MAP2 phosphorylation and visual plasticity in Xenopus.

Microtubule-associated protein 2 (MAP2) has been implicated in activity-dependent structural changes in dendrites. MAP2 regulates the assembly of cytoskeletal proteins such as microtubules and actin, and its function is phosphorylation-dependent. In hippocampus, MAP2 has been reported to be dephosphorylated by activation of the NMDA-type glutamate receptor, a key player in synaptic plasticity. In this work, we used a phospho-specific MAP2 antibody (Ab 305) that recognizes epitopes close to the microtubule-binding domain to investigate the possible role of MAP2 in the Xenopus visual system. The binocular system in Xenopus exhibits activity-dependent synapse rearrangement during a critical period of development. We have found that, in critical period animals, NMDA receptor activation leads to the dephosphorylation of MAP2 at sites recognized by Ab 305 in a dose-dependent manner. We compared the responses of MAP2 to NMDA treatment in animals with high binocular plasticity (critical period juveniles and dark-reared adults) and low plasticity (normal adults). Our results show that, in all groups, NMDA treatment induces the dephosphorylation of MAP2. Tecta from frogs with different degrees of plasticity show no differences in the baseline level of MAP2 phosphorylation or in the NMDA-induced MAP2 dephosphorylation response. These results suggest that activity may modify dendrite structure via the NMDA receptor--MAP2-cytoskeletal protein pathway, but this pathway does not seem to be a determinant of the degree of plasticity.

The development of abnormal axon trajectories after rotation of one eye in Xenopus.

The targeting of isthmotectal axons in the Xenopus binocular pathway is guided by both activity-dependent cues and activity-independent cues. Abnormal visual activity induced by unilateral eye rotation overrides activity-independent cues and causes isthmotectal axons to arborize at new locations during a critical period of development that ends approximately 3 months postmetamorphosis (PM). Horseradish peroxidase staining of isthmotectal axons reveals that they normally run rostrocaudally in the tectum; in contrast, those axons in animals with early eye rotation have circuitous trajectories. In this paper, by studying the trajectories and branching patterns of isthmotectal axons at different times after eye rotation, we aimed to investigate when and how activity cues determine the projection pattern of isthmotectal axons. As suggested by electrophysiological recording, isthmotectal axons initially grow normally and make arbors according to activity-independent cues despite the presence of abnormal visual input. Our findings demonstrate that the development of abnormal trajectories starts by 2 weeks PM in response to eye rotation and is a protracted process. It begins in the tectal regions in which the initial connections of isthmotectal axons are first formed according to activity-independent cues. At transitional stages (5 and 10 weeks), axons with arbors at two different locations are observed, with locations corresponding to the old and new termination sites, respectively. Later, at 10 weeks of age, the fainter horseradish peroxidase staining in arbors at old termination sites suggests that the older arbors are undergoing withdrawal.

Plasticity in the tectum of Xenopus laevis: binocular maps.

Xenopus frogs exhibit dramatic changes in the binocular projections to the tectum during a critical period of development. Their eyes change position in the head, moving from lateral to dorsal and creating an increasing region of binocular overlap. There is a corresponding shift of binocular projections to the tectum that keeps the two eyes' maps in register with each other throughout this period. The ipsilateral input is relayed via the nucleus isthmi. Two factors bring the ipsilateral projection into register with the contralateral projection. First, chemoaffinity cues establish a crude topographic map beginning when the shift of eye position begins. Approximately 1 month later, visual cues bring the ipsilateral map into register with the contralateral map. The role of visual input is demonstrated by the ability of the axons that bring the ipsilateral eye's map to the tectum to reorganize in response to a surgical rotation of one eye and to come into register with the contralateral eye's map. This plasticity can be blocked by NMDA receptor antagonists during the critical period. In normal adults, reorganization is minimal. Eye rotation fails to induce reorganization of the ipsilateral map. However, plasticity persists indefinitely in animals that are reared in the dark, and plasticity can be restored in normally-reared animals by treatment with NMDA. The working model to explain this plasticity posits that correlated input from the two eyes triggers opening of NMDA receptor channels and initiates events that stabilize appropriately-located isthmotectal connections. Specific tests of this model are discussed.

