Pathway choice by regenerating optic fibers following tectal lobectomy in the goldfish: Inferences from the study of gliosis in tectal efferent bundles

Cell counts in various fiber bundles of the goldfish brain have demonstrated a profound, but transient gliosis of tectal (and pretectal) efferent pathways following removal of a tectal lobe. In the majority of cases, the pathways which underwent gliosis were also those which were penetrated by regenerating optic fibers which had been sectioned by the tectal surgery. However, the dorsal trunk of the horizontal commissure on the intact side of the brain showed only a minimal gliotic response but was consistently innervated by the optic fibers. Conversely, the ansate commissure and the crossed tectobulbar tract invariably demonstrated a marked gliotic response but only rarely received more than minimal innervation by the regenerating fibers.These observations are discussed with regard to the modifications which they demand of the hypothesis that degenerating axon bundles in the goldfish brain are in some way attractive to regenerating axons.     

Anatomical evidence for the influence of degenerating pathways on regenerating optic fibers following surgical manipulations in the visual system of the goldfish

We have used [³H]proline radioautography to trace regenerating optic fibers in the goldfish following: (1) the removal of the right tectal lobe and the right eye, and (2) the removal of both tectal lobes. Our results indicate that following the removal of the right tectal lobe and the right eye, both the denervated tectal efferent pathways, and the denervated visual pathways and terminal zones of the enucleated eye were penetrated by the regenerating optic fibers. In addition, following bilateral lobectomy, the denervated tectal efferent pathways were bilaterally penetrated by the regenerating fibers. Since, in both types of operations, these denervated pathways and terminal zones should undergo degeneration, our results support the suggestion that the presence of degenerating axonal debris and proliferating glia may play an important role in guiding regenerating optic fibers in the visual system of the goldfish.     

A comparison of the normal and regenerated retinotectal pathways of goldfish

This is a light and electron microscopic study of the retinotectal pathway: intact and after regeneration of the optic nerve. The spatiotemporal pattern of axonal outgrowth and termination was studied with the methods of proline autoradiography, horseradish peroxidase (HRP) labeling, and fiber degeneration. The spatial order of optic fibers in the normal and regenerated pathways was assessed by labeling small groups intraretinally with HRP and then tracing them to the tectum. The labeled fibers occupied a greater fraction of the cross section of the regenerated than the normal optic tract. At the brachial bifurcation, roughly 20% of the regenerated fibers chose the incorrect brachium vs. less than 1% of the normals. In tectum, the regenerated optic fibers reestablished fascicles in stratum opticum, but they were less orderly than in the normals. The retinal origins of the fibers in the fascicles were established by labeling individual fascicles with HRP and then, following retrograde transport, finding labeled ganglion cells in whole-mounted retinas. Labeled cells were more widely scattered over the previously axotomized retinas than over the normal ones. A similar result was obtained when HRP was applied in the tectal synaptic layer. All of these results indicate that the pathway of the regenerated optic fibers is less well ordered than the intact pathway. Both autoradiography and HRP showed that the regenerating optic fibers invaded the tectum from the rostral end, and advanced from rostral to caudal and from peripheral to central tectum, along a front roughly perpendicular to the tectal fascicles. Synapses of retinal origin were noted electron microscopically in the tectum at the same sites where autoradiography indicated that the fibers had arrived. No retinal terminals were seen where grain densities were at background levels. Fiber ingrowth and synaptogenesis apparently occurred simultaneously. The synapses were initially smaller and sparser than in normals, but were in the normal tectal strata and contacted the same classes of post synaptic elements as in normals.     

