Tectothalamic visual projections in turtles: their cells of origin revealed by tracing methods

In two species of turtle (Emys orbicularis and Testudo horsfieldi), retrograde and anterograde tracer techniques were used to study projections from the optic tectum to the nucleus rotundus (Rot) and to the dorsal lateral geniculate nucleus (GLd). The ipsilateral Rot received the most massive tectal projections, stemming from numerous neurons located in the stratum griseum centrale (SGC). These neurons varied in size and shape, many of them having a wide zone of dendritic arborization within both the (SGC) and the stratum griseum et fibrosum superficiale (SGFS). Projections from the tectum to the GLd were ipsilateral, were extremely scarce, and arose from a small number of neurons of various shapes situated in the SGFS; these cells were, as a rule, smaller than those projecting to the Rot. For the most part, these neurons were radially oriented, with rather restricted dendritic arborizations in the most superficial sublayers of the SGFS; smaller numbers of projection neurons were horizontally oriented, with long dendrites branching throughout the layer. Some neurons located in the stratum griseum periventriculare (SGP) projected to both the Rot and the GLd. Most of these neurons had dendritic arborizations within the retinorecipient zone of the SGFS. We were unable to rule out the possibility that some cells projecting to the GLd were situated in the SGC. Both the GLd and the main body of the Rot did not contain neurons projecting to the optic tectum. Thalamic neurons projecting to the tectum were observed in the ventral lateral geniculate nucleus, the intergeniculate leaflet and the interstitial nuclei of the tectothalamic tract, and the nucleus of the decussatio supraoptica ventralis. The question of whether variation in the laminar organization of the tectorotundal and tectogeniculate projection neurons in reptiles, birds, and mammals may be related to different degrees of differentiation of the tectal layers is discussed.     

Fiber connections of the torus longitudinalis and optic tectum in holocentrid teleosts

Fiber connections of the torus longitudinalis (TL) and target(s) of toral recipient tectal neurons (pyramidal cells) in the optic tectum were examined by tract-tracing methods in holocentrids. Injections into the stratum marginale (SM) labeled neurons in the stratum opticum and stratum fibrosum et griseum superficiale (SFGS). They had superficial spiny dendrites, with a fan-shaped branching pattern in SM and a thick basal dendrite that gave rise to bushy horizontal branches at the boundary between the SFGS and the stratum griseum centrale (SGC), where an axon and a thin dendrite arose. The axon terminated in a middle cellular layer of the SGC, and the thin dendrite ramified slightly deeper to this cellular layer. The SM injections also labeled cells in the ipsilateral TL. Injections into either the lateral or the medial part of TL labeled terminals in the ipsilateral SM and neurons in the bilateral nucleus paracommissuralis (NPC) and nucleus subvalvularis and ipsilateral nucleus subeminentialis. Only medial TL injections labeled cells in the ipsilateral SGC. These neurons had a basal dendrite that branched in the middle cellular layer of SGC, suggesting that they receive inputs from the pyramidal cells and project back to the TL to form a closed circuit. Only lateral TL injections labeled terminals in the corpus cerebelli. A visual telencephalic portion projects to the NPC and sublayers of SGC, where dendrites of the pyramidal cells and SGC neurons ramify. The present results therefore suggest that the TL and SM are components of an intricate circuitry that exerts telencephalic descending visual influence on the optic tectum and corpus cerebelli.     

Distribution, laminar location, and morphology of tectal neurons projecting to the isthmo-optic nucleus and the nucleus isthmi, pars parvocellularis in the pigeon (Columba livia) and chick (Gallus domesticus): a retrograde labelling study.