Effects of choline and other nicotinic agonists on the tectum of juvenile and adult Xenopus frogs: a patch-clamp study.

We have used anatomical methods and whole-cell patch-clamp recording to assess the distribution of nicotinic receptors in the tectum of Xenopus frogs and to measure effects of nicotinic ligands (carbachol, cytisine and nicotine) on glutamatergic spontaneous miniature excitatory postsynaptic currents. Our results confirm that retinotectal axons account for the majority of nicotinic receptors in the tectum and that nicotinic agonists exert presynaptic effects that increase the rate of transmitter release on to tectal cells. The nicotinic blockers mecamylamine and methyllycaconitine reduced responses to carbachol and cytisine. A small percentage of cells also showed postsynaptic responses. We have assessed whether there are developmental changes in the frequency of occurrence of spontaneous miniature excitatory postsynaptic currents. The first three months post-metamorphosis fall within the critical period for the dramatic plasticity displayed by binocular inputs during development in Xenopus. During this period, visual activity governs the formation of orderly maps relayed from the ipsilateral eye via the cholinergic projection from the nucleus isthmi to the tectum. In this study, we have found that critical-period tecta (two to 12 weeks postmetamorphosis) tend to have higher spontaneous activity than do older tecta (two to 69 weeks postmetamorphosis), and that nicotinic agonists increase that activity in both groups, with the result that the peak rates in response to nicotinic agonists are higher during the critical period than later. We also investigated the possible role of choline as an agonist of nicotinic receptors in the tectum. We have found that choline, as well as carbachol and cytisine, can cause a reversible increase in the rate of miniature excitatory postsynaptic currents. This result may help to explain how the isthmotectal projection, which accounts for the overwhelming majority of cholinergic input to the tectum, can exert effects on retinotectal terminals even though there are no morphologically identifiable synapses between the two populations. We have examined the morphology of cells filled with biocytin during the patch-clamp experiments, and we find that cells with dendrites in the stratum zonale, a layer with particularly dense input from the contralateral nucleus isthmi, have higher spontaneous activity than cells with dendrites that do not extend into that layer. Nicotinic agonists increased the activity recorded in both classes of cells. In addition, four pretectal cells were identified. Nicotinic agonists increased the rate of spontaneous activity recorded in that population. The results indicate that retinotectal transmission in the superior colliculus can be increased presynaptically by activity of the cholinergic projections of the nucleus isthmi. This modulation may be the basis for observations that blocking of cholinergic input disrupts the formation of topographic retinotectal projections. Moreover, the ability of choline to activate these receptors suggests that this metabolite of acetylcholine may permit paracrine activation of presynaptic receptors even though the tectum contains high acetylcholinesterase activity.

Polysialylated neural cell adhesion molecule and plasticity of ipsilateral connections in Xenopus tectum.

The optic tectum of Xenopus offers a readily manipulated system for testing the hypothesis that polysialylation of the neural cell adhesion molecule is associated with axonal plasticity. Axons relaying input to the tectum from the ipsilateral eye employ visual input to establish a topographic map in register with the contralateral map, despite naturally-occurring or surgically-induced repositioning of the eyes. This capacity for activity-dependent refinement or re-organization of the ipsilateral map is normally confined to a period between about one and four months postmetamorphosis but can be restored in adults by local application of N-methyl-D aspartate to the tectum. In addition, dark-rearing prolongs plasticity indefinitely. We have used immunohistochemical staining with antibodies to polysialic acid to determine whether conditions of high plasticity are correlated with high levels of polysialylated neural cell adhesion molecule in the tectum. We find that the staining level is high in tecta from one to three month postmetamorphic frogs but is low both before and after this period. Thus, in normal Xenopus frogs, anti-polysialic acid staining is heavier in the period of high plasticity than in the preceding or following postmetamorphic periods. As a further test of this relationship, we examined brains of adults with experimentally-induced plasticity. Tecta of N-methyl-D-aspartate-treated adults and of dark-reared adults showed higher levels of staining than did the tecta of normally-reared adults. These results also support the hypothesis that the presence of high levels of polysialic acid on neural cell adhesion molecules is causally related to activity-related changes in axonal growth patterns.

