Tectal Projection Research Articles

The Visual Systems of Zebrafish.

The zebrafish visual system has become a paradigmatic preparation for behavioral and systems neuroscience. Around 40 types of retinal ganglion cells (RGCs) serve as matched filters for stimulus features, including light, optic flow, prey, and objects on a collision course. RGCs distribute their signals via axon collaterals to 12 retinorecipient areas in forebrain and midbrain. The major visuomotor hub, the optic tectum, harbors nine RGC input layers that combine information on multiple features. The retinotopic map in the tectum is locally adapted to visual scene statistics and visual subfield-specific behavioral demands. Tectal projections to premotor centers are topographically organized according to behavioral commands. The known connectivity in more than 20 processing streams allows us to dissect the cellular basis of elementary perceptual and cognitive functions. Visually evoked responses, such as prey capture or loom avoidance, are controlled by dedicated multistation pathways that-at least in the larva-resemble labeled lines. This architecture serves the neuronal code's purpose of driving adaptive behavior.

Afferent and efferent connections of the nucleus prethalamicus in the yellowfin goby Acanthogobius flavimanus.

The nucleus prethalamicus (PTh) receives fibers from the optic tectum and then projects to the dorsal telencephalon in the yellowfin goby Acanthogobius flavimanus. However, it remained unclear whether the PTh is a visual relay nucleus, because the optic tectum receives not only visual but also other sensory modalities. Furthermore, precise telencephalic regions receiving prethalamic input remained unknown in the goby. We therefore investigated the full set of afferent and efferent connections of the PTh by direct tracer injections into the nucleus. Injections into the PTh labeled cells in the optic tectum, ventromedial thalamic nucleus, central and medial parts of the dorsal telencephalon, and caudal lobe of the cerebellum. We found that the somata of most tecto-prethalamic neurons are present in the stratum periventriculare. Their dendrites ascend to reach the major retinorecipient layers of the tectum. The PTh is composed of two subnuclei (medial and lateral) and topographic organization was appreciated only for tectal projections to the lateral subnucleus (PTh-l), which also receives sparse retinal projections. In contrast, the medial subnucleus receives fibers only from the medial tectum. We found that the PTh projects to nine subregions in the dorsal telencephalon and four in the ventral telencephalon. Furthermore, cerebellar injections revealed that cerebello-prethalamic fibers cross the midline twice to innervate the PTh-l on both sides. The present study is the first detailed report on the full set of the connections of PTh, which suggests that the PTh relays visual information from the optic tectum to the telencephalon.

Direct Dopaminergic Projections from the SNc Modulate Visuomotor Transformation in the Lamprey Tectum

Dopamine neurons in the SNc play a pivotal role inmodulating motor behavior via striatum. Here, weshow that the same dopamine neuron that targets striatum also sends a direct branch to the optic tectum (superior colliculus). Whenever SNc neurons are activated, both targets will therefore be affected. Visual stimuli (looming or bars) activate the dopamine neurons coding saliency and also elicit distinct motor responses mediated via tectum (eye, orienting or evasive), which are modulated by the dopamine input. Whole-cell recordings from tectal projection neurons and interneurons show that dopamine, released by SNc stimulation, increases or decreases the excitability depending on whether they express the dopamine D1 or the D2 receptor. SNc thus exerts its effects on the visuomotor system through a combined effect directly on tectum and also via striatum. This direct SNc modulation will occur regardless of striatum and represents a novel mode of motor control.

Intralaminar and tectal projections to the subthalamus in the rat.