Retrograde transport of Phaseolus vulgaris leucoagglutinin (PHA-L), fluorogold, fast blue, rhodamine labelled microspheres, and horseradish peroxidase (HRP) was employed to study the distribution, laminar location within the optic tectum, and morphology of tectal cells projecting upon the isthmo-optic nucleus (ION) and the nucleus isthmi, pars parvocellularis (Ipc), in the pigeon and chick. Following injections into the ION, all retrograde markers labelled tecto-ION neurons and their dendrites in the ipsilateral tectum. The cells were located within a relatively narrow band at the border between layers 9 and 10 of the stratum griseum et fibrosum superficiale (SGFS). Retrogradely labelled neuronal somata were different in both dendritic branching and shape; however, tecto-ION neurons generally possessed non-spiny radially oriented and multi-branched dendrites. The apical processes extended into the retino-recipient layers (2-7) of the SGFS and basal dendrites extended into layers 12-14 of the SGFS. Positive neuronal somata were observed throughout the rostro-caudal extent of the optic tectum. The average distance between adjacent tecto-ION neurons varied from one region to another. Specifically, retrogradely labelled cells were more numerous in the caudal, lateral, and ventral tectum, and less numerous at rostro-dorsal levels. Approximately 12,000 tecto-ION neurons were labelled within the ipsilateral optic tectum following either PHA-L or fluorescent dye injections. While the regional distribution of tecto-Ipc neurons was not examined, the morphology indicated that the cells had a single radially oriented dendritic process. Therefore, the apical dendrites are more restricted than those of tecto-ION cells. Moreover, the dendrites were spiny and arborized within layers 3, 5, and 9 of the ipsilateral optic tectum. The axon of tecto-Ipc cells arise from the apical process as a shepherd's crook and descend into the deep layers of the optic tectum. These results indicate that 1) tecto-ION and tecto-Ipc neurons are possibly monosynaptically activated by retinal input, 2) tecto-ION neurons are heterogeneous in morphology, and 3) there is a differential distribution of the tecto-ION neurons throughout the rostro-caudal extent of the optic tectum, suggesting a greater representation of the caudo-ventral portion of the optic tectum within the ION. The discussion primarily concerns the organization of the retino-tecto-ION-retinal circuit in light of the distribution and morphology of tecto-ION neurons within the optic tectum.     

Spatial arrangement of radial glia and ingrowing retinal axons in the chick optic tectum during development

Neuroanatomical tracing of retinal axons and axonal terminals with the fluorescent dye, DiI, was combined with immunohistochemical characterization of radial glial cells in the developing chick retinotectal system. Emphasis was placed on the mode of the tectal innervation by individual retinal axons and on the distribution and fate of the tectal radial glial cells and their spatial relation to retinal axons. It was obvious from fluorescent images obtained from anterogradely filled axons that these axons deserted the superficial stratum opticum (SO) to penetrate the stratum griseum et fibrosum superficiale (SGFS) by making right-angled turns within the SO. Frequently, axons which had invaded the SGFS were bifurcated and had a superficial branch which remained within the SO. Terminal axonal arborization occurred at various depths within the SGFS. Characterization of the tectal glial cells and their radial fibers by means of the anti-filament antibody, R5, and post-mortem staining with the fluorescent dye, DiI, revealed the following. (a) At least from day E8 to P1, tectal glial fibers traversed all tectal layers from the periventricular location of their somata to the superficial interface between SO and pia mater. In this interface they enlarged and formed characteristic endfeet. (b) Glial endfeet covered the whole tectal surface. They showed at early ages anterior-posterior differences having a higher density in the posterior tectum. These differences disappeared at embryonic day E13. (c) After innervation, glial endfeet of the anterior tectal third were arranged in rows parallel to the retinal fibers within the SO. This arrangement was not observed in eyeless embryos. (d) Radial glial fibers could be stained with R5 from day E8 to late embryonic stages throughout their entire length. (e) At the first posthatching days, only the segments of the radial glial fibers restricted to the thickness of the SO were R5-positive, although the fibers still traversed throughout the depth of the tectum. The results are discussed in context to the genesis of the retinotectal projection.     