On the formation of eye dominance stripes and patches in the doubly-innervated optic tectum of the chick

By surgically dividing the region of the presumptive optic chiasm in chick embryos on the third day of incubation (around stage 15), we have been able to induce substantial numbers of optic nerve fibers to grow aberrantly into the ipsilateral optic tract. As a result, many of the visual centers that are normally innervated only by fibers from the contralateral retina received fibers from both eyes. The proportion of fibers going to each tectal lobe varied from case to case, but in about one-third of the animals the tectal lobes received approximately equal numbers of fibers from each eye. In animals that survived until embryonic days 17-19 (which is beyond the period of retinal ganglion cell death) labeling of the two eyes with WGA-HRP and [3H]proline respectively, revealed a pattern of sharply defined eye dominance stripes or patches in the stratum griseum et fibrosum superficiale (SGFS) of the optic tectum, and in the ventral lateral geniculate nucleus. Less clearly segregated eye dominance zones were seen in the ectomammillary nucleus and the nucleus externus. The size and distribution of the stripes varied depending on the number of fibers projecting from each eye to a given tectal lobe; the minimum size was about 75 micron, while the maximum was large enough to occupy almost the entire tectal lobe. In animals in which the tectal input from the two eyes was roughly equal, the stripes varied in width between 75 micron and about one-third of the surface of the tectal lobe. The orientation of the stripes was consistently orthogonal to the direction of fiber ingrowth from the optic tract. From the earliest stages of optic fiber ingrowth, the fibers from the two eyes are completely intermixed in the stratum opticum (SO). However, on embryonic day 12, shortly after they have begun to penetrate into the SGFS, they are already segregated into stripes, although the stripe borders are very fuzzy. This suggests that the fibers from the two eyes may overlap at this stage. The phase of stripe formation coincides with that of naturally occurring retinal ganglion cell death, and we suggest that the two processes are interlinked.     

Time course and pattern of optic fiber regeneration following tectal lobe removal in the goldfish

Following single tectal lobe removal in the adult goldfish, Carassius auratus, the pattern of regeneration of the optic fibers which had previously projected to that tectum was examined at 1, 2, 4, 6, 8, 10, and 12 weeks postoperative using ³H-proline radioautography. We found that regenerating optic fibers grew across the midline through the transverse, minor, horizontal, and posterior commissures to innervate the remaining tectum. At early postoperative times innervation of the tectum was continuous, while later, the regenerating fibers segregated into discrete patches in the superficial layers of the tectum. In addition, regenerating fibers also grew into non-optic centers/pathways such as the habenula, the fasciculus retroflexus, the forebrain, the torus semicircularis, the valvula and corpus cerebelli, the hypothalamus, and the medulla. While optic fibers were no longer apparent in the habenula and the fasciculus retroflexus after 2 weeks postoperative, all other structures were still occupied by the fibers at 12 weeks postoperative. Since most of the innervated pathways were either tectal efferent pathways, which should contain degenerating debris and proliferating glial cells after the tectal removal, or pathways closely associated with traumatized areas, we suggest that degenerating axonal debris and proliferating glia may play an important role in guiding regenerating fibers in this system.     

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.     

Visual recovery in goldfish following unilateral optic tectum ablation: Evidence of competition between optic axons for tectal targets

Anatomical studies suggest that regenerating optic axons which invade the ipsilateral lobe of the optic tectum following ablation of the contralateral lobe compete with resident optic axons for synaptic sites on tectal neurons. Invader optic axons are initially uniformly distributed over the entire tectal lobe. With time, the invader and resident optic axons progressively segregate so that the invaders are localized in bands or islands separated by areas that are innervated mainly by the residents. When the resident optic axons are destroyed by ablating the eye opposite to the experimental eye, the invader axons remain continuously distributed and the segregation process apparently does not occur. We investigated the relationship between the segregation process and the recovery of visual function by the invader axons. Visual recovery was measured with a behavioral method in which the index of vision was the occurrence of a branchial suppression response to a moving spot of red light that was classically conditioned to an electric shock stimulus. The minimum time to reappearance of vision following ablation of the contralateral lobe of the tectum in two-eye fish was similar to the reported time of onset of the segregation process. Visual recovery occurred sooner when the opposite eye was removed. The restored vision in both groups disappeared following subsequent ablation of the remaining lobe of the tectum. These results suggest that the goldfish optic tectum normally contains no free synaptic sites for anomalous optic afferents and that the invader axons must compete for targets with the resident optic afferents. The invader axons can apparently remain unconnected or non-functional for several weeks following their arrival in the ipsilateral tectal lobe.     

