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.     

Inaccuracies in initial growth and arborization of chick retinotectal axons followed by course corrections and axon remodeling to develop topographic order

The retinotectal projection is organized in a precise retinotopic manner. We find, though, that during development the growth and arborization of temporal retinal axons within the optic tectum of chick embryos is initially imprecise. Axonal targeting errors occur along the rostral-caudal and medial-lateral tectal axes, and arbors are formed at topographically inappropriate positions. Subsequent course corrections along both tectal axes and large-scale axonal remodeling lead to the retinotopic ordering of terminal arborizations characteristic of the mature projection. The trajectories and branching patterns of temporal retinal axons labeled with Dil or DiO were determined in whole mounts of retina and tectum from chicks ranging in age from embryonic day 9 to posthatching. Within the retina, labeled retinofugal axons travel in a compact bundle but do not maintain strict neighbor relations, as they course to the optic fissure. The axons enter the contralateral tectum at its rostral edge and grow caudally. Many extend well past their appropriate terminal zone within rostral tectum; a proportion of these later reverse their direction of growth. Many axons grow onto the tectum at incorrect positions along the medial-lateral tectal axis. Some correct this error in a directed manner by altering their trajectory or extending collateral branches at right angles. About 80% of the positional changes of this type are made in the direction appropriate to correct axon position, and thus are likely a response to tectal positional cues. After maturation of retinotopic order, about half of the axons that project to a mature terminal zone have made abrupt course corrections along one or both tectal axes, indicating that initially mistargeted axons can establish appropriately positioned arbors and survive. The development of temporal axons within the tectum is characterized by 3 phases: elongation, branch and arbor formation, and remodeling. After considerable rostrocaudal elongation, an axon typically develops numerous side branches and arbors, many at inappropriate locations. Most arbors are formed by side branches that develop as interstitial collaterals; few axons grow directly to their appropriate terminal zone and arborize. Aberrant arbors, and axons and axon segments that fail to form arbors in the appropriate terminal zone, are rapidly eliminated over about a 2 d period. Axon degeneration appears to play a role in this remodeling process.     

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.     

Embryogenesis of arborization pattern and topography of individual axons in N. laminaris of the chicken brain stem

This study examined the development of individual axon terminal fields in n. laminaris (NL) of the chicken brainstem. In their mature form axons from the nucleus magnocellularis (NM), second-order auditory neurons in the chicken brainstem, project bilaterally onto the NL. Axons from the ipsilateral and contralateral NM neurons form spatially segregated, elongated arbors in the dorsal and ventral neuropil of NL, respectively. The long axes of these arbors correspond to physiologically defined isofrequency bands. To assess the development of this stereotyped arborization pattern, 6-17-day embryonic chicken brain stems were maintained in vitro while injecting horseradish peroxidase into small groups of axons. Three-dimensional reconstructions were made from serial sections and projected onto a cartesian plane for quantitative analyses. At embryonic day 6 (E6), the ventral axons already course beneath the recently migrated NL neurons. The arrival of the dorsal NM axon branches is delayed and their paths are indirect. They first loop dorsally into the the ventricular layer, where they seem to make specific connections with migrating NL neurons and use these as guides to their appropriate positions in the NL. During the period from E9 to E17 the dorsal and ventral terminal fields become similar, each adopting properties of the other's initial pattern. The dorsal terminal fields extend to form bands similar to the early ventral terminal fields, while the ventral terminal fields narrow and appear to shift position in order to achieve the tonotopic specificity characteristic of the early dorsal terminal fields. The results show that a complex, mature pattern of neuronal connections can be formed during development by the combination and reorganization of two simple patterns--each shaped, in turn, by its respective axonal trajectory.     

