-BtxL Thus, it is likely that in goldfish tectum a-Btx binds to nicotinic cholinergic receptors and provides a useful ligand with which to study receptor dynamics associated with synaptogenesis. The utility of a-Btx as a probe for nicotinic receptors and the usefulness of the goldfish tectum as a model system would be further extended if it were known whether nicotinic cholinergic receptors are localized at retinotectal synapses. Our goal in this and in the companion paper 4° is to address this question using a combined biochemical, anatomical, and physiological approach. In the present study we have compared the localization of nAChR revealed by [125I]a-Btx radioautography, both to the distribution of optic fibers revealed by the cobalt and HRP methods, and to the distribution of acetylcholinesterase (ACHE) revealed by histochemistry. We have also compared the concentration of a-Btx binding sites and AChE in the normal tectum to that present following enucleation. In

115 order to correlate the observed loss of binding sites seen biochemically to that occurring anatomically after enucleation, we have made a semiquantitative analysis of changes in the histological distribution of toxin binding sites. The results show a selective loss of a-Btx binding sites in the layers of tectum containing retinotectal synapses. In the accompanying paper we present a more detailed analysis of the effects of various cholinergic ligands on retinotectal synaptic transmission. The results of both studies indicate that nicotinic cholinergic receptors constitute the predominate class of transmitter receptors at retinotectal synapses in the goldfish. METHODS

Biochemical studies Preparation of [125I]a-Btx. Crude lyophilized venom of Bungarus multicinetus (Miami serpentarium) was chromatographed on a CM-Sephadex C-50 column (Pharmacia) equilibrated in 50 mM ammonium acetate buffer (pH 5.0). The proteins were eluted with a gradient of 50 mM ammonium acetate buffer (pH 5.0) to 1 M ammonium acetate buffer (pH 6.8) 19. The second peak absorbing at 280 nm was pooled and rechromatographed on CM-Cellulose C-52 (Whatman) equilibrated in 0.1 M ammonium acetate. A 0.1--0.3 M ammonium acetate gradient was used to elute aBtx (peak 2.234). Polyacrylamide gel electrophoresis 35 and SDS-urea gel electrophoresis 46 revealed the presence of a single protein band. Sedimentation equilibrium experiments in a Beckman Model E analytical ultracentrifuge revealed that the preparation was monodisperse. a-Btx was iodinated using lactoperoxidase 30 and run on a Sephadex G-15 column to separate [12~l]a-Btx from free NalZSI. The specific activity of the eluted [125I]a-Btx ranged between 300 and 700 Ci/mmol. The physiological effectiveness of [125I]a-Btx in blocl~ing synaptic transmission was confirmed by intracellular recording from sartorius nmscle in toad. The binding activity was routinely checked with the DE81 filter disk assay of Schmidt and Raftery37 using the Emulphogene BC-720 solubilized nAChR from Narcine brasiliensis electroplax. Tissue preparation. Common goldfish ~Carassius auratus; Ozark Fisheries) were maintained at room temperature. Unilateral eye enucleations were performed on fish under ice anesthesia. At times ranging from 1 to 30 days following enucleation, animals were reanesthetized and the brains removed. For each condition, 8 tecta were blotted on filter paper, weighed, and homogenized (10 ~ w/v) in 0.7 M sucrose using a motor driven teflon pestle (12 passes at 800 rpm). The homogenate was then centrifuged at 1000 × g for I0 min, and the resultant pellet was washed once. The supernatants were combined and centrifuged at 30,000 × g for 30 min. The resulting crude synaptosomal pellet was resuspended in 0.7 N sucrose and used for particulate binding assays. Electron microscopy revealed that the fraction was highly enriched in synaptosomes (Fig. 1). Particulate binding assay. To 100 #1 of the resuspended synaptosomal fraction was added 50 #I of 50 mM sodium phosphate buffer (pH 7.4) or unlabeled a-Btx (4 ×