Persistence of graded EphA/Ephrin-A expression in the adult frog visual system

Many studies have demonstrated the involvement of the EphA family of receptor tyrosine kinases and their ligands, ephrin-A2 and -A5, in the development of the temporonasal axis of the retinotectal/collicular map, but the role of these molecules in optic nerve regeneration has not been well studied. Noting that the characteristic gradients of the EphA/ephrin-A family that are expressed topographically in the retina and tectum of embryonic chicks and mice tend to disappear after birth, we took as our starting point an analysis of EphA and ephrin-A expression in leopard frogs (Rana pipiens and utricularia), species capable of regenerating the retinotectal map as adults. For the EphA family to be involved in the regeneration, one would expect these topographic gradients to persist in the adult or, if downregulated after metamorphosis, to be reexpressed after optic nerve injury. Using EphA3 receptor and ephrin-A5 ligand alkaline phosphatase in situ affinity probes (RAP and LAP, respectively) in whole-mount applications, we report that reciprocally complementary gradients of RAP and LAP binding persist in the optic tract and optic tectum of postmetamorphic frogs, including mature adults. EphA expression in temporal retinal axons in the optic tract was significantly reduced after nerve section but returned during regeneration. However, ephrin-A expression in the tectal parenchyma was not significantly elevated by either eye removal, with degeneration of optic axons, or during regeneration of the retinotectal projection. Thus, the present study has demonstrated a persisting expression of EphA/ephrin-A family members in the retinal axons and tectal parenchyma that may help guide regenerating fibers, but we can offer no evidence for an upregulation of ephrin-A expression in conjunction with optic nerve injury.     

Optic fiber segregation in goldfish with two eyes innervating one tectal lobe

An ipsilateral retinotectal projection was induced by ablating one tectal lobe. Radioautography indicated that the ipsilateral projection initially spread out continuously over the remaining tectal lobe. With time, the continuous projection progressively changed into a rostrocaudally oriented banded projection that was comprised of high and low silver grain density bands. The undamaged native projection from the contralateral eye also became transformed into high and low density bands. The results indicate that foreign fibers displace portions of the native projection. Complete segregation of the two projections was found at any time point examined. Low density bands did not represent spillover of label from a high density band since cobalt-filled optic fibers were found in low density bands. Quantitative analysis indicated that the contralateral projection occupied more tectal area than the ipsilateral projection. Area occupied by a projection, band width and band frequency showed appreciable between-fish variability. Correlations between band width and per cent of area occupied by a projection approached unity, indicating that large projections were associated with wider bands. A model is proposed to account for induced banding in lower vertebrates.     

Relationship between isthmotectal fibers and other tectopetal systems in the leopard frog

We studied the relationship of isthmotectal input to other tectal afferent fiber systems in three ways. 1) Using horseradish peroxidase (HRP) histochemistry, we determined the nonretinal inputs to the superficial tectum. In different sets of animals we a) applied HRP to the tectal surface; b) inserted HRP crystals into the tectum; c) injected small volumes of HRP solutions into the superficial tectum. N. isthmi accounts for more than 65% of the nonretinal extrinsic input in the superficial tectal layers. One set of fibers from the contralateral n. isthmi projects to the most superficial layer. Fibers from posterior thalamus and tegmentum project to both superficial and deeper layers in the tectum, but not to the most superficial layer. The ipsilaterally projecting isthmotectal fibers terminate in the deeper superficial layers. 2) We investigated the relationship between retinofugal and contralaterally projecting isthmotectal pathways. We orthogradely labelled n. isthmi fibers by unilateral HRP injections into n. isthmi, and we also labelled retinal fibers by injecting tritiated l-proline into both eyes. In such animals contralaterally projecting isthmotectal fibers cross in the dorsal posterior region of the optic chiasm. From the chiasm to the tectum isthmotectal fibers and retinofugal fibers are admixed. 3) We determined whether other fiber systems cross with contralaterally projecting isthmotectal fibers. We cut the posterior part of the optic chiasm and applied HRP crystals to the cut. Only n. isthmi and retina are retrogradely labelled.     