Postnatal innervation of the rat superior colliculus by axons of late-born retinal ganglion cells

Rat retinal ganglion cells (RGCs) are generated between embryonic day (E) 13 and E19. Retinal axons first reach the superior colliculus at E16/16.5 but the time of arrival of axons from late-born RGCs is unknown. This study examined (i) whether there is a correlation between RGC genesis and the timing of retinotectal innervation and (ii) when axons of late-born RGCs reach the superior colliculus. Pregnant Wistar rats were injected intraperitoneally with bromodeoxyuridine (BrdU) on E16, E18 or E19. Pups from these litters received unilateral superior colliculus injections of fluorogold (FG) at ages between postnatal (P) day P0 and P6, and were perfused 1-2 days later. RGCs in 3 rats from each BrdU litter were labelled in adulthood by placing FG onto transected optic nerve. Retinas were cryosectioned and the number of FG, BrdU and double-labelled (FG+/BrdU+) RGCs quantified. In the E16 group, the proportion of FG-labelled RGCs that were BrdU+ did not vary with age, indicating that axons from these cells had reached the superior colliculus by P0/P1. In contrast, for the smaller cohorts of RGCs born on E18 or E19, the proportion of BrdU+ cells that were FG+ increased significantly after birth; axons from most RGCs born on E19 were not retrogradely FG-labelled until P4/P5. Thus there is a correlation between birthdate and innervation in rat retinotectal pathways. Furthermore, compared to the earliest born RGCs, axons from late-born RGCs take about three times longer to reach the superior colliculus. Later-arriving axons presumably encounter comparatively different growth terrains en route and eventually innervate more differentiated target structures.     

Development of topographic order in the mammalian retinocollicular projection.

We have used the anterograde axon tracer 1,1'-dioctodecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (Dil) to characterize the development of topographic order in the rat retinocollicular projection. Retinal axons were labeled by Dil injections covering 0.15-2% of peripheral temporal, nasal, superior, or inferior retina, or more central retina, in rats ranging in age from embryonic day 20 to postnatal day (P) 19. At P11-P12 and later, such injections label retinal axons that form overlapping arbors restricted to a topographically correct terminal zone covering about 1% of the superior colliculus (SC) area. At perinatal ages, though, axons labeled from each retinal site are distributed in the SC over much of its medial-lateral axis and extend caudally well beyond the rostral-caudal location of their correct terminal zone; some continue caudally into the inferior colliculus. Axons typically form side branches and often arborize at topographically incorrect positions throughout the SC; however, they appear to branch preferentially in a region that includes, but is much larger than, their correct terminal zone. The mature, retinotopically ordered projection emerges during an early postnatal remodeling period through the rapid remodeling of the early, diffuse projection. This process involves the large-scale removal of axons, axon segments, branches, and arbors from topographically inappropriate positions concurrently with a dramatic increase in branching and arborization at topographically correct locations. Quantitative measurements show that elimination of aberrant branches without loss of the primary axons contributes substantially to the development of order. By P6, fewer mistargeted axons persist, but those that do persist tend to branch or arborize more extensively in topographically inappropriate regions. By P8, the labeling patterns begin to approximate those seen at maturity. Further refinement leads to an adultlike topographic ordering of axonal arborizations by P11-P12. At maturity, some axons take very indirect routes to reach their correct terminal zone. However, such trajectory changes typically correct only small positional inaccuracies, indicating that axons and axon segments that make larger targeting errors do not survive the remodeling phase. Previous retrograde labeling studies indicate that some retinal axons make topographic targeting errors (O'Leary et al., 1986; Yhip and Kirby, 1990), but none have suggested the degree of diffuseness revealed by anterograde labeling with Dil. Our findings show that directed axon growth is inadequate as a mechanism to develop the topographic ordering of retinal axons in the rat SC. Rather, mechanisms that control the removal of mistargeted axons and promote the arborization of correctly positioned axons are critical for the development of retinotopic order.(ABSTRACT TRUNCATED AT 400 WORDS)     

Prenatal development of cat retinogeniculate axon arbors in the absence of binocular interactions

