Developing retinotectal projection in larval goldfish

The retinotectal projection in larval goldfish was studied with the aid of anterograde filling of optic fibers with HRP applied to the retina. The results show that optic fibers have already reached the tectum and begun to form terminal arbors in newly hatched fish. The projection is topographic in that fibers from local regions of the retina project to discrete patches of tectum, with the smallest patch covering 3.5% of the total surface area of tectal neuropil. Many fibers in young larvae have numerous short side branches along their length and only some of them show evidence of terminal sprouting. The arbors are approximately elliptical in shape and average about 1,500 microns 2. Growth cones are seen frequently. In older larvae, terminal arbors are larger and more highly branched, and they have begun to resemble those in adult fish. Fibers terminate in two strata; those in the upper layer are smaller (1,800 microns 2 on average) than those in the deeper stratum (4,000 microns 2 on average). The fraction of tectal surface area covered by individual arbors (the "tectal coverage") ranges from 1.5% to 3% of the total surface area of the tectal neuropil. In contrast, the tectal coverage of individual arbors in young adult goldfish is much smaller, ranging from 0.02% to 0.42% of tectal surface area (Stuermer, '84, and unpublished). This apparent increase in precision of the map in older animals is not due to retraction of arbors, which are slightly larger in adults, but is accounted for by overall tectal growth: the tectal neuropil in goldfish increases in area by about 250-fold during this period (Raymond, '86).     

Activity sharpens the map during the regeneration of the retinotectal projection in goldfish

In the regenerating retinotectal projection of goldfish, we have used intraocular injections of tetrodotoxin (TTX) to determine whether activity plays a role in organizing or refining the retinotopic map. Repeated injections produced a continuous 27-day block without producing extraocular effects or causing deleterious effects in the retinal ganglion cells⁸. The retinotectal maps regenerated in the TTX fish were normally organized but the multiunit receptive fields were grossly enlarged. In control regenerates, 1–3 units (arbors of retinal ganglion cell axons) were simultaneously recorded at each penetration and their combined receptive field averaged 11–12°, nearly the same as for single units. In TTX fish each penetration yielded at least 5–10 units whose receptive fields were clustered over a wider area averaging 27° across. Individual ganglion cell receptive fields were assessed both by tectal and by intraretinal recording and were not enlarged. Many fish were recorded up to 4 months after the release from TTX block, but no further refinement of the maps occurred. If the nerve was recrushed and regenerated a second time without TTX, a normal map was formed, ruling out any permanent changes in the retinal ganglion cells or in the tectum. Blocks during various portions of the regeneration process showed that lack of activity during the process of axonal elongation (first 2 weeks) does not cause enlargement of the multiunit receptive fields, but lack of activity during the period of synapse formation and maturation (14–34 days) does. The results are discussed in terms of an activity-dependent stabilization of synapses. Neighboring retinal ganglion cells are known to fire in a statistically correlated fashion and this could help in their elimination of incorrect branches following an early period of diffuse connections.     

Antibodies to ependymin block the sharpening of the regenerating retinotectal projection in goldfish

The regenerating optic nerve of goldfish first reestablishes a rough retinotopic map on the tectum, then goes through an activity dependent refinement that appears to involve the elimination of inappropriate branches from early regenerated arbors. Retinotopically appropriate branches and synapses may be stabilized because the normally correlated firing of neighboring ganglion cells could cause summation of their postsynaptic responses, making them more effective. Thus, refinement of the map may be similar in several ways to associative learning. In this study, we therefore tested whether ependymin, a major protein component of the extracellular fluid that has been implicated in synaptic changes thought to be associated with learning a simple task in goldfish, may also be involved in refinement of the retinotopic map. Goldfish that had undergone unilateral optic nerve crush received intraventricular infusion of antiependymin IgG or of control IgG's beginning at 21 days postcrush. Tectal recordings from these fish at 39-56 days postcrush showed that the projection had failed to sharpen, much as in the fish with activity blocked or synchronized; the average size of the multiunit receptive fields was 31 degrees vs 11 degrees normally. The field potentials elicited from these tecta by optic nerve shock were not significantly smaller than in controls, suggesting normal levels of synaptogenesis. Control projections, identically treated but infused with either unrelated IgG or Ringer's alone regenerated normally, giving multiunit receptive fields of 12 degrees. Intact (non-regenerating) projections of the experimental fish were not rendered abnormal by the IgG treatment. Histology showed the retinas and tecta of the infused fish to be normal in appearance. The results show a specific block of sharpening by antiependymin IgG. The ependymal glia of the tectum stain positively for ependymin in normal fish, particularly the cell bodies in the ependymal layer. The tectum, particularly the ependymal layer, stains more intensely during regeneration, which appears to trigger increased synthesis of ependymins in the ependymal glia. This increase and the block of sharpening by specific antibodies to ependymin suggest a possible role for ependymin in activity dependent synaptic stabilization, possibly through its polymerization when calcium is focally depleted at active synapses.     