Projections from the posterior intralaminar thalamic nuclei and the superior colliculus (SC) to the subthalamic nucleus (STN) and the zona incerta (ZI) have been described in the primate and rodent. The aims of this study was to investigate several questions on these projections, using modern neurotracing techniques in rats, to advance our understanding of the role of STN and ZI. We examined whether projection patterns to the subthlamus can be used to identify homologues of the primate centromedian (CM) and the parafascicular nucleus (Pf) in the rodent, the topography of the projection including what percent of intralaminar neurons participate in the projections, and electron microscopic examination of intralaminar synaptic boutons in STN. The aim on the SC‐subthalamic projection was to examine whether STN is the main target of the projection. This study revealed: (i) the areas similar to primate CM and Pf could be recognized in the rat; (ii) the Pf‐like area sends a very heavy topographically organized projection to STN but very sparse projection to ZI, which suggested that Pf might control basal ganglia function through STN; (iii) the projection from the CM‐like area to the subthalamus was very sparse; (iv) Pf boutons and randomly sampled asymmetrical synapses had similar distributions on the dendrites of STN neurons; and (v) the lateral part of the deep layers of SC sends a very heavy projection to ZI and moderate to sparse projection to limited parts of STN, suggesting that SC is involved in a limited control of basal ganglia function.

A GABAergic tecto-tegmento-tectal pathway in pigeons.

Previous studies have demonstrated that the optic tecta of the left and right brain halves reciprocally inhibit each other in birds. In mammals, the superior colliculus receives inhibitory γ-aminobutyric acid (GABA)ergic input from the basal ganglia via both the ipsilateral and the contralateral substantia nigra pars reticulata (SNr). This contralateral SNr projection is important in intertectal inhibition. Because the basal ganglia are evolutionarily conserved, the tectal projections of the SNr may show a similar pattern in birds. Therefore, the SNr could be a relay station in an indirect tecto-tectal pathway constituting the neuronal substrate for the tecto-tectal inhibition. To test this hypothesis, we performed bilateral anterograde and retrograde tectal tracing combined with GABA immunohistochemistry in pigeons. Suprisingly, the SNr has only ipsilateral projections to the optic tectum, and these are non-GABAergic. Inhibitory GABAergic input to the contralateral optic tectum arises instead from a nearby tegmental region that receives input from the ipsilateral optic tectum. Thus, a disynaptic pathway exists that possibly constitutes the anatomical substrate for the inhibitory tecto-tectal interaction. This pathway likely plays an important role in attentional switches between the laterally placed eyes of birds. J. Comp. Neurol. 524:2886-2913, 2016. © 2016 Wiley Periodicals, Inc.

Classification of nAChRβ2-immunoreactive retinal ganglion cells and their tectal projections in chicks

The relationship between the type of retinal ganglion cell (RGC) and the retinoreceptive layer of the tectum is investigated by the immunostaining of RGCs with nicotinic acetylcholine receptorβ2 (nAChRβ2) antibody and intracellular staining by DiI and also by anterograde degeneration and biotinylated dextran amine labeling of retinotectal fibers in chicks. The results strongly suggest that many of the RGCs that express immunoreactivity to nAChRβ2 send axons to tectal layer 7 and are mainly classified into the simple-type of Groups II and III, which contain the cells providing middle-sized to large dendritic fields with simple dendritic arborization. These nAChRβ2-immunoreactive RGCs receive visual information via the multiple sublayers of the inner plexiform layer.

Connections of the superior colliculus to shoulder muscles of the rat: a dual tracing study.

Previous investigations indicate that the superior colliculus (SC) is involved in the initiation and execution of forelimb movements. In the present study we investigated the tectofugal, in particular the tecto-reticulo-spinal projections to the shoulder and arm muscles in the rat. We simultaneously retrogradely labeled the premotor neurons in the brainstem by injection of the pseudorabies virus PrV Bartha 614 into the m. rhomboideus minor and m. acromiodeltoideus, and anterogradely visualized the tectofugal projections by intracollicular injection of the tracer FITC dextrane. Our results demonstrate that the connection of the SC to the skeletal muscles of the forelimb is at least trisynaptic. This was confirmed by long survival times after virus injections into the muscles (98–101 h) after which numerous neurons in the deep layers of the SC were labeled. Transsynaptically retrogradely labeled brainstem neurons connected disynaptically to the injected muscles with adjacent tectal terminals were predominantly located in the gigantocellular nuclear complex of the reticular formation. In addition, putative relay neurons were found in the caudal part of the pontine reticular nucleus. Both tectal projections to the nucleus gigantocellularis and the pontine reticular nucleus were bilateral but ipsilaterally biased. We suggest this projection to be involved in more global functions in motivated behavior like general arousal allowing fast voluntary motor activity.