During prenatal development of the cat's retinogeniculate projection, inputs from the ganglion cell axons of the two eyes are initially intermixed with each other within the lateral geniculate nucleus (LGN). As development proceeds, the inputs sort out to give rise to the eye-specific layers characteristic of the adult. During this sorting out process, individual axons undergo a stereotyped sequence of morphological changes that ultimately produce the characteristic pattern of arborization in which axon arbors are restricted in extent only to those layers of the LGN appropriate for the eye of origin (Sretavan and Shatz, 1984, 1986). Here, we examine whether binocular interactions between retinal ganglion cell axons from the two eyes are required for the formation of this restricted pattern of terminal arborization. To examine this question, one eye was removed at embryonic day 23 (E23), when ganglion cell axons have not yet reached the optic chiasm, and the ganglion cell axons from the remaining eye were allowed to develop in the complete absence of binocular interactions. At E59, when segregation into eye-specific layers is normally almost complete, the retinogeniculate projection from the remaining eye was then examined both by anterograde transport following intraocular injections of 3H-leucine and by the in vitro filling of individual ganglion cell axons with HRP. Results from the intraocular injections showed that in the absence of one eye, the remaining eye is still capable of forming both ipsilateral and contralateral optic tracts; however, the projection was distributed diffusely throughout each LGN, rather than being confined to normal eye-specific territories. When individual HRP-filled axons were reconstructed and examined, it was remarkable to find that the pattern of terminal arborization was virtually indistinguishable from normal axons. As usual, arbors were restricted to the distal portion of each axon trunk, and measurements showed that the total linear length of axon contributing to each arbor was within the normal range (enucleated, 2310 +/- 920 microns; normal, 2520 +/- 810 microns). Furthermore, the terminal arborizations of axons appeared to be organized into a series of tiers within the LGN in a pattern surprisingly similar to the pattern of eye-specific layers normally present by E59. Also unchanged was the normally occurring loss of axons from the remaining optic nerve: Counts at E59 showed that about 2.3 X 10(5) axons were present in enucleated animals as compared to 2.5 X 10(5) axons in controls.(ABSTRACT TRUNCATED AT 400 WORDS)     

Retinotopic organization of the developing retinotectal projection in the zebrafish embryo

Developing retinal axons in the zebrafish embryo were stained with HRP or with the fluorescent dyes dil and diO to study the formation of the retinotectal projection. Retinal axons leave the eye at 34-36 hr postfertilization (PF), invade the tectum at 46-48 hr PF, and innervate the tectal neuropil at 70-72 hr PF. Dorsal and ventral axons occupy separate aspects of the optic nerve and tract and pass into their retinotopically appropriate ventral and dorsal hemitectum, respectively. Nasal and temporal axons are segregated in the nerve, mixed in the tract, and are coextensive over the rostral half of tectum until 56 hr PF. They then segregate again, due to the progression of nasal axons into the open caudal tectum. Thus, at 70-72 hr PF, dorsal and ventral as well as temporal and nasal axons occupy their retinotopically appropriate tectal quadrants. After ablation of the temporal retina prior to the time of axonal outgrowth, the nasal axons bypass the vacant rostral tectum to terminate in the caudal tectal half. Temporal axons in the absence of nasal axons remain restricted to their appropriate rostral tectal half, suggesting that nasal and temporal axons possess a preference for their retinotopically appropriate tectal domains. Measurements of individual terminal arbors and the tectal areas in embryos and in adult zebrafish showed that individual arbors are large with respect to the embryonic tectum but are about 14-15 times smaller than in the adult. However, the proportion of tectum covered by embryonic arbors is about 7 times larger than in the adult, suggesting that a higher precision of the adult projection is achieved as a result of a greater enlargement of the tectum than of the arbors.     