Electrophysiologic evidence that retinotectal synaptic transmission in the goldfish is nicotinic cholinergic

A previous study identified, by conduction velocity following optic nerve shock, 3 classes of retinal fibers which project to 3 distinct laminae of the goldfish optic tectum. In the present study, the effect of various pharmacological agents on the synaptic efficacy of each of the 3 classes of retinal fibers was assessed by the use of current source-density analysis. All 3 classes of optic fibers appear to be nicotinic cholinergic. Six different nicotinic antagonists were tested. All 6 were effective in decrementing the responses of all 3 classes to a criterion level: alpha-bungarotoxin (10-8 M), alloferin (10-5 M), curare (10-4 M), metocurine (10-4 M), hexamethonium (10-4 M) and gallamine (10-3 M). Atropine, a muscarinic antagonist, had only a slight effect even at 10-3 M. Five nicotinic agonists tested also decremented synaptic responses: nicotine (10-5 M), carbamylcholine (10-4 M), acetylcholine (10-4 M), succinyl choline (10-4 M) and decamethonium (10-3 M), presumably via cellular depolarization and receptor desensitization. Two inhibitors of acetylcholinesterase prolonged the response at 10-4 M and decremented it as well at 10-3 M. Hemicholinium 3, an inhibitor of the high affinity uptake of choline, produced a gradual activity-dependent decrement in the responses. Beta-bungarotoxin, a presynaptically-acting toxin, abolished not only the postsynaptic components but also the presynaptic components at 10-6 M. In all other cases the presynaptic deflections were generally unaffected, and with the exception of the toxins, a return to at least 90% of the control value was achieved. In contrast, GABA (10-3 M) and bicuculine (10-4 M) both produced no discernible effect on the 3 classes of responses, and glutamate (10-3 M) produced only a slight decrement, which probably represents a non-specific effect.     

Reorganization of retinotectal projection following surgical operations on the optic tectum in goldfish

The pattern of neural reconnection between the retina and surgically operated tectum was studied in juvenile goldfish (8–11 cm long) with electrophysiological methods. The results confirm that the remaining rostral half-tectum reacquires a complete visual projection from the whole retina about 90 days after excision of the caudal half. The same reorganization of visual projection from the whole retina onto the rostral half-tectum was found to occur in the presence of the caudal half of the tectum, if the two halves were separated by a transverse surgical incision down to the level of the optic ventricle regardless of whether the contralateral optic nerve was left intact or crushed to regenerate. The reorganization of retinotectal projection was also found to occur biaxially along the mediolateral as well as the rostrocaudal axis of the tectum following excision of a caudomedial sector of the tectum. It is suggested that the reorganization of retinotectal projection is due to synaptic respecifications of individual tectal neurons in correct retinotopic order, and that the cellular discontinuity between the rostral and the caudal parts of the tectum is sufficient to induce the orderly synaptic respecifications in juvenile goldfish.     