Retinal neural progenitors express topographic map markers

Transplantation of neural stem cells for replacing neurons after neurodegeneration requires that the transplanted stem cells accurately reestablish the lost neural circuits in order to restore function. Retinal ganglion cell axons project to visual centers of the brain forming circuits in precise topographic order. In chick, dorsal retinal neurons project to ventral optic tectum, ventral neurons to dorsal tectum, anterior neurons to posterior tectum and posterior neurons to anterior tectum; forming a continuous point-to-point map of retinal cell position in the tectal projection. We found that when stem cells derived from ventral retina were implanted in dorsal host retina, the stem cells that became ganglion cells projected to dorsal tectum, appropriate for their site of origin in retina but not appropriate for their site of implant in retina. This led us to ask if retinal progenitors exhibit topographic markers of cell position in retina. Indeed, retinal neural progenitors express topographic markers: dorsal stem cells expressed more Ephrin B2 than ventral stem cells and, conversely, ventral stem cells expressed more Pax-2 and Ventroptin than dorsal stem cells. The fact that neural progenitors express topographic markers has pertinent implications in using neural stem cells in cell replacement therapy for replacing projecting neurons that express topographic order, e.g., analogous neurons of the visual, auditory, somatosensory and motor systems.

Topographic arrangement of the rotundo‐entopallial projection in the pigeon (Columba livia)

The tectofugal pathway (retina--optic tectum--nucleus rotundus--entopallium) is a prominent route mediating visual discrimination in diurnal birds. Several lines of evidence have shown that at the tecto-rotundal stage this pathway is composed of multiple parallel channels. Anatomical studies show that the nucleus rotundus is composed of at least four subdivisions, according to differences in cytoarchitectonic, histochemical, and hodological properties. Each of these subdivisions is in receipt of a highly convergent, nontopographic tectal projection, originating from a distinct subset of tecto-rotundal neurons. Physiological studies show that neurons of different subdivisions respond specifically to different visual dimensions, such as color, luminance, two-dimensional motion, and in-depth motion. At present it is less clear whether or to what extent this channel segregation is preserved at the telencephalic stage of the tectofugal pathway. The entopallium shows no obvious subdivisions or laminations. Nevertheless, tract-tracing experiments show that separate portions of the entopallium receive efferent projections from different rotundal subdivisions, in a way that maintains the rostrocaudal order of these subdivisions. In the present study we investigate in detail the topography of the rotundo-entopallial projection by means of anterograde and retrograde neuronal tracers. Our results confirm the zonal topography proposed by previous studies and indicate that each zone in the entopallium receives a direct and topographically organized projection from its corresponding rotundal subdivision. These results suggest that the spatial arrangement of the different rotundal functional modules is preserved at the entopallial level.

Morphological properties of chick retinal ganglion cells in relation to their central projections