Differential intertectal delay between Rana pipiens and Xenopus laevis: implications for species-specific visual plasticity.

In the frog Xenopus laevis, the isthmotectal projection, which relays input from the ipsilateral eye, exhibits anatomical reorganization following surgical eye rotation performed during tadpole stages while the isthmotectal projection in the frog Rana pipiens fails to show reorganization. This plasticity has been shown to be dependent upon activation of the N-methyl-D-aspartate (NMDA) receptor located on tectal cell dendrites. The reorganization process in Xenopus is hypothesized to employ a Hebbian mechanism requiring correlated firing of ipsilateral and contralateral inputs to a given tectal cell; when an ipsilateral axon synapses onto a tectal cell that receives input from a contralateral axon with a matching receptive-field location, the correlation in activity triggers stabilization of the ipsilateral synapse. However, in neither Xenopus nor Rana do ipsilateral and contralateral inputs begin to fire simultaneously in response to a given visual stimulus; the ipsilateral input is delayed because it reaches the tectum indirectly, through a polysynaptic relay via the opposite tectum and nucleus isthmi. The objective of this experiment was to test whether there is a significant difference in this intertectal delay between Xenopus laevis and Rana pipiens in order to determine whether intertectal delay could be a contributing factor in this species-specific ability to exhibit visual plasticity. We have found that intertectal delay is 26.16 ms longer in Rana pipiens (36.53 ms) than in Xenopus laevis (10.37 ms).

Concanavalin A reduces habituation in the tectum of the frog.

In the tectum of Rana pipiens, responses to repeated flashes of light to the ipsilateral eye display considerable habituation. We have employed the plant lectin concanavalin A (Con A), which can diminish desensitization of glutamate receptors in vitro, in order to examine whether desensitization of glutamate receptors contributes to this habituation. The ipsilateral eye's projection reaches each tectal lobe indirectly, being relayed from the opposite tectal lobe via the tecto-isthmo-tectal projection. One of the sites along this pathway at which habituation may take place is the retinotectal synapse, where glutamate is a putative transmitter. Pretreatment of one lobe of the tectum with Con A significantly diminished the habituation of responses recorded in the other tectal lobe to light offset, with less of an effect on responses to light onset. These data suggest that OFF habituation may reflect glutamate receptor desensitization at the retinotectal synapse. In contrast, recordings from retinotectal terminals indicate that a primary site of habituation of ON responses is within the retina.

Ultrastructure and GABA immunoreactivity in layers 8 and 9 of the optic tectum of Xenopus laevis.