116

Fig. 1. Electron micrograph of the crude synaptosomal fraction. The pellet was obtained as described under Methods and was fixed for 4 h in 2 ~ glutaraldehyde buffered with 100 mM sodium phosphate, pH 7.4. The pellet was postfixed in 1 ~ OsO4, dehydrated in a series of alcohols, and embedded in Araldite. The sections were stained with uranyl acetate and lead citrate. 10-SM). After 10 min, 50 /zl of phosphate buffer containing 1 pmol (5 nM, final concentration) of [125I]a-Btx, 5 mM NaNa, 0.1 mM a-toluene-sulfonyl fluoride (PMSF), 1 mM ethylene-diaminetetraacetic acid (EDTA), and 0.25 ~ bovine serum albumin (BSA) were added to the synaptosomal fraction and incubated for I h at room temperature with shaking. Incubations were terminated by the addition of 8 ml of PBS (50 mM sodium phosphate buffer, pH 7.4, with 0.25 M NaCI) and filtration through EGWP millipore filters presoaked in 0.25% BSA and 10-.5 M a-Btx. The filters were washed 5 times with 8 ml aliquots of PBS and counted at 68 ~ efficiency in a gamma scintillation spectrometer. Specific binding was defined as the difference between samples containing no cold a-Btx and those containing 10-a M a-Btx.

Anatomical studies Cobalt filling of the optic nerve. In order to demonstrate the laminar pattern of the optic projection in the goldfish, the optic nerve was filled with cobalt using a suction stalk placed over the cut end of the nerve 39. The fish were maintained at 4 °C for 2-4 days before sacrifice. The brains were removed and cobalt was precipitated with ammonium sulfide. The brains were then post-fixed for 12 h in Carnoy's

117 fixative ta, embedded in paraffin and sectioned at 10 #m. The sections were deparaffinized, and the cobalt reaction intensified with Timm's silver sulfide method 47 before coverslipping. Horseradish peroxidase injections. The optic nerve was exposed in the orbit, and a small incision in the sheath was made. A micropipette (20 #m tip diameter) filled with a 20 ~o solution of HRP (Type VI, Sigma) in H20 was inserted into the nerve. A total volume of 1/zl was injected over a period of 10 rain. After 3 days, the fish were perfused with 10~o formalin, and the brains were removed and postfixed in 1 ~o glutaraldehyde for 6 h at 4 °C. The brains were placed in 0.1 M sodium phosphate buffer, pH 7.4, containing 30~ sucrose for 12 h and then sectioned at 50 #m in a cryostat. After air drying, the sections were incubated in 0.1 M Tris.HC1 buffer, pH 7.6, containing 2.8 mM p-phenylenediamine dihydrochloride, 2.3 mM pyrocatechol, and 0.01 ~ hydrogen peroxide for 15 min to localize the HRP 15. After staining, the sections were rinsed, dehydrated, and coverslipped. Autoradiographic localization of [125I]a-Btx. For the autoradiographic localization of [125I]a-Btx, tecta in normal animals (8 fish) or in animals unilaterally enucleated (4 fish) were injected intraventricularly with 300 fmol of [125I]a-Btx in a volume of 6/zl using a micropipette. Control injections included 0.1 mM nicotine in addition to [125I]a-Btx. After 24 h, the brains were removed and fixed for 4 h in 1 glutaraldehyde buffered with 0.1 M sodium phosphate buffer (pH 7.4). Brains were postfixed for 12 h in 1 ~ glutaraldehyde, 5 Y/ooglacial acetic acid, and 80~ ethanol. After dehydration through a series of graded alcohols, the brains were embedded in paraffin and sectioned at 10 #m. Sections were deparaffinized, rehydrated, and coated with Kodak NTB-2 emulsion (1:1 with H20). After 3-7 days, sections were developed in Dektol (1:1 dilution with H20) for 3 rain at 12 °C and stained with cresyl violet. Acetylcholinesterase localization. Unilateral enucleations were performed on goldfish from 1 week to 6 months before sacrifice. For the demonstration of acetylcholinesterase (ACHE), brains were fixed in 10~ buffered formalin (0.1 M sodium phosphate buffer, pH 7.4) for 12 h at 4 oC, rinsed in phosphate buffer, and sectioned in a cryostat at 40 #m. The sections were air-dried and then incubated for 1-3 h at 32 °C in the Hanker et al. 14 modification of the Karnovsky and Roots t7 medium. As controls, some sections were incubated in mediums containing no substrate, or containing 10-5 M BW284C51, a specific AChE inhibitor. Following staining, sections were rinsed and coverslipped. Densitometric scanning. In order to compare semiquantitatively the relative distibutions of a-Btx binding, AChE staining and optic nerve fibers, we employed a specially constructed densitometric scanning device consisting of a computer driven light source focused onto a sensitive photodetector through a 200 /zm diameter aperture. The scanning assembly was coupled to a Tektronics 4662 Digital Incremental Plotter, in turn coupled to a PDP-12 computer. Successive scans of 256 points/scan were made through darkfield photomicrographs of the rectum in a direction normal to the tectal surface. The optical densities from corresponding points in adjacent scans were then averaged for each photomicrograph.