Morphological properties of chick retinal ganglion cells (RGCs) were studied in relation to their central projections in 23 chicks. A total of 217 RGCs were retrogradely labeled by applying a carbocyanine dye (DiI) to the thalamus and optic tectum. The labeled RGCs were classified into six groups on the basis of their somal areas, dendritic fields, and branching patterns. The dendrites of these RGCs extended horizontally in the inner plexiform layer (IPL) forming eight dendritic strata. The RGCs in each group showed certain specificities in their central projections. Group Ic predominantly projected to the tectum. Groups IIs and IIIs showed a high thalamic dominance. Groups Is and IIc were nonspecific with regard to their tectal and thalamic projections. Group IVc showed tectal-specific projections. Occurrence rates of the dendritic strata increased progressively toward the inner part of the IPL, i.e., DSs (dendritic strata) 1-4 were scantily distributed, DSs 5 and 6 were moderately distributed, and DSs 7 and 8 were the most frequently distributed. A total of 42 dendritic stratification patterns were identified, and of these, 18 patterns were common to the tectal RGCs (tec-RGCs) and thalamic RGCs (tha-RGCs). The common patterns were detected very frequently in the tec- and tha-RGCs (approximately 85%), and the dendritic strata were largely distributed in the inner part of the IPL (DSs 5-8). In contrast, the remaining 24 noncommon stratification patterns showed low occurrence rates (approximately 15%); however, these dendritic strata were widely distributed in both the outer (DSs 1-4) and inner (DSs 5-8) IPL.

Organization of last-order premotor interneurons related to the protraction of tongue in the frog, Rana esculenta

Moving visual stimuli elicit a sequence of coordinated activity of muscles including tongue protraction. Morphological and physiological studies fail to reveal any direct tectal projections to hypoglossal motoneurons suggesting that the last-order premotor interneurons (LOPI) are the direct recipients of neural activities generated in the optic tectum. The aim of this study is to analyze the topographical organization of the last-order premotor interneurons related to protractor muscles of the tongue. In Rana esculenta, biotinylated dextran amine (BDA) was injected by iontophoresis into the subnucleus of the hypoglossal nerve containing the motoneurons of protractor muscles of the tongue. For visualizing BDA, sections were treated with avidin–biotin complex and a nickel-enhanced DAB chromogen reaction. The position of labeled neurons was reconstructed with a Neurolucida equipment. Morphologically heterogeneous populations of neurons were detected bilaterally, the majority of them were distributed ipsilateral to the site of injection and extended 1200 μm in rostral and 500 μm in caudal directions. Labeled neurons were found in the rhombencephalic reticular formation, the vestibular nuclei, the nucleus prepositus hypoglossi, the nucleus of solitary tract, the spinal nucleus of trigeminal nerve and the dorsal column nuclei. Our results indicate that the majority of last-order premotor interneurons related to protractor muscles of the tongue are located in the reticular formation of the brainstem. Since this area also receives a significant input from the vestibular system and from proprioceptive fibers, the last-order premotor interneurons presented here may be the target of convergence of sensory modalities involved in prey-catching behavior.

Dystroglycan regulates structure, proliferation and differentiation of neuroepithelial cells in the developing vertebrate CNS

In the developing CNS α- and β-dystroglycan are highly concentrated in the endfeet of radial neuroepithelial cells at the contact site to the basal lamina. We show that injection of anti-dystroglycan Fab fragments, knockdown of dystroglycan using RNAi, and overexpression of a dominant-negative dystroglycan protein by microelectroporation in neuroepithelial cells of the chick retina and optic tectum in vivo leads to the loss of their radial morphology, to hyperproliferation, to an increased number of postmitotic neurons, and to an altered distribution of several basally concentrated proteins. Moreover, these treatments also altered the oriented growth of axons from retinal ganglion cells and from tectal projection neurons. In contrast, expression of non-cleavable dystroglycan protein in neuroepithelial cells reduced their proliferation and their differentiation to postmitotic neurons. These results demonstrate that dystroglycan plays a key role in maintaining neuroepithelial cell morphology, and that interfering with dystroglycan function influences proliferation and differentiation of neuroepithelial cells. These data also suggest that an impaired dystroglycan function in neuroepithelial cells might be responsible for some of the severe brain abnormalities observed in certain forms of congenital muscular dystrophy.