Topographic projections between the nucleus isthmi and the optic tectum in a teleost,Navodon Modestus

Following horseradish peroxidase injections into the optic tectum of a teleost,Navodon modestus, reciprocal and topographic projections between the nucleus isthmi and the ipsilateral optic tectum were determined. The isthmo-tectal fibers diverge to the optic tectum while maintaining the spatial arrangements of the isthmic cells from which the fibers originate. The tecto-isthmic projections also keep the spatial arrangements in the optic tectum. The tectal fibers converge near the nucleus isthmi and terminate in the non-cellular portion of the nucleus. The reciprocal topography is apparent in the combined results of 9 experiments with one tectal injection in each region. No labeled cells and fibers were found in the contralateral nucleus isthmi.     

Spatiotemporal profile of synaptic activation produced by the electrical and visual stimulation of retinal inputs to the optic tectum: a current source density analysis in the pigeon (Columba livia)

The optic tectum of the pigeon is a highly organized, multilayered structure that receives a massive polystratified afference of at least five different populations of retinal ganglion cells and gives rise to various anatomically segregated efferent systems. The synaptic organization of retino-tectal circuitry is, at present, mostly unknown. To investigate the spatiotemporal profile of synaptic activation produced by differential (electrical and visual) stimulation of the retinal inputs, we performed a high-spatial-resolution current source density analysis in the optic tectum of the anaesthetized pigeon. Electrical stimuli consisted of brief pulses of different durations applied to the optic nerve head, while visual stimuli consisted of light flashes of different intensities. Electrical stimulation generated sinks confined to retinorecipient layers. The temporal structure, spatial location and thresholds of these sinks indicated that they are all due to primary tectal synapses of retinal fibers with different conduction velocities. Sinks evoked by the fastest retinal axons were more superficially located than sinks produced by slower retinal fibers. Visual stimulation, on the other hand, resulted in a more complex pattern of current sinks, with various sinks located in the retinorecipient layers and also well below. Visual stimulation induced action potentials at superficial as well as deep tectal levels. We conclude that electrical stimulation activates most of the populations of ganglion cells as well as their primary tectal synapses, but is unable to elicit a significant activation of secondary tectal synapses. Visual stimulation, on the contrary, activates just some of the incoming retinal populations, but in a way that produces noticeable secondary activation of intratectal circuits. Laminar segregation of retinally evoked tectal activity, as reported here, has also been found in other vertebrates. Similarities and differences with previous studies are discussed.     

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

When one tectum of an adult goldfish is removed, the severed retinal fibers regenerate ipsilaterally into the remaining tectal lobe. Initially fibers from the two eyes overlap in the tectum but EM-HRP data suggest that few mature retinal synapses are formed between the ipsilateral eye and tectum at this time. At longer time periods, when some fibers appear to segregate into eye-specific termination bands, our data suggest that a significant number of synapses from the ipsilateral eye are present. These findings have important implications for how eye-specific termination bands are formed in doubly innervated tecta.     

Synaptic transmission of excitation from the retina to cells in the pigeon's optic tectum

Intracellular recordings were used to study the synaptic excitation of optic tectum neurons in the pigeon. Electrical stimulation of both contralateral optic nerve and ipsilateral optic tract evoked in the tectal neurons EPSPs which in most cases were followed by an IPSP. An extrapolation procedure based on response latency was used to reveal that the EPSPs were mediated by way of mono-, di- and polysynaptic connections with the retinal endings. The laminar location of the recorded cells was estimated according to the field potential and the recording depth with the exception of the cell which was intracellulary stained with HRP. Monosynaptic EPSPs were recorded from cells in the retinorecipient region (sublayers IIa-f) as well as in the non-retinorecipient region (sublayers IIg-j and layer III) of the tectum, while di- and polysynaptic EPSPs were never recorded from the input layers. Tectofugal projections arise largely from layer III neurons. Thus, these results indicate that retinal excitation is transmitted to the output tectal cells by way of mono-, di- or polysynaptic pathways. The conduction velocities of most retinal fibers mediating the EPSP ranged from 4 to 22 m/s (average 12 m/s). However, in a number of retinal fibers the conduction velocities were in a faster range, up to 36 m/s.     