Mapping the normal and regenerating retinotectal projection of goldfish with autoradiographic methods

In normal goldfish, lesions of various size were made in nasal or temporal retina immediately prior to retinal labeling with tritiated proline. The resulting gaps in retinal innervation of tectum indicated that the projection is retinotopographically ordered to a precision of about 50 μm. Similarly, acute tectal incisions transecting the optic pathways were combined with immediate retinal labeling. The resulting tectal denervation confirmed that most fibers follow highly ordered paths through the stratum opticum of tectum; but a few fibers were found to follow unusual paths to their appropriate tectal positions. In other fish, the optic nerve was crushed. At various times afterwards, retinotopography and pathway order were similarly analyzed by making retinal lesions or tectal incisions just prior to labeling. For up to 40 days after crush, the projection lacked any refined retinotopic order. Only a gross topography could be demonstrated. Over several months, retinotopography gradually improved eventually approaching that of normals. Correlated with this was an initial stereotypic growth through the pathways of the stratum opticum followed by a long period of highly anomalous growth through the innervation layer. Evidently, many regenerated fibers grew in through inappropriate routes to the wrong region of tectum but subsequently arrived at their appropriate locus by circuitous routes within the innervation layer.     

Activity-dependent sharpening of the regenerating retinotectal projection in goldfish: relationship to the expression of growth-associated proteins

During regeneration of the optic nerve in goldfish, manipulations that disrupt the transmission of patterned visual information, if applied within the so-called 'sensitive period', lead to the formation of a diffuse retinotopic map (Schmidt, Cell. Mol. Neurobiol., 5 (1985) 65). The present study examined: (a) whether the sensitive period (14-50 days postcrush) coincides with the period in which specific 'growth-associated proteins' are present in the regenerating optic nerve terminals; and (b) whether manipulations that alter physiological activity during the sensitive period influence the expression of these proteins. Following bilateral optic nerve crush, goldfish regenerated their optic nerves either under normal illumination conditions (control), in total darkness, or with physiological activity suppressed in the nerve by intraocular injections of tetrodotoxin (TTX). At various times postcrush, proteins conveyed from the retina to the developing nerve endings were visualized by labeling the eye with [35S]methionine and then analyzing, by 2-dimensional gel electrophoresis and fluorography, radiolabeled proteins present in the optic tectum 15 h later. Rapidly-transported proteins that underwent large, specific increases during regeneration included the previously described 48 kDa growth-associated protein (GAP-48); labeling of GAP-48 was maximal during axonal outgrowth and then declined, but still remained well above background levels throughout the 'sensitive period'. Another group of rapidly-transported proteins, mol. wt. = 110-140 kDa (HMW), followed a similar time course, while levels of a 28 kDa protein peaked at 2 weeks and then declined rapidly. Thus, activity-dependent 'sharpening' processes occur during a period in which the levels of GAP-48 and HMW remain elevated in the nerve terminals.(ABSTRACT TRUNCATED AT 250 WORDS)     

Behavioral tests of compression of retinotectal projection after partial tectal ablation in goldfish

The course of functional recovery of vision during the compression of the retinotectal projection that follows hemitectal ablations was mapped by behavioral methods in goldfish (Carassius auratus). Differential suppression of respiration to red or green stimuli was used as a behavioral measure. Results show that vision was restored throughout the temporal half-field, originally blinded by tectal lesion, within 84 to 112 days of surgery. The scotoma diminished in an orderly fashion, starting from the central edge and progressing caudalward. Color discriminability was concomitant with visual recovery. The return of color vision indicates prespecification of retinal and tectal cells for color and selective reconnection of neurons according to their specificities. Functional topographic reorganization of the retinotectal projection implies that locus-specific affinities between retinal and tectal neurons, which may play a prominent role in the direction of nerve growth and formation of synapses under more normal circumstances, are not permanently fixed even in mature goldfish and may yield to compensatory developmental pressures created by the size disparity.     

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

The optic tectum of the goldfishCarassius auratus is a rich source of α-bungarotoxin (α-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 [¹²⁵Iα-Btx radioautography to the distribution of optic nerve terminals revealed by the use of cobalt and horseradish peroxidase (HRP) techniques. The majority of α-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 α-Btx binding in the contralateral tectum, observed both by radioautography and by a quantitative binding assay of α-Btx binding. Approximately 40% of the α-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 enucleation. These results are discussed in light of recent studies which show that the α-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.     