Retinotectal projection in reeler mutant mice: relationships among axon trajectories, arborization patterns and cytoarchitecture

The distribution of axons in the midbrain and thalamus of homozygous reeler mutant mice is anomalous. The cytoarchitecture of these regions is normal. In the normal mouse SC there is a distinct SO in which fascicles of retinotectal axons pass caudally before terminating in the overlying SGS. In reeler, by contrast, fascicles of retinotectal axons are distributed through the entire thickness of SGS as well as through SO. There are also abnormalities of fiber pattern in the thalamus, most notably in the region of the dorsal nucleus of the LGd. Retinotectal axon trajectory and patterns of terminal arborization in reeler and normal animals were compared by single-fiber HRP axonography. In normal mice, two distinct morphological classes of retinotectal axons form focal terminal arborizations at different radial levels in the superficial layers of the SC. Class U axons are of relatively small diameter and terminate in upper portions of SGS. Class L1 axons are of larger diameter and form terminal arbors which are confined to SO and deeper regions of SGS. Axons of both classes ascend to their terminal zones from parent axons which course through SO. Similarly, in reeler mice axons of both large and small diameter can be distinguished. However, many axons of both classes pass caudally in anomalous fascicles distributed through the full thickness of SGS and descend to terminate. Other axons pass in normal fashion in SO and ascend to terminate in SGS. Regardless of their trajectories, the small axons terminate superficially in SGS while the thick axons terminate deeper in SGS and/or SO, as in normal mice. These findings suggest that the ingrowth of afferents and the formation of terminal arbors are regulated by different mechanisms and that fiber architecture and cytoarchitecture are regulated by different mechanisms. It is not known if the anomalous fiber pattern in reeler adults arises in development through a defect in initial patterns of axon fasciculation or from a failure of axon elimination.     

Mode of growth of retinal axons within the tectum of Xenopus tadpoles, and implications in the ordered neuronal connection between the retina and the tectum

Retinal axons of Xenopus tadpoles at various stages of larval development were filled with horseradish peroxidase (HRP), and their trajectories and the patterns of branching within the tectum were analyzed in wholemount preparations. To clarify temporal and spatial modes of growth of retinal axons during larval development, special attention was directed to labeling a restricted regional population of retinal axons with HRP, following reported procedures (H. Fujisawa, K. Watanabe, N. Tani, and Y. Ibata, Brain Res. 206:9-20, 1981; 206:21-26, 1981; H. Fujisawa, Dev. Growth Differ 26:545-553, 1984). In developing tadpoles, individual retinal axons arrived at the tectum, without clear sprouting. Axonal sprouting first began when growing tips of each retinal axon had arrived at the vicinity of its site of normal innervation within the tectum. Thus, the terminals of the newly added retinal axons were retinotopically aligned within the tectum. The retinotopic alignment of the terminals may be due to an active choice of topographically appropriate tectal regions by growth cones of individual retinal axons. The stereotyped alignment of the newly added retinal axons was followed by widespread axonal branching and preferential selection of those branches. Each retinal axon was sequentially bifurcated within the tectum, and old branches that had inevitably been left at ectopic parts of the tectum (owing to tectal growth) were retracted or degenerated in the following larval development. The above mode of axonal growth provides an adequate explanation of cellular mechanisms of terminal shifting of retinal axons within the tectum during development of retinotectal projection. Selection of appropriate branches may also lead to a reduction in the size of terminal arborization of retinal axons, resulting in a refinement in targeting.     

Topographic specificity in the retinocollicular projection of the developing ferret: An anterograde tracing study