Remodeling of sensory nerve projection onto Mauthner cell during development in zebrafish larva

Although visually evoked Mauthner cell (M-cell) startle responses occur in the goldfish, the afferent projections underlying these reactions have not been previously studied. We have recorded from the M-cell while stimulating the left optic nerve and/or right optic tectum and have traced projections of the optic nerve and restricted areas of the optic tectum using HRP histochemistry and autoradiography. Tectal stimulation elicits similar postsynaptic potentials (PSPs) in both M-cells. The responses recorded in the right (ipsilateral) cell were localized to its ventral dendrite. The existence of uncrossed tectal projections to the ventral dendrite was confirmed morphologically following application of horseradish peroxidase (HRP) to the optic tectum. The PSPs contained both inhibitory and excitatory components, but with adequate stimulus strength, excitation of either M-cell dominated. Thus, this pathway is probably sufficient to trigger visually evoked startle responses mediated by the M-cell. Stimulation of the left optic nerve also evoked PSPs capable of bringing both M-cells to threshold. The blockage of this response by conditioning stimulation of the right tectum suggests that the visual information is relayed to the M-cells through this structure. In support of these findings, no label was found near any portion of the M-cell after either intraocular injection of tritiated proline or application of HRP to the cut end of the optic nerve. In summary, visual input to the M-cell is mediated via projections from the tectum, is segregated onto the ventral dendrite, and is capable of bringing this neuron to threshold. This pathway presumably accounts for the demonstrated behavioral efficacy of visual stimuli in evoking a startle response.

Serotonergic reticular formation cells in Rana pipiens: Categorization, development, and tectal projections

The reticular formation contributes serotonin to many brain regions, including the optic tectum. We examined the organization and development of its serotonergic neurons in the leopard frog. Serotonin-immunoreactive (5-HT-ir) cells in adult frogs were organized into 10 distinct populations that were identified on the basis of their location and cellular morphology. Populations ranged in size from 16 to 2,066 cells and sometimes spanned more than one previously identified nuclear region. Four of the ten populations were absent in tadpoles. The remaining populations, though present, had two contrasting patterns of development. Half of the populations were established early and showed little change in numbers during tadpole stages but increased in size in juvenile and adult frogs. The other half increased dramatically during tadpole stages but failed to add many more cells in juveniles and adults. Three populations provided 90% of the serotonergic projections from the reticular region to the adult optic tectum. These projections were established early in development and likely originated from the dorsal raphe, median raphe, raphe pontis, raphe magnus, and reticularis pontis oralis. Termination sites were located in midtectal layers and were not topographically organized. We conclude that serotonergic cells within the reticular formation of the leopard frog have an organization similar to that found in mammals, that the overall increase in numbers of these cells is attributable to growth in different cell populations at different stages, and that input from this region changes activity levels in the optic tectum in a global rather than a site-specific manner.

Competitive interactions between retinal ganglion axons for tectal targets explain plasticity of retinotectal projection in the servomechanism model of retinotectal mapping

The mechanism of topographic mapping of retinal ganglion cells to the midbrain was previously elucidated by the servomechanism model, which is based on the fact that cells expressing Eph-receptors respond specifically to surface expressing membrane-bound ephrin-ligands at a critical level. The retina has increased nasal-to-temporal gradient of Eph receptor-density, and the optic tectum/superior colliculus has increased rostral-to-caudal gradient of membrane-bound ephrin-ligand. An axon from the retina has an identification tag of a certain level of Eph-receptor density depending on its retinal position, and adheres to the site on the tectum/superior colliculus expressing ephrin-ligands at a critical ligand-density level. The servomechanism model rigidly defines positions of axon terminals on the midbrain. However, optic nerve regeneration experiments combined with halved retina or tectum show a plastic or flexible mapping (expansion, compression and transposition of tectal projections). To reconcile the discrepancy between the rigid model and the plastic behavior, competition between retinal axon terminals for a target site was introduced to the servomechanism. The servomechanism/competition model succeeded in computer simulations of the plastic mapping of retinal axons on the tectum. Recent experiments of upregulated ligand-density on the tectum during nerve regeneration and the role of axonal competition are discussed.