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Fig. 2. The binding of [t~5I]a-Btxas a function of mg of tissue added to the incubation mixture. The error bars represent the standard error of the mean, and the line is a linear least-squares fit through the origin. RESULTS Biochemical studies In order to use the binding of [125I]a-Btx to quantitate the number of binding sites present, the [125I]a-Btx concentration should be sufficient to saturate the binding sites, and the receptor concentration should be within the linear portion of its toxin binding isotherm. As shown previously 2s, a [125I]a-Btx concentration of 5 nM is sufficient to saturate 85-90 ~ of the binding sites. Fig. 2 demonstrates that the binding of [125I]a-Btx is a linear function of the amount of tissue added between 5 and 25 rag. Routine measurements were made using between 15 and 20 mg of tissue per assay, values well within the linear portion of the binding curve. Following enucleation, a-Btx binding sites are lost in the denervated tectum compared to control tecta as shown by the decrease in the amount of binding of [125I]a-Btx. Fig. 3 shows that two days following enucleation there is a rapid fall in [~25I]a-Btx binding, and after two weeks [125I]a-Btx binding in the denervated tectum levels off to approximately 60 ~ of the control values. The decrease in the binding of [x25I]a-Btx is apparent when binding is expressed in terms of binding/mg tissue or binding/tectum. Histological studies Fig. 4 compares the distribution of optic nerve terminals as revealed by cobalt filling of the optic nerve with the laminar patterns of a-Btx binding sites revealed using radioautography and with AChE staining. Fig. 5 shows a plot of optical density scans

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Fig. 3. The binding of [z~'5I]a-Btxin enucleatedrelative to control tecta as a function of time following denervation. The data are expressedin binding per mg of tissue. The error bars represent the standard error of the mean. of photographs showing cobalt, [125I]a-Btx and AChE bands in normal and enucleated goldfish tecta as a function of depth below the tectal surface. A numbering system is included in Fig. 5 to clarify the discussion of the histological banding pattern. As shown in both Figs. 4A and 5A, cobalt filling of the optic nerve results in a wide densely staining band in the stratum opticum (SO) and the superficial gray and white (SGW) layers (la and la') as well as revealing sparse fiber bands in the central gray (CG, 2a) and deep white layers (DW, 4a). Diffuse label is also seen between the fiber bands in the CG and DW (3a). This distribution of optic terminals in goldfish tectum has been described previously using both cobalt injections39 and current source density analysisl0,as,~9. Following incubation with [125I]a-Btx, the tectum shows a dense multi-peaked banding pattern of silver grains (lc and lc') distributed in the regions corresponding to the optic projections in the SO and SGW layers (Figs. 4B and 5C). In addition, 3 smaller bands are present in the middle CG (2c), the extreme lower portion of the CG (3c) and in the DW layer (4c). Two of these bands of silver grains (2c and 4c) correspond in depth to the bands of optic nerve preterminals filled with cobalt (2a and 4a). The prominent middle band (3c) showing [1251]a-Btx binding corresponds roughly in depth to a band (3a) of diffuse cobalt labeling (Fig. 5A, C). Although no distinct AChE band occurs at this depth (3b), the immediately adjacent wide dense AChE band (2b) in the CG layer extends into this area (Fig. 5B). The presence of AChE activity can be demonstrated in the region corresponding to both the SO and SGW layers (Figs. 4C and 5B; lb,2b' and lb"). Three distinct bands of activity were apparent in this area. They included (l) a dense narrow band in the marginal layer (1 b); (2) a thicker, less dense band in the superficial portion of the SGW (lb'); and (3) a narrow dense band at the lower border of the SGW (l b"). Two bands