To assess the degree of order exhibited during development by crossed and uncrossed retinocollicular pathways, focal deposits of 1,1′-dioctodecyl-3,3,3′3′-tetramethylinodocarbocyanine perchlorate (DiI) were made into the temporal or nasal retina of prenatal and postnatal ferrets. This procedure revealed that the first retinal fibers (from the ipsilateral temporal retina) grow into the superior colliculus at embryonic (E) day 30. Both crossed and uncrossed fibers innervate the colliculus by E34. At this age, terminal arbors were lacking, and there was no evidence of extensive axonal branching. Retinocollicular arbors first appeared at E38, with both the crossed and uncrossed projections forming well-defined terminal zones that appeared to be localized to topographically appropriate regions. At E38, the ipsilateral terminal zone was significantly larger but notably less dense than the contralateral zone. At this and later ages (postnatal day [P] 0 and P7), a few crossed and uncrossed fibers extended beyond the terminal zone. Four days later, at P0, the terminal zone of the uncrossed projection was reduced in size in comparison with that of earlier ages, whereas the crossed projection became substantially larger. By P7, the few misprojecting fibers seen in younger ferrets had been virtually eliminated. When focal retinal deposits of tracer were made into the nasal retina of E36 and E40 ferrets, crossed fibers were found to innervate the caudal segment of the superior colliculus. These crossed nasal cells appear to project to the topographically appropriate region of the superior colliculus (caudal segment) but on the wrong side of the brain. Collectively, the present findings indicate that throughout development the ferret retinocollicular pathway is characterized by a remarkable degree of topographic precision as evident by the paucity of axonal branches and the low number of grossly misprojecting axons. J. Comp. Neurol. 392:35–47, 1998. © 1998 Wiley-Liss, Inc.     

Postnatal changes in arborization patterns of murine retinocollicular axons

The growth and arborization of murine retinocollicular axons have been studied by means of HRP axon filling during postnatal development. Transformations in arborization patterns have been correlated with changes in synaptic density in the superficial collicular neuropil and with the formation of synapses by HRP-filled axons. At all postnatal ages axons of the optic projection are fasciculated and most follow a rostrocaudally aligned path. On the day of birth the axons course through both stratum griseum superficiale (SGS) and stratum opticum (SO); during the following 4 days the axon trunks disappear from SGS and are subsequently found only in SO. From postnatal day (P) 0 to P3, the majority continue far caudally in the colliculus, giving rise to small ascending collaterals at multiple points along their course. Ultimately, usually by P3, one or two collaterals begin to branch profusely and by P5 the majority of axons give rise to a focal terminal ascending arborization. The general configuration of most arborizations at P3 approximates that of the mature axon. However, the richness of terminal branching increases from P3 through the first 2 postnatal weeks. Synaptic density is relatively low in the first postnatal week, and no synapses involving HRP-filled optic axons were identified in this interval. Subsequently, after elaboration of definitive arbors has begun, synaptogenesis in the surrounding neuropil accelerates. Synaptic density in the upper SGS approximates adult values early in the third postnatal week. By this time synaptic junctions involving the terminal arborizations of optic axons are abundant.     

DSCAM differentially modulates pre- and postsynaptic structural and functional central connectivity during visual system wiring

Background

Proper patterning of dendritic and axonal arbors is a critical step in the formation of functional neuronal circuits. Developing circuits rely on an array of molecular cues to shape arbor morphology, but the underlying mechanisms guiding the structural formation and interconnectivity of pre- and postsynaptic arbors in real time remain unclear. Here we explore how Down syndrome cell adhesion molecule (DSCAM) differentially shapes the dendritic morphology of central neurons and their presynaptic retinal ganglion cell (RGC) axons in the developing vertebrate visual system.

Methods

The cell-autonomous role of DSCAM, in tectal neurons and in RGCs, was examined using targeted single-cell knockdown and overexpression approaches in developing Xenopus laevis tadpoles. Axonal arbors of RGCs and dendritic arbors of tectal neurons were visualized using real-time in vivo confocal microscopy imaging over the course of 3 days.

Results

In the Xenopus visual system, DSCAM immunoreactivity is present in RGCs, cells in the optic tectum and the tectal neuropil at the time retinotectal synaptic connections are made. Downregulating DSCAM in tectal neurons significantly increased dendritic growth and branching rates while inducing dendrites to take on tortuous paths. Overexpression of DSCAM, in contrast, reduced dendritic branching and growth rate. Functional deficits mediated by tectal DSCAM knockdown were examined using visually guided behavioral assays in swimming tadpoles, revealing irregular behavioral responses to visual stimulus. Functional deficits in visual behavior also corresponded with changes in VGLUT/VGAT expression, markers of excitatory and inhibitory transmission, in the tectum. Conversely, single-cell DSCAM knockdown in the retina revealed that RGC axon arborization at the target is influenced by DSCAM, where axons grew at a slower rate and remained relatively simple. In the retina, dendritic arbors of RGCs were not affected by the reduction of DSCAM expression.