Periventricular efferent neurons in the optic tectum of rainbow trout

The efferent connections and axonal and dendritic morphologies of periventricular neurons were examined in the optic tectum of rainbow trout to classify periventricular efferent neurons in salmonids. Among the target nuclei of tectal efferents, tracer injections to the following four structures labeled periventricular neurons: the area pretectalis pars dorsalis (APd), nucleus pretectalis superficialis pars magnocellularis (PSm), nucleus ventrolateralis of torus semicircularis (TS), and nucleus isthmi (NI). Two types of periventricular neurons were labeled by injections to the APd. One of them had an apical dendrite ramifying at the stratum fibrosum et griseum superficiale (SFGS), with an axon that bifurcated into two branches at the stratum griseum centrale (SGC), and the other had an apical dendrite ramifying at the SGC. Two types of periventricular neurons were labeled after injections to the TS. One of them had an apical dendrite ramifying at the boundary between the stratum opticum (SO) and the SFGS, and the other had dendritic branches restricted to the stratum album centrale or stratum periventriculare. Injections to the PSm and NI labeled periventricular neurons of the same type with an apical dendrite ramifying at the SO and a characteristic axon that split into superficial and deep branches projecting to the PSm and NI, respectively. This cell type also possessed axonal branches that terminated within the tectum. These results indicate that periventricular efferent neurons can be classified into at least five types that possess type-specific axonal and dendritic morphologies. We also describe other tectal neurons labeled by the present injections.     

Rostral Cerebellum Originates from the Caudal Portion of the So-Called 'Mesencephalic' Vesicle: A Study Using Chick/Quail Chimeras

Homotopic and isochronic transplantations of the right dorsal half of the mesencephalic vesicle have been performed between chick and quail embryos at the stage of 10 - 14 somites. Analysis of the extension of the graft, by means of the quail nucleolar marker, combined with cytoarchitectonic analysis has disclosed that the transplanted neuroepithelium gives rise to isthmic nuclei and to a portion of rostral cerebellum, in addition to the optic tectum and mesencephalic dorsal grisea. These results show that the rostral portion of the cerebellar primordium is located in the so-called 'mesencephalic' alar plate, thus considerably more rostrally than previously supposed. This has been confirmed by two other types of chimeric embryos resulting from homotopic transplantation of either: (i) the quail right alar plate of the first rhombencephalic vesicle, which gives rise to caudal but not rostral cerebellum in the operated side, or (ii) the right alar portion of a segment of the quail neural tube including both the caudal third of the mesencephalic vesicle and the rostral half of the first rhombencephalic vesicle, which gives rise to the whole hemicerebellum in the operated side. Moreover, in chimeric embryos with transplants restricted to the mesencephalic alar plate, the grafted portion of the cerebellar primordium gives rise both to deep cerebellar neurons and to all types of cortical neurons. Among the quail cortical neurons, the Purkinje cells, although intermingled with host Purkinje cells, are organized, at E18, in a characteristic longitudinal band which is strongly reminiscent of the longitudinal functional and morphological organization of the cerebellum. Other types of quail cortical neurons, that is, Golgi cells, granule cells, and molecular layer interneurons, are also observed within this sagittal band. In addition, quail granule cells and molecular layer interneurons as well as quail glial cells, extend over a larger territory on both sides of the longitudinal band containing quail Purkinje cells and even cross the midline and invade the contralateral hemicerebellum. In all types of chimeric embryos, the proliferation, migration, and differentiation of quail transplanted neurons, both in the isthmic region and in the cerebellum, evolve asynchronously from the host homologous ones, following a more precocious and faster developmental schedule. This asynchrony in the development of grafted and host isthmic and cerebellar homologous areas confirms and extends previous findings concerning the proliferation and migration of quail tectal cells in chick quail chimeric embryos (Senut and Alvarado-Mallart, 1987).     

Comparative neurology of the optic tectum in ray-finned fishes: patterns of lamination formed by retinotectal projections

Retinotectal projections were studied in 33 different species of Actinopterygii, the ray-finned fishes, with horseradish peroxidase and cobalt tracing techniques. The distribution of retinorecipient layers in the contralateral optic tectum was analyzed. In addition, the degree of differentiation of the stratum periventriculate, and the presence of ipsilateral retinotectal projections was examined. Retinofugal fibers are labeled in the stratum opticum (SO), stratum fibrosum et griseum superficiale (SFGS), stratum griseum centrale (SGC), stratum album centrale (SAC) and stratum periventriculare (SPV). Some species lack the projection to the SO, others lack the projection to the SGC, and a third group of fishes lack both projections. Five different patterns of retinorecipient tectal strata are distinguished. These patterns correlate with the species' taxonomic position. Evolutionary trends of tectal lamination and retinotectal innervation are described. The retinotectal projection patterns provide a useful indicator of phylogenetic relationships. Some of our data suggest different relationships between actinopterygian species than hitherto believed.     