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Fig. 4. Normal histology of the optic tectum. A: cobalt staining of the optic projection. B: dark-field photomicrograph of the radioautographic localization of [125I]a-Btxbinding. C: AChE stain shown with Nomarski optics. All sections are transverse. The laminae of the tectum are labeled as follows: M, marginal layer; SO, stratum opticum; SGW, superficial gray and white; CG, central gray; DW, deep white. of AChE activity were found in the CG layer (2b) and one band was found in the DW (4b). The latter bands correspond in position to optic nerve fiber bands (2a and 4a) shown using cobalt staining and two of the three deeper bands of [125I]a-Btx binding sites (2c and 4c). Unilateral enucleation results in a loss of [125I]a-Btx binding sites (shown radioautographically) from the major bands in the contralateral tectum. The 3 bands in the CG and DW layers (2c, 3c, and 4c) are entirely lost while only a small remnant of the superficial multi-modal band (lc and Ic') in the SO and SGW layers remains (Figs. 5E and 6). The scan in Fig. 5e is taken through the portion of the enucleated tectum (same animal as Fig. 5c) where the remnant of the superficial band is largest. Scans through sections of the enucleated tectum not containing remnants of the superficial band revealed no lamination. The scan in Fig. 5 is semiquantitative in that only relative densities of silver grains are shown. Except for the marginal layer, the entire extent of both normal and enucleated tecta was diffusely labeled to a higher degree than nicotine controls. Normal tecta demonstrated a lamination corresponding to the layers of optic projection in addition to this diffuse label. This lamination was selectively lost in enucleated tecta. The nature of the band (3c) of [125I]a-Btx binding in the CG, lost after enucleation, corresponding to the diffuse cobalt labeling (3a) was investigated further using H R P labeling of the optic projection. The injection of H R P into the optic nerve results in Golgi-like staining and seems to fill much finer processes than cobalt. Fibers originating in the middle CG and the DW (layers having optic fibers visualized by

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Fig. 6. Effect of enucleation on [12~I]a-Btxlocalization. A dark-field photomicrograph of a transverse section of the brain through both optic tecta is shown. The right tectum (N) is normally innervated, and the band in the superficial tectum is clearly visible. The left tectum (E) is the enucleated tectum which clearly shows the loss of [a2SI]a-Btx.

cobalt) seem to branch and terminate in the lower CG (the layer of the middle [1~5I]aBtx band, 3c) as well as their layer of origin. Fig. 7 shows thick fibers that originate in both the middle CG and the DW that send fine processes into the lower CG, as well as what seems to be HRP-filled terminal boutons within the lower CG. Thus, the middle p25I]a-Btx band (3c) seems to correspond to a tectal layer containing retinal terminals whose axons originate in the middle CG and the DW. In contrast to the loss of a-Btx binding sites following enucleation, nol oss of AChE activity could be demonstrated in the contralateral optic tectum up to 6 months following denervation (Fig. 5D) using light microscopic techniques. Biochemical techniques, particularly microassay techniques for quantitation of ACHE, are currently being explored to determine if subtle changes in the amount of AChE occurs following enucleation. DISCUSSION We have observed a loss of [125I]a-Btx binding sites in the goldfish optic tectum following enucleation, both biochemically (using a quantitative binding assay) and histologically (using light microscopic radioautography). The binding assay revealed