Conclusions

Together, our observations implicate DSCAM in the control of both pre- and postsynaptic structural and functional connectivity in the developing retinotectal circuit, where it primarily acts as a neuronal brake to limit and guide postsynaptic dendrite growth of tectal neurons while it also facilitates arborization of presynaptic RGC axons cell autonomously.

     

Glial environment in the developing superior colliculus of hamsters in relation to the timing of retinal axon ingrowth

We have examined the developmental changes of glial cell organization in the superior colliculus of embryonic and neonatal hamsters in reference to the known sequence of retinal axon ingrowth and arborization in the midbrain. Immunolocalization of vimentin, a marker for neuronal and glial cell precursors, reveals a uniform distribution of radially oriented cells, with perikarya located at the ventricular surface and thin, elongated processes fanning out toward the pia. These vimentin-positive cells, referred to as the lateral radial cells, are present in the tectum from embryonic day (E) 10 (earliest day examined) until approximately postnatal day (P) 5. Vimentin expression in the lateral radial cells decreases markedly during the second week of postnatal life: application of DiI to the ventricular surface reveals that the pial attachment of the lateral radial cells is withdrawn and that the radial processes are gradually pulled back toward the ventricular zone. By P14, virtually no vimentin-positive radial cells are detectable in the superior colliculus. At no time during development are the lateral radial cells immunopositive for the glial fibrillary acidic protein (GFAP); however, shorter, vimentin-positive astrocytic profiles can be seen in the tectum, around the time the radial fibers have been withdrawn, suggesting that at least some radial cells are transformed into astrocytes that will colonize the mature colliculus. At approximately E12, a second group of cells, referred to as the midline radial glia, is detected at the tectal midline. These cells are tightly bundled, forming a raphe in the tectum. They are intensely vimentin positive from E13 until at least P14. From the time of birth, the midline radial cells also exhibit intense immunoreactivity for GFAP. The lateral radial cells are present in the superior colliculus prior to and during the period of neurogenesis but remain well past the time when collicular neuronal migration is completed. Pial processes of the lateral radial cells are present within the superficial tectal layers during the time retinal axons are entering this target; they may be involved in directing the growth and initial collateralization of retinotectal axons. Their withdrawal from retinorecipient collicular zones begins at about the time arbors are being elaborated on retinal axons. In constrast, the midline glia become distinct just prior to the time retinal axons enter the superior colliculus and persist during the time retinotectal projections are being fully established. These raphe glia may be involved in maintaining the laterality of the retinotectal projection. © 1995 Wiley-Liss, Inc.     

Local neurotrophin effects on central trigeminal axon growth patterns

In dissociated cell and wholemount explant cultures of the embryonic trigeminal pathway NGF promotes exuberant elongation of trigeminal ganglion (TG) axons, whereas NT-3 leads to precocious arborization [J. Comp. Neurol. 425 (2000) 202]. In the present study, we investigated the axonal effects of local applications of NGF and NT-3. We placed small sepharose beads loaded with either NGF or NT-3 along the lateral edge of the central trigeminal tract in TG-brainstem intact wholemount explant cultures prepared from embryonic day 15 rats. Labeling of the TG with carbocyanine dye, DiI, revealed that NGF induces local defasciculation and diversion of trigeminal axons. Numerous axons leave the tract, grow towards the bead and engulf it, while some axons grow away from the neurotrophin source. NT-3, on the other hand, induced localized interstitial branching and formation of neuritic tangles in the vicinity of the neurotrophin source. Double immunocytochemistry showed that axons responding to NGF were predominantly TrkA-positive, whereas both TrkA and TrkC-positive axons responded to NT-3. Our results indicate that localized neurotrophin sources along the routes of embryonic sensory axons in the central nervous system, far away from their parent cell bodies, can alter restricted axonal pathways and induce elongation, arborization responses.     