This study presents an ultrastructural analysis of layers 8 and 9 in the optic tectum of Xenopus laevis. Retinotectal axons were labelled with horseradish peroxidase and tectal cells were labelled with antibody to GABA. Four distinct axonal and dendritic structures were identified. GABA-negative axon terminals formed asymmetric synapses and were categorized as type a-1 (which included retinotectal axons), characterized by medium size synaptic vesicles and pale mitochondria, and type a-2 (non-retinotectal) with large vesicles and dense mitochondria. GABA-negative dendrites (type d) contained dense mitochondria, microtubules in the dendritic shafts, and dendritic spines devoid of microtubules. GABA-positive structures contained small synaptic vesicles and dense mitochondria. Some dendrites (type D) were not only postsynaptic but were also presynaptic elements, as defined by the presence of vesicles and distinct synaptic clefts with symmetric specializations. GABA-positive presynaptic structures were mostly located in vesicle-filled, bulbous extensions of dendritic shafts and usually terminated onto dendritic spines. Some type D dendrites were the middle element in serial synapses, with input from either GABA-positive or GABA-negative structures and output to GABA-negative structures. Retinotectal terminals were identified as one of the synaptic inputs to GABA-positive processes. Glia were characterized by granular cytoplasm and large mitochondria, often displaying a crystalline matrix structure. These results indicate that GABA-positive neurons are a prominent component of circuitry in the superficial layers of the tectum of Xenopus and that, as in mammals, they participate in serial synaptic arrangements in which retinotectal axons are the first element. These arrangements are consistent with complex processing of visual input to the tectum and a central role for inhibitory processes in the shaping of tectal responses.

Acceleration by NMDA treatment of visually induced map reorganization in juvenile Xenopus after larval eye rotation.

Each tectal lobe of Xenopus frogs receives two topographic maps, one via the ipsilateral eye and one via the contralateral eye. The alignment of the ipsilateral map with the contralateral map depends upon binocular visual input during a critical period that extends from late tadpole to early juvenile stages. Rotation of one eye during the critical period leads to reorganization of the ipsilateral map, which eventually comes back into alignment with the contralateral map despite the abnormal eye position. The ipsilateral eye's map initially develops as if there had been no alteration in eye position; there is a delay of 4-6 weeks before reorganization can be detected by electrophysiological mapping. In this paper, the possible role of the NMDA receptor in the delay in reorganization is addressed. The degree of NMDA receptor activation may need to be above some threshold level to trigger reorganization. If NMDA receptor activation normally is below that level until after the first month postmetamorphosis, then exogenous NMDA might boost the process sufficiently to start the reorganization process sooner than usual. In order to test this possibility, the left eye of tadpoles was rotated and NMDA was applied to the right tectal lobe for 3-5 weeks, starting at 1 week postmetamorphosis. Electrophysiological mapping demonstrated that reorganization takes place more rapidly than in untreated frogs or frogs treated with vehicle only. This result is consistent with the interpretation that the activation of the NMDA receptor is a rate-limiting step in the activity-dependent matching of binocular maps in Xenopus tectum.

Xenopus exhibits seasonal variation in retinotectal latency but not tecto-isthmo-tectal latency.

1. The tectum of Xenopus receives visuotopic input from both eyes. The contralateral eye's projection reaches the tectum directly, via the optic nerve. The ipsilateral eye's projection reaches the tectum indirectly, via the nucleus isthmi and isthmo-tectal projection. 2. Because of the multi-synaptic nature of the ipsilateral pathway, there is an inherent delay between the time that information from the contralateral eye reaches the tectum and the time that information from the ipsilateral eye arrives at the tectum. The length of the intertectal delay is a function of the latencies of the contralateral and ipsilateral pathways. 3. The length of this intertectal delay has functional, as well as developmental, implications with regard to the role of N-methyl-D-aspartate receptors in tectal cell activity and development of orderly synaptic connections. 4. We have found that the latencies of the contralateral and ipsilateral pathways exhibit a seasonal variation, increasing during the winter months. The increases of both latencies during the winter were of similar magnitude, indicating that there were no significant changes in intertectal delay. The seasonal alteration in contralateral latency was not affected by dark-rearing and was affected to only a minor extent by a week-long alteration of ambient temperature.

Isthmotectal axons make ectopic synapses in monocular regions of the tectum in developing Xenopus laevis frogs.