123

Fig. 7. HRP labeling of the optic projection in the CG and DW. Fibers originating in the DW (1) seem to send branches up into the lower CG layer, whereas fibers originating in the mid-CG (2) send branches into the lower CG. Also shown are punctate profiles (3) labeled with HRP which may correspond to optic terminals. that, two weeks following enucleation, 40 % of the [125I]a-Btx binding sites are lost. The radioautographic studies revealed that the majority of a-Btx binding sites are lost from those tectal layers that contain optic nerve fibers. Before considering these results in more detail, we should like to consider the probable identity of a-Btx binding sites in the goldfish optic tectum. Electrophysiological studies10,40, 42 of the goldfish optic tectum have demonstrated that highly purified preparations of a-Btx are capable of blocking visually evoked responses identified by current source density analysis 9. Concentrations of aBtx on the same order of magnitude as the biochemically measured equilibrium dissociation constant (10 -9 M) 2s effectively inhibited visual responses. These results, in conjunction with similar findings in the optic rectum of the toad 6,a,10 and the turtle al, suggest that the nAChR and the a-Btx binding site are likely to be the same in these animals. Investigators studying the properties of cultured parasympathetic a4 and sympathetic a,1s,2~,31,32 neurons, however, have questioned the use ofa-Btx as a marker for the nAChR due to the apparent inability of a-Btx to block agonist-induced activation of the nAChR in these systems. In addition, Patrick and Stallcup a~ have demonstrated that the a-Btx binding protein from cultured sympathetic neurons is not immunologically reactive with anti-eel electroplax AChR, although the antibody does block

124 AChR activation by nicotinic cholinergic agonists. Thus it appears that, unlike goldfish, toad and turtle optic rectum, a-Btx and ACh do not bind to identical sites in autonomic ganglia. A distinction between the a-Btx binding site in autonomic ganglia and that in toad and goldfish optic tecta can also be made biochemically, a-Btx dissociates from its binding site in sympathetic neurons with a half-time of dissociation of approximately 4 h 12,32, whereas dissociation of a-Btx from the binding component of toad and goldfish brains have much longer half-times of 452s and 10027 h, respectively. These slower dissociation rates are similar to that of nAChRs of muscle z where a-Btx is also effective. The loss of a-Btx binding sites in denervated tecta is somewhat surprising in light of the findings in autonomic ganglia 11 and at the neuromuscular junction 16. There transsection and degeneration of the presynaptic axon does not lead to a decrease in the number of postsynaptic receptors; in fact, at the neuromuscular junction, it leads to an increase in extrajunctional receptors. On the other hand, transection of postganglionic fibers leads to a retrograde loss of receptors from the cell bodies within the ganglion 11. There exists a class of cells in the fish tectum which send their axons out the optic nerve 48. However, since those sending axons to each eye are bilaterally distributed 39, retrograde response to axotomy could not account for the differences seen in the two tecta here. The most likely explanation is that the loss of binding represents an actual loss of acetylcholine receptors on the postsynaptic membrane of primary optic synapses. Since this would indicate a difference in the regulation of central receptors as opposed to those in the periphery, we have taken care to exclude other possible explanations. These include (1)an increase in mass of the tectal tissue rather than a loss of receptors (thereby decreasing the number of a-Btx binding sites per mg tissue), (2) a secondary, transneuronal loss of cells having intratectal cholinergic synaptic connections, and (3) a loss of receptors on the presynaptic optic terminals. The first possibility is unlikely because no increase in enucleated tectal volume was apparent in any histological section of an enucleated goldfish brain. In addition, although the results here are expressed as binding per mg tissue, similar results are obtained when binding per tectum is used. The second possibility, that of a secondary, transneuronal cell loss, is unlikely due to the rapid loss of binding sites. In a quantitative electron microscopic study of the synaptic effects of enucleation in the optic tectum of Xenopus laevis, Ostberg and Norden z6 and Norden et al. 23,24 have found no evidence for transneuronal degeneration before two months post-enucleation. In fact, the rate of loss of a-Btx binding sites seen in the present study appears to follow the same time course as the loss of optic synapses as determined by electron microscopy in both Xenopus 2~,24,26 and Carassius 2°,21. With respect to the third possibility mentioned above, the data presented here do not distinguish between the localization of the binding protein on the presynaptic optic terminal or on the membrane postsynaptic to the optic terminal. The accompanying paper, however, describes a selective inhibition of visual responses by a-Btx and several other nicotinic ligands that appears to be due to a direct effect on first order tectal cells postsynaptic to retinal ganglion cell terminals, rather than to a secondary effect of a postulated axo-