Binocular competition does not regulate retinogeniculate arbor size in fetal monkey

Binocular interactions play a prominent role in shaping the axonal arbors of geniculocortical fibers and the arbors of Y cells in the retinogeniculate pathway of the fetal cat. Fiber interactions between the two eyes have also been suggested to regulate the formation of retinal projections to the dorsal lateral geniculate nucleus (dlgn) of the fetal monkey, but whether this reflects structural refinements of retinal arbors has not been established. To address this issue, we quantified the morphologic properties of individual fibers in two macaque monkeys at embryonic day (E) 110 and E121 that had an eye removed at E69 and E61, respectively. Fibers were labeled by DiI crystals into the fixed optic tract and were visualized by confocal microscopy. Three measurements were made: the number of branch points within the axon terminal arbor, the total arborization length, and the incidence of axonal side branches on the preterminal axon within the confines of the geniculate. There were no significant differences with respect to these parameters between the prenatal enucleates and normal monkeys of comparable age. This was the case for retinal fibers innervating the magnocellular and the parvocellular segments of the dlgn. The arbors stemming from the remaining eye were widely distributed in the dlgn, with some terminating in territories normally innervated by the other (enucleated) eye. These results lend support to the hypothesis that the expanded projection from the remaining eye to the lateral geniculate nucleus of the prenatally enucleated monkey is due to the maintenance of a contingent of retinal fibers normally eliminated by ganglion cell death.     

Axon trajectories and pattern of terminal arborization during the prenatal development of the cat's retinogeniculate pathway

In this study we have examined the trajectories taken by populations of ganglion cell axons and the spatial gradients of terminal arbor maturity within the lateral geniculate nucleus (LGN) during the prenatal development of the cat's visual system. To do so, an in vitro method of labeling optic tract axons from fetal brains between embryonic day 37 (E37) and postnatal day 2 (P2) with horseradish peroxidase (HRP) was used. At the earliest ages studied (E37-E53), optic axons leave the optic tract to run across the LGN toward their sites of termination in straight trajectories parallel to each other. At later ages (E57-P2), however, axons with abrupt changes in their course across the nucleus can be clearly identified. When the detailed terminal arbor morphology of the set of retinogeniculate axons filled with HRP at a given age was examined, two different spatial gradients of maturation could be detected. The terminal arbors of axons within LGN layer A are always more mature than those ending in layer A1, an observation consistent with previous findings that axons from the contralateral eye arrive within the LGN several days before those from the ipsilateral eye. Moreover, the terminal arbors of axons projecting to the medial portions of each layer are always more mature than their more lateral counterparts. These gradients are likely to be a direct reflection of the central-first, peripheral-last gradient associated with the neurogenesis of the retinal ganglion cells themselves. In the oldest animals studied (E58-P2), a remarkable periodic pattern of terminal arbor labeling was seen following a localized HRP injection into the optic tract. Within the labeled portions of the LGN, densely filled axon terminal arbors are separated by unlabeled gaps of similar width. This pattern of labeling could reflect local topographic disorder within the optic tract or could arise if axons of different classes of retinal ganglion cells run in separate portions of the optic tract. Taken together, all of these observations suggest that there may be a fair degree of topographic order in the retinogeniculate projection within the cat's LGN early on in development. However, when topographic errors are present, some can be corrected by minor readjustments in axonal trajectories.     

Regulation of retinal ganglion cell axon arbor size by target availability: Mechanisms of compression and expansion of the retinotectal projection