During the development of binocular maps in the tectum of Xenopus laevis, axons that relay input from the ipsilateral eye via the nucleus isthmi undergo a prolonged period of shifting connections. This shifting accompanies the dramatic change in eye position that takes place as the laterally placed eyes of the tadpole move dorsofrontally. There is a concomitant expansion of the proportion of tectum that receives contralateral retinotectal input corresponding to the binocular portion of the visual field. Electrophysiological recording demonstrates that ipsilateral units are present in those rostral tectal zones, and anatomical methods show that the isthmotectal axons arborize densely in the rostral region but also extend sparser branches into the caudal zone, which is occupied by contralateral inputs with receptive fields in the monocular zone of the visual field. A mechanism that aligns the ipsilateral and contralateral maps is activity-dependent stabilization of isthmotectal axons that exhibit firing patterns correlated with those of nearby retinotectal axons. In order for activity patterns to function in stabilizing correct connections and promoting the withdrawal of incorrect connections, synaptic communication of some sort is hypothesized to be essential. We have investigated whether isthmotectal axons make morphologically identifiable synapses during development and where such synapses are located. We find evidence for morphologically identifiable synapses in all regions of the tectum, along with many growth cones and structures that are probably immature synapses. As in the adult, the synapses contain round, clear vesicles, have asymmetric specializations, and terminate on structures that appear to be dendrites. In both adult and tadpole, the rarity of serial synapses involving isthmotectal terminals suggests that the interactions between retinotectal and isthmotectal inputs are mediated by postsynaptic dendrites.

Physiological effects of chronic and acute application of N-methyl-D-aspartate and 5-amino-phosphonovaleric acid to the optic tectum of Rana pipiens frogs.

Visually elicited activity contributes to the formation of orderly connections in the optic tectum of frogs. Glutamate receptors of the N-methyl-D-aspartate class participate in this process. Blocking those receptors interferes with activity-dependent refinement of maps in normal frogs and of ocular dominance bands in surgically produced animals with three eyes. Chronic application of N-methyl-D-aspartate sharpens the bands. The possibility that 5-amino-phosphonovaleric acid depresses tectal responsiveness was motivation for studying the effects of 5-amino-phosphonovaleric acid and N-methyl-D-aspartate applied both chronically and acutely. We evaluated tectal responsiveness to visual input by presenting flashes of light to one eye and recording responses in the ipsilateral tectal lobe. This method reveals the output of the tectal cells contralateral to the stimulated eye. These cells project via the nucleus isthmi to the opposite tectal lobe. We also mapped the receptive field dimensions of the crossed isthmotectal axons. Our results show that acute topical application of 500 microM or 1 mM N-methyl-D-aspartate dramatically increases spontaneous activity, while 100 microM N-methyl-D-aspartate causes little change. Chronic treatment with N-methyl-D-aspartate at a low dose (estimated to be in the micromolar range) shown to influence retinotectal mapping, reduces response latencies but produces no statistically significant changes in tectal cell firing rates or receptive field size. Acute application of 5-amino-phosphonovaleric acid produces complex results: 10 microM produces no changes in firing, 100 microM 5-amino-phosphonovaleric acid decreases firing, and doses of 500-100 microM increase the firing.(ABSTRACT TRUNCATED AT 250 WORDS)

Chronic effects of NMDA and APV on tectal output in Xenopus laevis.

In the South African clawed-toed frog Xenopus laevis, visual experience plays a crucial role in the formation of matching binocular maps in the tectum. The ipsilateral eye's projection, relayed through the crossed isthmotectal projection, displays marked plasticity in response to altered visual input during a critical period of development. This plasticity and the events responsible for the end of the critical period are mediated by N-methyl-D-aspartate (NMDA) receptor function. We have previously reported that chronic blockade of tectal NMDA receptors with the NMDA antagonist 5-amino-phosphonovaleric acid (APV) prevents plasticity of the crossed isthmotectal projection during the critical period, while chronic treatment with NMDA restores this plasticity after the end of the critical period. These results raise the question of whether the effects on plasticity are due to changes in electrical responsiveness of the treated tissue. In this study, we have quantitatively assessed the actions of APV and NMDA on certain aspects of tectal cell activity in Xenopus during and after the critical period by recording the output of the nucleus isthmi cells that are activated by the tectum after three weeks of treatment. We have found that chronic APV treatment does not alter tectal output, as indicated by the firing of isthmotectal axons, during the critical period and that chronic NMDA treatment increases tectal output in postcritical period Xenopus. Tectal output does not differ between normal Xenopus during and after the end of the critical period.(ABSTRACT TRUNCATED AT 250 WORDS)