Identification of retinotectal projection pathway in the deep tectal laminae in the chick

The optic tectum is a visual center of nonmammalian vertebrates that receives retinal fibers in a retinotopic manner. It has been accepted that retinal fibers project to some superficial laminae of the tectum, but do not go through lamina g of stratum griseum et fibrosum superficiale (SGFS). By a novel fiber-tracing method, we found a novel pathway of retinal fibers that run through deep laminae of the tectum. The retinal fibers that would run through the newly identified pathway first run caudally along the medial edge after invading the tectum, turn laterally, and extend toward the lateral side through the deep pathway. The deep pathway runs through stratum album centrale and stratum fibrosum periventriculare. The fibers that run through the deep pathway do not enter the stratum opticum, where the conventional retinal fibers run. As development proceeds, these fibers decrease and disappear by the adult stage. By the new method, we found that some of the conventional retinal fibers transiently run through lamina g of SGFS and invade laminae h/i. In conclusion, we found distinct but transient retinal fiber pathway in the deep tectal laminae, which have not been thought to be retinorecipient.     

Transplanted mesencephalic quail cells colonize selectively all primary visual nuclei of chick diencephalon: a study using heterotopic transplants

A portion of the quail mesencephalic alar plate (10-12 somites embryos, second day of incubation) was heterotopically transplanted to replace a portion of the diencephalic alar plate of a similar stage chick embryo. Analysis of the chimeric embryos on day 18 of incubation was performed both by Feulgen and Rossenbeck histochemical staining to recognize the transplanted cells, and by cytoarchitectonic methods. The heterotopically transplanted neuroepithelia were integrated in the host pretectal area, although their precise location, substituting some missing host pretectal nuclei, varied slightly from case to case. The cytoarchitecture of the graft and its extension allowed to distinguish two types of transplants: in 50% of the cases the graft developed a laminated, tectal-like structure appearing as a supernumerary optic tectum, whereas in the other 50% of the cases it gave rise to a smaller, not well-defined, non-laminated structure, which could not be recognized as tectal. Independent of the extension and cytoarchitecture of the grafts, in all cases numerous transplanted quail cells were observed beyond the limits of the graft spreading along the optic tract, into all the retino-recipient diencephalic nuclei and into the mesencephalic tectal gray. Conversely, the host optic tectum and the non-primary visual nuclei, even those in close apposition to the transplant, were always devoid of transplanted cells. Analysis of 5- to 10-day-old chimeric embryos has shown that the ectopically located mesencephalic quail cells start migrating from the transplant on day 7 of incubation and follow a tangential pathway at the surface of the diencephalon, throughout the optic tract and between the optic tract and the incipient primary visual nuclei. On day 10, many of these cells have already invaded most of the host retino-recipient nuclei. These observations are discussed with respect to both the phenotypic expression of the transplanted primordium and the tangential migration of tectal cells previously observed in homotopically transplanted chimeric embryos. The possible significance of these results is also discussed.     

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.     

Tectal projection neurons to the retinopetal nucleus in the filefish

Following horseradish peroxidase (HRP) injections into the preoptic retinopetal nucleus (PRN), neurons in the ipsilateral optic tectum were labeled retrogradely. Labeled neurons exhibited a 'Golgi-like' appearance, somata of these neurons were pyriform or round, and most of them were located in the stratum album centrale (SAC) or the stratum periventriculare (SPV). These neurons had a long apical dendrite, which ramified in the upper-half of SGC into horizontally arborized dendritic fields. The main trunk of the apical dendrites also gave off several branches in the stratum fibrosum et griseum superficiale (SFGS) and reached the stratum opticum (SO). These neurons resemble the 'large pyriform neurons' of Vanegas et al. (Vanegas, H., Laufer, M. and Amat, J., The optic tectum of a perciform teleost. I. General configuration and cytoarchitecture, J. Comp. Neurol., 154 (1974) 43-60) except that in the tecto-PRN neurons the axons originates from the apical dendritic shaft at or near the level of the SAC. Judging from their dendritic patterns, the tectal neurons projecting indirectly to the retina may receive non-retinal inputs besides the retinal input.     