The ability of pre- and postsynaptic populations to achieve the proper convergence ratios during development is especially critical in topographically mapped systems such as the retinotectal system. The ratio of retinal ganglion cells to their target cells in the optic tectum can be altered experimentally either by early partial tectal ablation, which results in an orderly compression of near-normal numbers of retinal projections into a smaller tectal area, or by early monocular enucleation, which results in the expansion of a reduced number of axons in a near-normal tectal volume. Our previous studies showed that changes in cell death and synaptic density consequent to these manipulations can account for only a minor component of this compensation for the population mismatch. In this study, we examine other mechanisms of population matching in the hamster retinotectal system. We used an in vitro horseradish peroxidase labeling method to trace individual retinal ganglion cell axons in superior colliculi partially ablated on the day of birth, as well as in colliculi contralateral to a monocular enucleation. We found that individual axon arbors within the partially lesioned tectum occupy a smaller area, with fewer branches and fewer terminal boutons, but preserve a normal bouton denstiy. In contrst, ipsilaterally projecting axon arbors in monoculary enucleated animals occupy a greater area than in the normal condition, with a much larger arbor length and greater number of boutons and branches compared with normal ipsilaterally projecting cells. Alteration of axonal arborization of retinalganglion cells is the main factor responsible for matching the retinal and tectal cell populations within the tectum. This process conserves normal electrophysiological function over a wide range of convergence ratios and may occur through strict selectivity of tectal cells for their normal number of inputs. © 1994 Wiley-Liss, Inc.     

Terminal arborizations of retinotectal axons in the bullfrog

In the optic tectum of Rana catesbeiana four laminae of myelinated fibers in the superficial zone of the optic tectum (laminae B, D, F, and G: Potter, ′69) are identified as retinal axons on evidence from patterns of degeneration following contralateral eye removal. After survival times of 5 to 22 days Cajal's block method II shows either fragmentation or abnormal beading of the axons in the four laminae and the paraffin-Nauta method shows coarse granules, representing axonal debris, in these laminae. Golgi impregnations of terminal arborizations arising from fibers in laminae B, D, F, and G show three major types of branching patterns. The arbors are horizontally flattened and are elongated in the same direction as the parent fibers. Densely branched (DB) arbors have stem fibers in laminae B and D and are distributed within the same and adjacent laminae. Widely branched (WB) and thin, straight (TB) arbors have stem fibers in laminae F and G and are distributed mainly in the two laminae of origin with some overlap into adjacent layers of cells and fibers. The horizontal dimensions of DB arbors are 30 μ–70 μ by 100 μ–200 μ, whereas the WB arbors are more variable in overall dimensions. The TB arbors consist of long thin stem axons that give off infrequent terminal specializations in the form of forked or spiny appendages. Numerous terminal forks are present on the WB arbors but are rare on the DB arbors. Swellings and varicosities, that probably represent synaptic specializations, are particularly numerous on the long tertiary branches of DB arbors.     

Development of the retinotectal projection in zebrafish embryos under TTX-induced neural-impulse blockade

The influence of neural activity on the morphology of retinal-axon-terminal arbors and the precision of the developing retinotectal projection in zebrafish embryos was explored. Terminal-arbor morphology and their distribution in the tectum was determined with anatomical fiber-tracing methods using the fluorescent dyes dil and diO. To allow development under activity-deprived conditions, TTX was injected into the eyes of 30-38-hr-old zebrafish embryos at concentrations that effectively blocked neural activity both in retinal ganglion cells and throughout the CNS. Much like axons with normal neural-activity patterns, activity-deprived axons from dorsal and ventral and from temporal and nasal regions in the retina terminated over retinotopically appropriate and nonoverlapping regions of the tectum. Even after ablation of 1 hemiretina at the time of axonal outgrowth, activity-deprived axons from the remaining hemiretina grew directed toward and arborized selectively within their retinotopically appropriate tectal half in the same way as would nondeprived axons. Besides being retinotopic, the area over which small populations of activity-deprived axons from neighboring ganglion cells arborize is as small as that of active axons. The size of terminal arbors of retinal ganglion cell axons was unaffected by blockade of neural activity. The mean terminal-arbor size was 27 x 18 microns for the TTX-injected and 31 x 22 microns for the control embryos. The tectal coverage of TTX-blocked and control axons was equally small, with values of 1.4% and 1.6%, respectively. These data show that a precisely organized retinotopic map in developing zebrafish forms independent of neural-impulse activity.