Latency and temporal overlap of visually elicited contralateral and ipsilateral firing in Xenopus tectum during and after the critical period.

The mechanisms underlying the development of proper topographic registration of binocular maps in the tectum of Xenopus laevis involve correlation of activity patterns of ipsilateral and contralateral inputs. Recent evidence implicates NMDA-type glutamate receptors in this process. In general, NMDA receptors are considered to function optimally when there are multiple, simultaneous excitatory inputs to a dendrite. In the binocular system of the frog, however, the ipsilateral eye's response to a visual stimulus reaches the tectum later than the contralateral eye's response. The reason for this delay is that the ipsilateral pathway to the tectum is indirect, involving a relay in the opposite tectum and nucleus isthmi. In this paper, we evaluate the duration of the delay between arrival of contralateral and ipsilateral input in response to cessation of light and we also gauge the extent of temporal overlap in responses of the two inputs. We find that the average delay is about 10 ms and that this delay is not significantly different during the critical period vs later in development. The temporal overlap is 40-60 ms in duration. We conclude that the intertectal delay does not prevent a substantial period of simultaneous firing of ipsilateral and contralateral inputs in response to sudden changes in illumination. Therefore, the firing patterns of these afferents are compatible with a mechanism of activity-dependent alignment of binocular maps in the tectum.

Restoration of the plasticity of binocular maps by NMDA after the critical period in Xenopus.

Visual input during a critical period of development plays a major role in the establishment of orderly connections in the developing visual system. In Xenopus laevis, the matching of visual maps from the two eyes to the optic tectum depends on binocular visual input during the critical period, which extends from late tadpole to early juvenile stages. Alterations in eye position, which produce a mismatch of the tectal maps, normally evoke a compensatory adjustment in the map of the ipsilateral eye only during the critical period. However, continuous application of the glutamate receptor agonist N-methyl-D-aspartate (NMDA) after the normal end of the critical period restores this ability to realign the visual map.

Ultrastructure of the crossed isthmotectal projection in Xenopus frogs.

The nucleus isthmi (NI) of frogs is a relay for input from the eye to the ipsilateral tectum; each NI receives retinotopic input from one tectum and sends retinotopic output to both tecta. The crossed isthmotectal projection in Xenopus displays tremendous plasticity during development. Physiological and anatomical studies have suggested that the location at which a developing isthmotectal axon will terminate is determined by the correlation of its visually evoked activity with the activity of nearby retinotectal terminals. What structures could mediate such communication? We have examined quantitatively the ultrastructural characteristics of crossed isthmotectal axons and synapses in order to determine whether retinotectal axons communicate directly with isthmotectal axons via axo-axonic synapses or whether the communication is indirect, e.g., via common postsynaptic dendrites. Our results support the conclusion that isthmotectal axons interact with retinotectal axons indirectly and that tectal cell dendrites are the critical site of interaction.

Plasticity in the ipsilateral visuotectal projection persists after lesions of one nucleus isthmi in Xenopus.