Restoration of vision in genetically eyeless axolotls (Ambystoma mexicanum).

Eyes grafted into genetically eyeless axolotls at embryonic stages 26 or 27 (early tailbud stage) are capable of establishing retinotectal connections and restoring near normal vision. Normal vestibulo-ocular reflexes are also present in most of the eyeless mutants having grafted eyes. The animals are capable of accurately localizing objects in visual space and demonstrate following movements in an optokinetic drum. Evoked potentials can be recorded from the surfaces of the tectal lobes of eyeless mutants having a right eye graft which do not differ significantly from those recorded from a normal animal, except that recordings can still be obtained from the ipsilateral tectal lobe in the former following section of the intertectal fibers. This indication of direct retinotectal connections to the ipsilateral tectum was confirmed by histological examination which also showed that the optic fibers entering the diencephalon high on the lateral wall are initially directed toward the normal optic tract position before proceeding to be tectum.     

Normal and regenerating optic fibers in goldfish tectum: HRP-EM evidence for rapid synaptogenesis and optic fiber-fiber affinity

The distribution of normal and regenerating retinal fibers and synapses was studied on tectum in goldfish by light (LM) and electron microscopy (EM). Since labeling of the early regenerating fibers was previously reported to be difficult, a new 'cold-fill' HRP labeling protocol was developed, which labeled regenerating optic fibers and terminals on tectum as early as 14 days after nerve crush when they first arrive on tectum. In order to characterize the laminar distribution of optic afferents in normal fish and in fish regenerating for 14-240 days, EM photomontages of areas 14 microns wide by 160 microns deep through the HRP-labeled primary optic innervation layer (S-SO-SFGS) were constructed. The time points in regeneration that were examined spanned the period in which others have shown that an initially diffuse retinotopic map becomes spatially restricted. At the LM level regenerating optic fibers were restricted to the optic lamina. They reinnervated tectum in an anterior to posterior sequence as previously seen with autoradiography. In addition, at 14 days, some "pioneer" optic fascicles were found to have already grown to posterior tectum where they gave rise to branches with boutonlike terminations and growth-cone-like processes. Form the ultrastructural analysis it was clear that optic fibers and terminals observed strict laminar boundaries as they partitioned themselves in the optic laminae (S, SO and SFGS) in both normal and regenerating fish. The behavior of optic fibers was lamina specific with respect to synapse formation and the orientation of fiber outgrowth. As early as 14 days regeneration, optic fibers made synapses onto the four types of postsynaptic profiles observed in normal fish. Numerous optic terminals were labeled at 14 days, and there appeared to be no waiting period between fiber ingrowth to the SO and synapse formation in the S and SFGS. At 14-60 days, atypical synaptic contacts which appear to be nascent synapses were made by labeled optic fibers in fascicles and by growth-cone-like processes. By 21-30 days, the density of optic terminals was high and there were many more fasciculated optic fibers in the SFGS than normal as late as 350 days. These findings suggest that optic fiber lamination is highly constrained by tectal cues, that fibers rapidly regenerate many synaptic terminals before retinotopic map refinement is complete, and that fibers have a strong affinity for each other.     