Glutamate-immunoreactivity in the retina and optic tectum of goldfish

Glutamate was immunohistochemically localized in the goldfish retina and tectum at the light and electron microscopic (E.M.) levels using double affinity purified antisera against glutaraldehyde conjugated L-glutamate. In retina, glutamate-immunoreactivity (Glu+) was observed in cone inner segments, cone pedicles, bipolar cells, a small number of amacrine cells and the majority of cells in the ganglion cell layer. The latter were shown to be ganglion cells by simultaneous retrograde labeling. Centrally, Glu+ was observed in axons in the optic nerve and tract, and in stratum opticum and stratum fibrosum et griseum superficialis (SFGS) of the tectum. The Glu+ in the optic pathway disappeared four days after optic denervation and was restored by regeneration without affecting the Glu+ of intrinsic tectal neurons. In tectum, Glu+ was also observed in torus longitudinalis granule cells, toral terminals in stratum marginale, some pyramidal neurons in the SFGS, multipolar and fusiform neurons in stratum griseum centrale, large multipolar and pyriform projection neurons in stratum album centrale, and many periventricular neurons. Glu+ was also localized within unidentified puncta throughout the tectum and within radially oriented dendrites of periventricular neurons. At the E.M. level, a variety of Glu+ terminals were observed. Glu+ toral terminals formed axospinous synapses with dendritic spines of pyramidal neurons. Ultrastructurally identifiable Glu+ putative optic terminals formed synapses with either Glu+ or Glu- dendritic profiles, and with Glu- vesicle-containing profiles, presumed to be GABAergic. These findings are consistent with the hypothesis that a number of intrinsic and projection neurons in the goldfish retinotectal system, including most ganglion cells, may use glutamate as a neurotransmitter.     

Rapid onset of neuronal death induced by blockade of either axoplasmic transport or action potentials in afferent fibers during brain development.

We have investigated how neurons in the optic tecta of embryonic day 16 chick embryos depend for survival on their afferents from the retina. To distinguish between activity-mediated effects and other, "trophic," ones, we compared the effects on the tectal neurons of blocking intraocular axoplasmic transport (with colchicine) or action potentials (by means of TTX). Both interventions rapidly induced the appearance of dying (pyknotic) neurons in the tectum, with major increases in their number occurring within 13 hr post-colchicine and within 9 hr post-TTX. Following both drugs, the dying neurons were morphologically similar, and in both cases the cell death depended on protein synthesis. However, the effects of colchicine and of TTX could be dissociated, since the most superficial tectal neurons became pyknotic only in response to colchicine, and, with a sufficiently short survival time (9 hr), the deep cells of the stratum griseum centrale became pyknotic only in response to TTX. We hence argue that the survival of the tectal neurons depends on their ongoing maintenance by substances released from retinotectal axon terminals, the release being activity dependent in the case of the deep neurons but independent of activity in the case of the superficial ones.     

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.     

The optic tectum of the carp: pyramidal neurons of the SFGS

We have studied the stratum fibrosum et griseum superficiale (SFGS) of the carp optic tectum with optic and electron microscopy. This stratum is a dense neuropil with disordered appearance, in which numerous neuronal bodies of different characteristics and variable distribution according to the tectal regions are intercalated, more abundant in the dorsomedial zones of the tectum. Within these neuronal types, the most characteristic of SFGS are the large pyramidals of vertical development. Such neurons shows an ascendant dendritic shaft, very developed in the stratum marginale, a thinner dendritic shaft in the basal pole and a descending axon that reaches the internal zones of the stratum griseum centrale. Glial elements are highly associated to the pyramidal neuron bodies. The synaptic contacts are abundant and of various types, specially on the spinous dendritic branches which lie in the stratum marginale.     