125 axonal inhibitory synapse (for which there is no anatomical evidence). Thus, the loss of a-Btx binding following denervation probably reflects the loss of nAChRs from the post-synaptic membrane of tectal neurons receiving primary optic synapses. The study of Ostberg and Norden 2a offers a possible ultrastructural correlate of the loss of toxin binding sites. They found that the glial cells engulfed and removed not only the degenerating optic terminals but also the post-synaptic specializations at the same time. The post-synaptic specialization is postulated to be the portion of the postsynaptic membrane that contains the neurotransmitter receptor. Thus, the removal of this structure suggests the removal of the post-synaptic receptor as well. As shown in Fig. 5D, there appears to be very little loss of AChE activity following enucleation. The reason why the loss of a-Btx binding is not accompanied by a loss of AChE activity remains unclear. One possibility is that the majority of both AChE and a-Btx binding protein is synthesized by the cell post-synaptic to retinal ganglion cells. When the optic terminals degenerate, the a-Btx binding protein may be degraded without appreciable new synthesis. ACHE, on the other hand, may continue to be synthesized by the post-synaptic cell regardless of the presence or absence of presynaptic input. In contrast to the work reported here, other workers have found a loss of AChE following denervation. For example, Storm-Mathisen 45 showed a loss of AChE activity in the rat hippocampus following interruption of the cholinergic septal input. Wawrzyniak 49 showed a loss of AChE activity in the optic rectum of Tinca vulgaris following enucleation, and we have also found a loss of AChE activity in the optic tectum of Bufo marinus ~9 following enucleation. Another possibility is that cholinergic fibers sprouted to occupy the sites vacated by the degenerating optic terminals. Such an explanation is unlikely because there is no evidence for sprouting in the optic tectum of Xenopus following enucleation ~3,z4,z6 and Murray 21 observed no unoccupied post-synaptic specializations in the goldfish optic tectum following nerve crush. Furthermore, in Carassius, enucleation produces a rapid glial response, and following long-term (225 days) enucleation the denervated rectum is also severely shrunken, indicating that little if any sprouting has occurred 25. Thus the relationship between AChE and the a-Btx binding protein in the goldfish optic tectum remains unclear. A definitive answer will require quantitative electron microscopic histochemistry. We conclude that the loss of a-Btx binding from the layers of optic projection very likely indicates that a substantial number of optic nerve fibers are cholinergic and that the post-synaptic receptor at these synapses is the nAChR. These conclusions are supported by the results presented in the accompanying paper, which shows a selective inhibition of retinotectal synaptic transmission induced by nicotinic antagonists. ACKNOWLEDGEMENTS This work was supported in part by a grant from the National Institutes of Health, EY01117, to J.A.F. In addition, generous support was provided by Dr. Robert N. Brady (USPHS Grant NS-11439). R.E.O. was supported by NIH predoctoral grant 5T32 EY07007 and J.T.S. by NIH Postdoctoral Fellowship 5F32 NS05437.