The effect of TTX-activity blockade and total darkness on the formation of retinotopy in the goldfish retinotectal projection

In the normal goldfish, neighboring retinal ganglion cells terminate in one small tectal locus to produce the precise retinotopy characteristic of this projection. This can be directly demonstrated by labeling neighboring ganglion cells with small "spot" injections of WGA-HRP, which yield a single small patch of product at the retinotopically appropriate part of the tectum. When the optic nerve is crushed, label from these spot injections was previously found to be widely dispersed during the early phase of regeneration. With time, label subsequently condensed, typically into several discrete patches reminiscent of ocular dominance columns. In this study, we tested whether the formation of these patches required impulse activity by injecting tetrodotoxin (TTX) into the eye during regeneration. We found that impulse blockade completely inhibited the formation of discrete patches while permitting considerable condensation of the label. This implies that these patches are generated by activity but that some map "refinement" utilized cellular processes that are activity independent. This activity-independent condensation progressed at a noticeably slower rate than the equivalent condensation seen with activity, thus suggesting that activity normally participates as a "helper factor," even though it is not strictly required. Since the formation of discrete patches during regeneration provides a sensitive measure of activity-dependent refinement, this was used to further address two controversial questions concerning the role of impulse activity. One is whether there is a chronologically defined critical period for activity-dependent refinement. This was tested by blocking impulse activity for 2 to 4 months, much longer than the activity-dependent refinement is thought to last, and then permitting activity to resume. We found that multiple patches were formed following this period of late activity, thus indicating that synaptic plasticity extends for several months beyond the supposed critical period. The other question was whether spontaneous retinal activity was sufficient for activity-dependent ordering. To test this, fish were kept in constant darkness during optic nerve crush and labelled with retinal spot injections at various times during regeneration. Condensation of label with the final formation of multiple patches formed at about the same time as fish with normal visual experience. This implies that the amount and extent of correlation of spontaneous activity in retina is adequate for driving activity-dependent refinement.     

Rules of order in the retinotectal fascicles of goldfish

Individual fascicles of retinal axons were labeled in the goldfish tectum with horseradish peroxidase (HRP). The contralateral retina was later processed for HRP histochemistry to mark the cells that had axons in the fascicles. Labeled cells were found in a partial half anulus in ventral hemiretina, centered on the optic disk. The distance of the partial anulus from the disk depended on which tectal fascicle had been labeled; the more rostrocentral the fascicle, the smaller was the annular radius. The angular subtense of the partial anulus with respect to the disk depended on where (along its tectal course) the fascicle had been labeled; the more rostral the label site, the longer was the angular subtense. These results were interpreted in the context of retinotectal growth, and it was inferred that the axons followed two rules: (1) grow in along the edge of the tectum and (2) exit and terminate in order, axons from temporal retina first, nasal retina last. These rules would produce a retinotopic projection in peripheral tectum, but they require that some of the terminals already in place must shift as the tectum grows.     

Long-term potentiation and activity-dependent retinotopic sharpening in the regenerating retinotectal projection of goldfish: common sensitive period and sensitivity to NMDA blockers

The regenerating retinotectal projection in goldfish goes through an activity-driven refinement that appears to involve the elimination of inappropriate branches from early arbors. Retinotopically appropriate branches may be stabilized because the normally correlated firing of neighboring ganglion cells causes summation of their postsynaptic responses and increases their effectiveness by a Hebbian mechanism. In this study, I report that the regenerating projection has an increased capacity for long-term potentiation (LTP) that may be related to the activity-driven sharpening. In the normal projection, field potentials, reflecting currents from EPSPs elicited by optic nerve shock, are large (greater than 4 mV) and very stable. In newly regenerated projections, field potentials are initially small (less than 1 mV), but a train of 20 stimuli at 0.1 Hz results in a large (100-200%) increase in amplitude that is stable for at least 8 hr, and in 3 cases overnight. The capacity for potentiation is greatest from 20 to 40 d postcrush, the time just after arrival of the optic fibers, and during the period of retinotopic sharpening. A greater-than-normal capacity for potentiation persists for many months. Topical application of NMDA receptor blockers AP5 or AP7 at 25 microM prevents potentiation without decrementing ongoing responses. The closely related agent AP6, which is not an NMDA receptor blocker, does not prevent potentiation. In addition, infusion of the NMDA receptor blockers AP5 or AP7 into the tectal ventricle (4 microliters/d of 500 microM solution) for 2-3 weeks during regeneration prevented retinotopic sharpening, as assessed by electrophysiological mapping. At each tectal point, responsive areas in the visual field were enlarged to 28 degrees vs 11-12 degrees in control regenerates and normals. This was comparable to data from fish regenerating with activity blocked with intraocular tetrodotoxin or synchronized by stroboscopic illumination and indicates uncorrected errors in targeting of regenerated arbors (Schmidt, 1985). The results support the involvement of NMDA receptors in sharpening and suggest that the initial step in stabilizing appropriate branches may be a long-lasting increase in synaptic gain.     