Optic tectum of the eastern garter snake, Thamnophis sirtalis. III. Morphology of intrinsic neurons

Extracellular iontophoretic injections of horseradish peroxidase and Golgi preparations were used to study the distribution and morphology of intrinsic neurons of the garter snake optic tectum. Four morphologically distinct classes of neurons were identified. The type A neuron is found throughout the retinorecipient tectal layers. It has a large, fusiform soma and infrequently branching dendrites that radiate in the horizontal plane and are studded with varicose appendages. An axon arises from the soma or proximal dendrite and gives rise to widely spreading branches that overlap the cell's dendritic field. The type B neuron has a small, spherical soma in sublayer b of the stratum fibrosum et griseum superficiale. Thick, varicose dendrites ascend from the soma and form a bushy arbor in the overlying sublayer a. A thin axon descends vertically from the soma and arborizes in vertical alignment with the cell's dendritic field in sublayer c of the stratum fibrosum et griseum superficiale and the upper third of the stratum griseum centrale. The type C neuron is a bipolar cell with a small, vertically fusiform soma situated at the upper border of the stratum griseum centrale. Thin, sparsely branching dendrites extend vertically into the superficial and central gray layers. An axon arises from the soma and courses ventrally into the stratum griseum centrale where it gives rise to a plexus of widely spreading branches that extend medially from the cell's dendritic field. The type D neuron is a small, stellate cell with a spherical soma and fine, appendage-laden dendrites that are restricted to the stratum griseum centrale. The axon of the type D cell courses in the central gray where it gives rise to widely spreading branches that extend laterally from the cell's dendritic field.     

Evidence for shifting connections during development of the chick retinotectal projection

The pattern in which optic axons invade the tectum and begin synaptogenesis was studied in the chick. The anterogradely transported marker, horseradish peroxidase, was injected into one eye of embryos between 5 and 16 days of development (E5 to E16). This labeled the optic axons in the brain. The first retinal axons arrived in the most superficial lamina of the tectum on E6. They entered the tectum at the rostroventral margin. During the next 6 days of development the axons grew over the tectal surface. First they filled the rostral tectum, the oldest portion of the tectum, and then they spread to the caudal pole. Shortly after the first axons entered the tectum on E6, labeled retinal axons were found penetrating from the surface into deeper tectal layers. In any given area of the tectum, optic axons were seen penetrating deeper layers shortly after arriving in that area. Electron microscopic examination showed that at least some of the labeled axons in rostral tectum formed synapses with tectal cells by E7. These results show two things which contrast with results from previous studies. First, there is no delay between the time the retinal axons enter the tectum and the time they penetrate into synaptic layers of the tectum. Second, the first retinotectal connections are formed in rostral tectum and not central tectum. Retrograde tracing showed the first optic axons that arrived in the tectum were from ganglion cells in central retina. Previous studies have shown that the ganglion cells of central retina project to the central tectum in the mature chick. This opens the possibility that the optic axons from central retina, which connect to rostral tectum in the young embryo, shift their connections to central tectum during subsequent development. As they enter the tectum the growth cones of retinal axons appear to be associated with the external limiting membrane. During the time that connections would begin to shift in the tectum a second population of axons appears at the bottom of stratum opticum, some with characteristics of growth cones. This late-appearing population may represent axons shifting their connections. These results have implications for theories on how the retinotopic pattern of retinotectal connections develops.     

Optic tectum of the eastern garter snake, Thamnophis sirtalis. II. Morphology of efferent cells