Development of the retinotectal system in normal quail embryos: cytoarchitectonic development and optic fiber innervation

The development of the optic tectum and the establishment of retinotectal projections were investigated in the quail embryo from day E2 to hatching day (E16) with Cresyl violet-thionine, silver staining and anterograde axonal tracing methods. Both tectal cytodifferentiation and retinotectal innervation occur according to a rostroventral-caudodorsal gradient. Radial migration of postmitotic neurons starts on day E4. At E14, the tectum is fully laminated. Optic fibers reach the tectum on day E5 and cover its surface on day E10. 'Golgi-like' staining of optic fibers with HRP injected in vitro on the surface of the tectum reveals that: growing fronts are formed exclusively by axons extending over the tectal surface; fibers penetrating the outer tectal layers are always observed behind the growing fronts; the penetrating fibers are either the tip of the optic axons or collateral branches; as they penetrate the tectum, optic fibers give off branches which may extend for long distances within their terminal domains; the optic fiber terminal arbors acquire their mature morphology by day E14. The temporal sequence of retinotectal development in the quail was compared to that already established for the chick, thus providing a basis for further investigation of the development of the retinotectal system in chimeric avian embryos obtained after xenoplastic transplantation of quail tectal primordia into the chick neural tube.     

Systematic analysis of the visual projection neurons of Drosophila melanogaster. I. Lobula-specific pathways

In insects, visual information is processed in the optic lobe and conveyed to the central brain. Although neural circuits within the optic lobe have been studied extensively, relatively little is known about the connection between the optic lobe and the central brain. To understand how visual information is read by the neurons of the central brain, and what kind of centrifugal neurons send the control signal from the central brain to the optic lobe, we performed a systematic analysis of the visual projection neurons that connect the optic lobe and the central brain of Drosophila melanogaster. By screening approximately 4,000 GAL4 enhancer-trap strains we identified 44 pathways. The overall morphology and the direction of information of each pathway were investigated by expressing cytoplasmic and presynapsis-targeted fluorescent reporters. A canonical nomenclature system was introduced to describe the area of projection in the central brain. As the first part of a series of articles, we here describe 14 visual projection neurons arising specifically from the lobula. Eight pathways form columnar arborization in the lobula, whereas the remaining six form tangential or tree-like arborization. Eleven are centripetal pathways, among which nine terminate in the ventrolateral protocerebrum. Terminals of each columnar pathway form glomerulus-like structures in different areas of the ventrolateral protocerebrum. The posterior lateral protocerebrum and the optic tubercle were each contributed by a single centripetal pathway. Another pathway connects the lobula on each side of the brain. Two centrifugal pathways convey signals from the posterior lateral protocerebrum to the lobula.     

N‐methyl‐D‐aspartate receptors strongly regulate postsynaptic activity levels during optic nerve regeneration

During development, neuronal activity is used as a cue to guide synaptic rearrangements to refine connections. Many studies, especially in the visual system, have shown that the N‐methyl‐D‐aspartate receptor (NMDAr) plays a key role in mediating activity‐dependent refinement through long‐term potentiation (LTP)‐like processes. Adult goldfish can regenerate their optic nerve and utilize neuronal activity to generate precise topography in their projection onto tectum. Although the NMDAr has been implicated in this process, its precise role in regeneration has not been extensively studied. In examining NMDAr function during regeneration, we found salient differences compared with development. By using field excitatory postsynaptic potential (fEPSP) recordings, the contribution of the NMDAr at the primary optic synapse was measured. In contrast to development, no increase in NMDAr function was detectable during synaptic refinement. Unlike development, LTP could not be reliably elicited during regeneration. Unexpectedly, we found that NMDAr exerted a major effect on regulating ongoing tectal (postsynaptic) activity levels during regeneration. Blocking NMDAr strongly suppressed spontaneous activity during regeneration but had no significant effect in the normal projection. This difference could be attributed to an occlusion effect of strong optic drive in the normal projection, which dominated ongoing tectal activity. During regeneration, this optic drive is largely absent. Optic nerve stimulation further indicated that the NMDAr had little effect on the ability of optic fibers to evoke early postsynaptic impulse activity but was important for late network activity. These results indicate that, during regeneration, the NMDAr may play a critical role in the homeostatic regulation of ongoing activity and network excitability. © 2013 Wiley Periodicals, Inc.