Antagonists of glutaminergic neurotransmission block retinotectal transmission in goldfish

The hypothesis that excitatory retinotectal transmission is mediated primarily by a glutamate or glutamate-related transmitter-receptor system was examined by recording extracellular field potentials in isolated sections of goldfish tectum while stimulating the optic tract and applying antagonists of excitatory amino acid (EAA) neurotransmission via the tissue bath. Three antagonists of EAA receptors produced greater than 90% reduction in the postsynaptic components of these evoked potentials. In order of potency, these were (with the concentrations that produced 50% block): kyurenic acid (0.15 mM), γ- -glutamyglycine (0.33 mM), and cis-2,3-piperidine dicarboxylic acid (0.47 mM). All 3 log concentration-effect curves were parallel, symmetrically sigmoidal, and somewhat steeper than non-cooperative single-site binding isotherms. All antagonist actions stabilized within 15 min and were completely reversible. An EAA antagonist potent and selective for the N-methyl- -aspartate (NMDA) subtype of receptor, 2-amino-5-phosphonovalerate, had little or no effect in either normal, low [Ca²⁺]/high [Mg²⁺], or Mg²⁺-free media. These data indicate that an excitatory amino acid receptor not of the NMDA subtype plays an essential role in fast excitatory retinotectal transmission, and would be most consistent with the mediation of most or all excitatory retinotectal transmission by a single class and subtype of glutamate receptor.     

Topography of regenerating optic fibers in goldfish traced with local wheat germ injections into retina: evidence for discontinuous microtopography in the retinotectal projection

A small injection of wheat germ agglutinin conjugated to horseradish peroxidase (WGA-HRP) was injected into dorsal or ventral peripheral retina in normal goldfish and in goldfish with prior optic nerve crush. Serial sections of tectum were subsequently taken for horseradish peroxidase (HRP) histochemistry 18 hours after injection and studied with light microscopy and densitometric reconstructions. In normals, a small, sharply delineated patch of product 200-300 microns wide was observed at the appropriate medial or lateral periphery of tectum. This product filled the entire SFGS, the main optic termination layer, and fell off abruptly at its edges. No labelling was detected in the optic pathways. In regenerates at about 20 days after nerve crush, these retinal injections yielded product that was dispersed across 1,000 microns or more of tectum but not in a uniform fashion. The densest product was biased toward the appropriate tectal position while product of intermediate density was mainly distributed along a path from the anterior end of tectum to this region. Product in the inappropriate half of tectum was much lighter and typically fiberlike in appearance. By about 40 days, product had condensed considerably at roughly the correct region of tectum but it was not as sharply delimited as in normals. Dense label occupied a single area about twice that of normals and exhibited flanking regions of light label extending for several hundred micrometers. At 59-148 days, a further condensation was observed but into more than one patch of product. The patches were of variable size and consisted of sharply delimited dense product which filled the entire SFGS at each position. Morphologically, these patches bore a remarkable resemblance to the ocular dominance columns previously seen in this system.     

Activity-dependent refinement in the goldfish retinotectal system is mediated by the dynamic regulation of processes withdrawal: an in vivo imaging study

Imaging of regenerating optic fibers in living adult goldfish was used to visualize arbor restructuring during activity-dependent refinement. A small number of neighboring retinal ganglion cells were labeled with DiI and observed in the tectum of the living animal for 5-7 hours during the period of activity-dependent refinement. In contrast to earlier stages of regeneration, many optic arbors were surprisingly stable, showing little or no change. The observed changes were mainly retractions, and these were affected by retinotopic position and activity. Axon branches in retinotopic positions changed by much smaller amounts than ectopic axons, but in fish with retinal tetrodotoxin impulse blockade, no systematic difference was observed as a function of tectal position. Otherwise, impulse blockade had no notable effects.