Tectal efferent neurons were retrogradely filled from extracellular injections of horseradish peroxidase (HRP) into pathways efferent from the tectum. Tectorotundal neurons have cylindrical dendritic trees, 80-100 microns in diameter, that extend vertically across the central and superficial tectal layers. Apical and basal dendrites are laden with complex appendages. The axon gives rise to an intratectal, collateral arbor that extends horizontally into the stratum griseum centrale beyond the cell's dendritic tree. The parent axon exits the tectum laterally in the tectothalamic tract. Tectogeniculate neurons also have narrow, radially oriented, and highly branched apical dendrites, but their basal dendrites are infrequently branched and lack appendages. An intratectal axon collateral forms a small, spherical arbor overlapping the apical dendrites in sublayer c of the stratum fibrosum et griseum superficiale. The parent axon ascends vertically and just below the stratum opticum turns rostrad to follow the optic fibers to the diencephalon. Tectoisthmi neurons have small somata and thin, radial dendrites that arborize below the pial surface in the stratum zonale. An intratectal axon collateral forms a spatially restricted arbor ventral to the soma in register with the dendritic tree. Tectoisthmobulbar neurons have dendrites that arborize extensively in sublayer a of the stratum fibrosum et griseum superficiale. The axon exits the tectum without collateralizing and joins a small-caliber component of the ventral tectobulbar tract. Ipsilateral tectobulbar neurons have stellate dendritic fields, 150-250 microns in diameter, that are restricted to the deep layers of the tectum. Sparsely branched dendrites are appendage-free but bear many short, fine spicules. The axon initially ascends from the soma and recurves into the stratum album centrale without collateralizing before joining a medium-caliber component of the ventral tectobulbar tract. Crossed tectobulbar neurons have large, stellate dendritic trees with diameters ranging from 200 to 500 microns. Like ipsilateral tectobulbar neurons, their dendrites are appendage-free but bear spicules. Their thick-caliber axons exit the tectum without collateralizing and course deep in the stratum album centrale to reach the dorsal tectobulbar tract.     

The visual connections of the adult flatfish, Achirus lineatus.

Metamorphosis in the flatfish is characterized by the migration of one eye around the dorsal surface of the head to a position adjacent to the other eye on the new top side of the animal. The visual connections of the adult flatfish, Achirus lineatus, were examined. Either the migrating or non-migrating eye was removed and the animal allowed to survive for one to three weeks. Alternate sections of the brain were stained by a modification of the Fink-Heimer technique, or with cresyl violet. The diencephalic visual connections of the flatfish were similar to those of other teleosts with contralateral projections to the nuclei corticalis, dorsomedialis thalami, pretectalis, and the corpus geniculatum laterale. The distribution of the retinal efferents to the optic tectum is unique in the flatfish. In the medial one-third of the tectum, terminal degeneration was found in three bands in the stratum opticum (SO) and the stratum griseum et fibrosum superficiale (sgfs). In the middle part of the tectum, two bands of degeneration remained over the sgfs. The lateral part of the tectum has only a very small amount of degeneration distributed radomly in scattered clusters over the deep SO and superficial sgfs. The Nissl preparations also reflected the differences between the medial and lateral parts of the tectum. Distinct layer was lacking in the medial tectum with a conspicuously absent large cell layer in the stratum griseum centrale (sgc). In contrast, the lateral tectum had a typical tectal stratification. Most notable were the large neurons of the sgc.     

Sonic Hedgehog Regulation of the Neural Precursor Cell Fate During Chicken Optic Tectum Development

During nervous system development, neurons project axons over long distances to reach the appropriate targets for correct neural circuit formation. Sonic hedgehog (Shh) is a secreted protein and plays a key role in regulating vertebrate embryogenesis, especially in central nervous system (CNS) patterning, including neuronal migration and axonal projection in the brain and spinal cord. In the developing ventral midbrain, Shh is sufficient to specify a striped pattern of cell fates. Little is known about the molecular mechanisms underlying the Shh regulation of the neural precursor cell fate during the optic tectum development. Here, we aimed at studying how Shh might regulate chicken optic tectum patterning. In the present study, in ovo electroporation methods were employed to achieve the overexpression of Shh in the optic tectum during chicken embryo development. Besides, the study combined in ovo electroporation and neuron isolation culturing to study the function of Shh in vivo and in vitro. The fluorescent immunohistochemistry methods were used to check the related indicators. The results showed that Shh overexpression caused 87.8% of cells to be distributed to the stratum griseum central (SGC) layer, while only 39.3% of the GFP-transfected cells resided in the SGC layer in the control group. Shh overexpression also reduced the axon length in vivo and in vitro. In conclusion, we provide evidence that Shh regulates the neural precursor cell fate during chicken optic tectum development. Shh overexpression impairs neuronal migration and may affect the fate determination of transfected neurons.