126 REFERENCES 1 Attardi, D. G. and Sperry, R. W., Preferential selection of central pathways by regenerating optic fibers, Exp. Neurol., 7 (1963) 46-64. 2 Brockes, J. P. and Hall, Z. W., Acetylcholine receptors in normal and denervated rat diaphragm muscle. I. Purification and interaction with 125I-a-bungarotoxin, Biochemistry, 14 (1975) 2092-2099. 3 Carbonetto, S. T., Fambrough, D. M. and Muller, K. J., Nonequivalence of a-bungarotoxin receptors and acetylcholine receptors in chick sympathetic neurons, Proc. nat. Acad. Sci. (Wash.), 75 (1978) 1016-1020. 4 Changeux, J. P. and Danchin, A., Selective stabilization of developing synapses as a mechanism for the specification of neuronal networks, Nature (Lond.), 264 (1976) 705-712. 5 Foders, R. and Salvaterra, P. M., Comparative aspects of nicotinic and muscarinic acetylcholine receptors in brain, Neurosci. Abstr., 3 (1977) 90. 6 Freeman, J. A., Possible regulatory function of acetylcholine receptor in maintenance of retinotectal synapses, Nature (Lond.), 269 (1977) 218-222. 7 Freeman, J. A., Intracellular responses and receptor localization of neurons in slices of goldfish tectum, Invest. OphthaL, Suppl. (1979) 228. 8 Freeman, J. A., Lutin, W. A. and Brady, R. N., Possible molecular mechanisms associated with retino-tectal synapse formation in Bufo marinus, revealed by the use of labelled snake neurotoxin, Neurosci. Abstr., 5 (1975) 138. 9 Freeman, J. A. and Nicholson, C., Experimental optimization of current source density technique: application to anuran cerebellum, J. NeurophysioL, 38 (1975) 369-382. 10 Freeman, J. A., Schmidt, J. T. and Oswald, R. E., Effect of a-bungarotoxin on retino-tectal synaptic transmission in goldfish and toad, Neuroscience, in press. 11 Fumagalli, L., De Renzis, G. and Miani, N., a-Bungarotoxin-acetylcholine receptors in the chick ciliary ganglion: Effects of deafferentation and axotomy, Brain Research, 153 (1978) 87-98. 12 Greene, L. A., Binding of a-bungarotoxin to chick sympathetic ganglia: properties of the receptor and its rate of appearance during development, Brain Research, 111 (1976) 135-145. 13 Gude, W. D., Autoradiographic Techniques, Prentice-Hall, New Jersey, 1968. 14 Hanker, J. S., Thornburg, L. P., Yates, P. E. and Moore, H. G., The demonstration ofcholinesterases by the formation of osmium blacks at the sites of Hatchett's brown, Histochemie, 37 (1973) 223-242. 15 Hanker, J. S., Yates, P. E., Metz, C. B. and Rustioni, A., A new specific, sensitive and non-carcinogenic reagent for demonstration of horseradish peroxidase, Histochem. J., 9 (1977) 789-792. 16 Harris, A. J., Inductive functions of the nervous system, Ann. Rev. Physiol., 36 (1974) 251-305. 17 Karnovsky, M. J. and Roots, L., A 'direct-colouring' thiocholine method for cholinesterase, J. Histochem. Cytochem., 12 (1964) 219-221. 18 Kouvelas, E. D., Dichter, M. A. and Greene, L. A., Chick sympathetic neurons develop receptors for a-bungarotoxin in vitro, but the toxin does not block nicotinic receptors, Brain Research, 154 (1978) 83-93. 19 Lee, C. Y., Chang, S. L., Kau, S. T. and Shing-Hui, L., Chromatographic separation of the venom of Bungarus multicinctus and characterization of its components, J. Chrom., 72 (1972) 71-82. 20 Marotte, L. R. and Mark, R. F., Ullrastructural localization of synaptic input to the optic lobe °f the carp ( Carassius carassius), Exp. Neurol., 49 (1975) 772-789. 21 Murray, M., Regeneration of retinal axons into the goldfish optic tectum, J. comp. NeuroL, 168 (1976) 175-196. 22 Nurse, C. A. and O'Lague, P. H., Formation of cholinergic synaspses between dissociated sympathetic neurons and skeletal myotubes of the rat in cell culture, Proc. nat. Acad. Sci. (Wash.), 72 (1975) 1955-1959. 23 Norden, J. J., Ostberg, A.-J. C. and Freeman, J. A., A quantitative EM investigation of changes in the synaptic organization of the optic tectum following enucleation in Xenopus, Neurosci. Abstr., 3 (1977) 571. 24 Norden, J. J., Ostberg, A.-J. C. and Freeman, J. A., Descriptive and quantitative EM studies of the optic tectum of Xenopus following enucleation, Neurosci. Abstr., 4 (1978) 639. 25 Norden, J. J., unpublished results. 26 Ostberg, A.-J. C. and Norden, J. J., Ultrastructural study of degeneration and regeneration in the amphibian tectum, Brain Research, 168 (1979) 441~,55. 27 Oswald, R. E. and Freeman, J. A., Amphibian optic nerve transmitter: ACh, yes; GABA and glutamate, no, Neurosci. Abstr., 3 (1977) 411.