A Pallial Visual Area in the Telencephalon of the Bony Fish Polypterus

The dorsal (P2) and lateral (P3) pallial zones of bichirs receive a substantial projection from nucleus medianus of the posterior tuberculum. Although nucleus medianus does not receive a direct retinal input, its close proximity to ascending tectal efferents suggests that it might receive a tectal input and form a segment of a retinotectal-tubercular pathway to the pallium. In order to test this possibility, evoked responses to light flashes were electrophysiologically recorded and tectal efferents were experimentally determined in bichirs. These experiments suggest that a single visual field exists across the dorsal and lateral pallial zones and that this field is mediated by nucleus medianus, which does receive a direct tectal projection. This visual pathway appears to be uniquely derived and not homologous to any other known pathway in tetrapods. Furthermore, these results support the contention that the P2 and P3 pallial zones in bichirs are subdivisions of a single pallial zone.

Pretectal and tectal afferents to the dorsal lateral geniculate nucleus of the turtle: an electron microscopic axon tracing and gamma-aminobutyric acid immunocytochemical study.

The pretectal and tectal projections to the dorsal lateral geniculate nucleus (GLd) of two species of turtle (Emys orbicularis and Testudo horsfieldi) were examined under the electron microscope by using axonal tracing techniques (horseradish peroxidase or biotinylated dextran amine) and postembedding gamma-aminobutyric acid (GABA) immunocytochemistry. After injection of tracer into the pretectum, two types of axon terminals were identified as those of pretectogeniculate pathways. Both contained pleomorphic synaptic vesicles and were more numerous in the inner part of the nucleus. They could be distinguished on the bases of size and shape of their synaptic vesicles, type of synaptic contact, and level of GABA immunoreactivity. One type had a higher density of immunolabeling and established symmetric synaptic contacts, whereas the other, less densely immunolabeled, made asymmetric synaptic contacts. In both cases, synaptic contacts were mainly with relay cells and occasionally with interneurons. We suggest that these two types of pretectogeniculate terminals originate in two separate pretectal nuclei. After injection of tracer into the optic tectum, a single population of GABA-immunonegative tracer-labeled terminals was identified as belonging to the tectogeniculate pathway. These were small, had smooth contours, contained very small round synaptic vesicles, and established asymmetric synaptic contacts with long active zones, predominantly with relay cells and less frequently with interneurons, in the inner part of the nucleus. In addition, a population of GABA-negative and occasionally GABA-positive terminals, labeled by tracer injected into either the pretectum or the tectum, was identified as retinal terminals; these were presumably labeled by the retrograde transport of tracer in collateral branches of visual fibers innervating both the GLd and the pretectum or tectum. Comparison of the present ultrastructural findings in turtles with those previously reported in mammals shows that the cytological features, synaptic morphology, and immunochemical properties of the pretectogeniculate and tectogeniculate terminals of both groups share many similarities. Nevertheless, the postsynaptic targets of these two categories of terminals display some pronounced differences between the two groups, which are discussed in terms of their possible functional significance.

Control of orienting movements: role of multiple tectal projections to the lower brainstem

In movement neuroscience this past decade, a conceptual approach that puts emphasis on population coding was clearly dominant. The purpose of numerous studies has been to define presumably homogeneous groups of neurons on the basis of the correlation of their discharges with sensory and motor events. The goal of this chapter is to stress the importance of taking into account individual properties of neurons, this being an essential prerequisite for a biologically meaningful definition of neuron populations. Taking as an example the executive limb of the neural network controlling gaze movements, we demonstrate the functional and anatomical diversity of tectal and reticular neurons, which are generally considered as homogeneous populations and used, accordingly, as lumped elements in models. We argue that the extraction of effector-specific signals from the global command of gaze displacement is based not on the interplay between discrete neural modules, but rather on a gradual process of signal specification at all levels of the executive network. An eventual accurate description of this network will require knowledge of the unique combinations of afferent inputs and efferent connections for as many subsets of its constituent neurons as is conceivably possible.