Visual input has a profound effect on the development of binocular maps in the tectum of the frog Xenopus laevis. Input from the ipsilateral eye, which is relayed to the tectum via the opposite nucleus isthmi, is normally in register with the retinotectal map from the contralateral eye. However, if one eye is rotated during larval stages while the other eye is left in normal orientation, then the resulting mismatched visual input induces the crossed isthmotectal axons to change their trajectories and to establish a reoriented ipsilateral visuotectal map in register with the contralateral retinotectal map. The major cue which aligns the two maps is the correlation of visually-evoked activity from the two eyes. This experiment was designed to determine whether the uncrossed isthmotectal projection is necessary to organize the map transmitted by the crossed isthmotectal axons. Each NI receives a topographic map from the tectum on the same side of the brain and therefore carries the same topographic information as the retinotectal projection, and each NI transmits that map not only to the opposite tectum but also back to the same tectum from which it received its input. Thus, the uncrossed isthmotectal axons provide each tectum with a map which is essentially topographically identical to the retinotecal map but which is slightly delayed temporally. The uncrossed isthmotectal axons therefore could provide topographic cues to the guide the alignment of the crossed isthmotectal axons as they establish the ipsilateral visuotectal map. In order to determine whether the uncrossed isthmotectal projection is an important source of topographic cues for the crossed isthmotectal axons, the right nucleus isthmi was ablated and one eye was rotated by 90 degrees-150 degrees in midlarval tadpoles.(ABSTRACT TRUNCATED AT 250 WORDS)

N-methyl-D-aspartate antagonists prevent interaction of binocular maps in Xenopus tectum.

Glutamate receptors appear to play a key role in several forms of experience-dependent modification of both the strength of synapses and synaptic connectivity. In developing Xenopus frogs, the connections made by isthmotectal axons relaying visual input from the eye to the ipsilateral tectum are markedly influenced by the visual activity of contralateral retinotectal axons, and normal binocular visual input is necessary in order for the ipsilateral visuotectal map to come into register with the contralateral map. We have tested whether NMDA receptors play a role in establishment of the topographic matching of binocular maps during development. We have examined the effects of chronic treatment of tectum with either the receptor agonist NMDA or the antagonists APV or CPP applied throughout early postmetamorphic life using subpial implants of drug-impregnated elvax. Both antagonists blocked the matching of the ipsilateral map to the contralateral map, while NMDA permitted such matching. Our data therefore indicate that NMDA receptors are involved in the experience-dependent establishment of matching binocular maps during development.

Development of the nucleus isthmi in Xenopus, II: Branching patterns of contralaterally projecting isthmotectal axons during maturation of binocular maps.

The tectum of Xenopus frogs receives input from both eyes. The contralateral eye's projection reaches the tectum directly, via the optic nerve, and the ipsilateral eye's projection reaches the tectum indirectly, via the nucleus isthmi. Under normal conditions, the topography of the ipsilateral map relayed from the nucleus isthmi is in register with the topography of the retinotectal map from the contralateral eye. During development, the process of aligning the two maps is complicated by the dramatic changes in binocular overlap of the two eyes' visual fields which take place during late tadpole and juvenile stages. The goal of this study is to determine the branching patterns of contralaterally projecting isthmotectal axons before, during, and after the period of rapid eye migration. Isthmotectal axons were filled by anterograde transport of horseradish peroxidase (HRP) from the nucleus isthmi. The results show that crossed isthmotectal axons enter the entire extent of the tectum before binocular overlap begins to increase. Therefore, binocular overlap is not necessary for the initial isthmotectal projection to span the tectum. The density of isthmotectal branches rises dramatically at the same time that the eyes begin to shift. During the period when eye migration is most rapid, many isthmotectal axons form arbors which resemble adult arbors but which extend over greater proportions of the tectal surface. The axons appear to be directed toward appropriate mediolateral positions as they enter the tectum. Their trajectories are roughly rostocaudal, with relatively little change along the mediolateral dimension. These data, when combined with available physiological data, suggest that mediolateral order is initially established by vision-independent mechanisms but can be altered by vision-dependent mechanisms. Rostrocaudal order becomes discernable only at the time when binocular visual cues become available and appears to be established primarily on the basis of the activity of the retinotectal and isthmotectal axons.