127 28 Oswald, R. E. and Freeman, J. A., Characterization of the nicotinicacetylcholine receptor isolated from goldfish brain, J. bioL Chem., 254 (1979) 3419-3426. 29 Oswald, R. E., Schmidt, D. E. and Freeman, J. A., Assessment of acetylcholine as an optic nerve neurotransmitter in Bufo marinus, Neuroscience, 4 (1979) 1129-1136. 30 Papaionannou, S. and Gospodarowicz, D., Comparison of the binding of human chorionic gonado tropin to isolated bovine luteal cells and bovine luteal plasma membranes, Endocrinology, 97 (1975) 114-124. 31 Patrick, J. and Stallcup, W. B., Immunological distinction between acetylcholine receptor and the a-bungarotoxin-bindingcomponent on sympathetic neurons, Proc. nat. Acad. Sei. (Wash.), 74 (1977) 4689--4692. 32 Patrick, J. and Stallcup, B., a-Bungarotoxin binding and cholinergic receptor function on a rat sympathetic nerve line, J. bioL Chem., 252 (1977) 8629-8633. 33 Raftery, M. A., Schmidt, J., Clark, D. G. and Wolcott, R. G., Demonstration of a specific a-bungarotoxin binding component in Electrophorus electricus electroplax membranes, Biochem. biophys. Res. Commun., 45 (1971) 1622-1629. 34 Ravdin, P. M. and Berg, D., Inhibition of neuronal acetylcholine sensitivity by a-toxins from Bungarus multicinctus venom, Proc. nat. Acad. Sci. (Wash.), 76 (1979) 2072-2076. 35 Reisfeld, R. A., Lewis, U. J. and Williams,D. E., Disk electrophoresis of basic proteins and peptides in polyacrylamide gels, Nature (Lond.), 195 (1962) 281-283. 36 Schechter, N., Francis, A., Deutsch, D. G. and Gazzaniga, M. S., Recovery of tectal nicotiniccholinergic receptor sites during optic nerve regeneration in goldfish, Brain Research, 166 (1979) 57-64. 37 Schmidt, J. and Raftery, M. A., A simple assay for the study of solubilized acetylcholine receptors, Analyt. Biochem., 52 (1973) 349-354. 38 Schmidt,J.T.,Thesynapticorganizationofopticafferentstothenormalgoldfishtectum,Neurosci. Abstr., 3 (1977) 94. 39 Schmidt, J. T., The laminar organization of optic nerve fibres in the tectum of goldfish, Proc. roy. Soc. B, 205 (1979) 287-306. 40 Schmidt, J. T. and Freeman, J. A., Electrophysiological evidence that retinotectal synaptic transmission in the goldfish is nicotinic cholinergic, Brain Research, 187 (1980) 129-142. 41 Schmidt, J. T. and Freeman, J. A., Comparative effects of a-bungarotoxin on retinotectal transmission in different vertebrate phyla, Invest. OphthaL, Suppl. (1979) 280. 42 Schmidt, J. T. and Oswald, R. E., Electrophysiologic evidence that retinotectal transmission in the goldfish is nicotinic cholinergic, Neurosci. Abstr., 4 (1978) 520. 43 Springer, A. D. and Agranoff, B. W., Effect of temperature on rate of goldfish optic nerve regeneration: a radioautographic and behavioral study, Brain Research, 128 (1977) 405-415. 44 Springer, A. D., Heacock, A. M., Schmidt, J. T. and Agranoff, B. W., Bilateral tectal innervation by regenerating optic nerve fibers in goldfish: a radioautographic, electrophysiological and behavioral study, Brain Research, 128 (1977) 417--427. 45 Storm-Mathisen, J., Localization of transmitter candidates in the brain: the hippocampal formation as a model, Progr. Neurobiol., 8 (1977) 119-181. 46 Swank, R. T. and Munkres, K. D., Molecular weight analysis of oligopeptides by electrophoresis in polyacrylamide gel with sodium dodecyl sulfate, Analyt. Biochem., 39 (1971) 462-477. 47 Tyrer, N. M. and Bell, E. M., The intensification of cobalt-filled neurone profiles using a modification of Timm's sulphide-silver method, Brain Research, 73 (1974) 151-155. 48 Vanegas, H., Amat, J. and Essayag-Millan, E., Electrophysiological evidence of tectal efferents to the fish eye, Brain Research, 54 (1973) 309-313. 49 Wawrzyniak, M., Cbemoarchitektonische studien am tectum opticum yon teleostiern unter normalen und experimentellen beingungen, Z. Zellforsch., 58 (1962) 234-264.