Spatial organization of the pigeon tectorotundal pathway: an interdigitating topographic arrangement.

The retinotectofugal system is the main visual pathway projecting upon the telencephalon in birds and many other nonmammalian vertebrates. The ascending tectal projection arises exclusively from cells located in layer 13 of the optic tectum and is directed bilaterally toward the thalamic nucleus rotundus. Although previous studies provided evidence that different types of tectal layer 13 cells project to different subdivisions in Rt, apparently without maintaining a retinotopic organization, the detailed spatial organization of this projection remains obscure. We reexamined the pigeon tectorotundal projection using conventional tracing techniques plus a new method devised to perform small deep-brain microinjections of crystalline tracers. We found that discrete injections involving restricted zones within one subdivision retrogradely label a small fraction of layer 13 cells that are distributed throughout the layer, covering most of the tectal representation of the contralateral visual field. Double-tracer injections in one subdivision label distinct but intermingled sets of layer 13 neurons. These results, together with the tracing of tectal axonal terminal fields in the rotundus, lead us to propose a novel "interdigitating" topographic arrangement for the tectorotundal projection, in which intermingled sets of layer 13 cells, presumably of the same particular class and distributed in an organized fashion throughout the surface of the tectum, terminate in separate regions within one subdivision. This spatial organization has significant consequences for the understanding of the physiological and functional properties of the tectofugal pathway in birds.

Tracing developing pathways in the brain: a comparison of carbocyanine dyes and cholera toxin b subunit

The present study examined the efficiency of fluorescent carbocyanine dye 1,1′-dioctadecyl-3,3,3′,3′-tetramethylinodocarbocyanine perchlorate and cholera toxin B subunit in tracing the crossed tectal projection to the nucleus rotundus of the thalamus (tectorotundal pathways) of paraformaldehyde-fixed and living chick embryos. The tracers were injected into the optic tectum under three experimental conditions (carbocyanine postfix, carbocyanine in vivo, and cholera toxin B subunit in vivo) and the anterograde transport of the nucleus rotundus was monitored and compared. In the carbocyanine postfix method, small crystals of carbocyanine dye were inserted into the tectum of paraformaldehyde-fixed embryos. A 6-month post-insertion period was required to label the crossed tectorotundal pathway. Results showed that tectal neurons did not begin to innervate the ipsilateral nucleus rotundus until embryonic day 9 and the contralateral nucleus rotundus until embryonic day 17. This slow progression of labeling through the crossed tectal projection resulted in significant contrast of the labeling between the ipsilateral and contralateral nuclei rotundus. In the carbocyanine in vivo method, a small volume of carbocyanine dye solution was injected into the tectum of living embryos. A 8- to 12-h survival period was sufficient enough to label the tectorotundal pathway. By embryonic day 8, the labeled axons terminated in the ipsilateral nucleus rotundus and the crossed tectorotundal projection was first detected by embryonic day 10. Similarly, in the cholera toxin B subunit in vivo method, a small volume of cholera toxin B subunit solution was injected into the tectum of living embryos. After a 6- to 10-h survival period, heavily labeled axons were found to innervate bilaterally the nucleus rotundus by embryonic day 8. This appeared to be the earliest schedule for detecting the crossed tectorotundal projection, compared with that of both the postfix and in vivo methods of carbocyanine dye. Based on the differences in the detectability of the crossed tectorotundal projection between the postfix and in vivo methods, the present data suggest that the former method is of limited purpose for labeling tectal collaterals during embryogenesis. Moreover, given the rapid transport rate and absence of photobleaching, which is often seen when using carbocyanine dye, the cholera toxin B subunit in vivo method appears to be the tracer of choice for investigating embryonic pathways.