Search

The Nile grass rat (Arvicanthis niloticus) has a high proportion of cone photoreceptors (∼30-40%) compared with that in the common laboratory mouse and rat (∼1-3%) and may prove a preferable murine model with which to study cone-driven information processing in retina and primary visual centers. However, other than regions involved in circadian control, little is known about the retinorecipient structures in this rodent. We undertook a detailed analysis of the retinal projections as revealed after intravitreal injection of the anterograde tracer cholera toxin subunit B. Retinal efferents were evaluated in 45 subcortical structures. Contralateral projections were always dominant. Major contralateral inputs consisted of the suprachiasmatic nucleus, dorsolateral geniculate nucleus (dLGN), intergeniculate leaflet, ventral geniculate nucleus (magnocellular part), lateroposterior thalamic nucleus, all six pretectal nuclei, superficial layers of the superior colliculus (SC), and the main nuclei of the accessory optic system. Terminals from the contralateral eye were also localized in an unnamed field rostromedial to the dLGN as well as in the subgeniculate thalamic nucleus. Ipsilateral inputs were found mainly in the suprachiasmatic nucleus, dLGN, intergeniculate leaflet, internal sector of the magnocellular part of the ventral geniculate nucleus, olivary pretectal nucleus, and SC optic layer. Retinal afferents were not detected in the basal forebrain or the dorsal raphe nucleus. Morphometric measurements revealed that the superficial layers of the SC are disproportionately enlarged relative to other retinorecipient regions and brain size compared with rats and mice. We suggest that this reflects the selective projection of cone-driven retinal ganglion cells to the SC., (Copyright © 2013 Wiley Periodicals, Inc.)

Retinal ganglion cells (RGCs), which transfer information from the eye to the brain, are heterogeneous in structure and function, but developmental studies have generally treated them as a single group. Here, we investigate the development of RGC axonal and dendritic arbors using four mouse transgenic lines in which nonoverlapping subsets of RGCs are indelibly labeled with a fluorescent protein. Each subset has a distinct functional signature, size, and morphology. Dendrites of each subset are restricted to specific sublaminae within the inner plexiform layer in adulthood, but acquire their restriction in different ways: one subset has lamina-restricted dendrites from an early postnatal stage, a second remodels an initially diffuse pattern, and two others develop stepwise. Axons of each subset arborize in discrete laminar zones within the lateral geniculate nucleus or superior colliculus, demonstrating previously unrecognized subdivisions of retinorecipient layers. As is the case for dendrites, lamina-restricted axonal projections of RGC subsets develop in different ways. For example, while axons of two RGC subsets arborize in definite zones of the superior colliculus from an early postnatal stage, axons of another subset initially occupy a deep layer, then translocate to a narrow subpial zone. Together, these results show that RGC subsets use a variety of strategies to construct lamina-restricted dendritic and axonal arbors. Taking account of these subtype-specific features will facilitate identification of the molecules and cells that regulate arbor formation.

BDNF contributes to the activity-dependent establishment and refinement of visual connectivity. In Xenopus, BDNF applications in the optic tectum influence retinal ganglion cell (RGC) axon branching and promote synapse formation and stabilization. The expression patterns of BDNF and TrkB suggest that BDNF specifically regulates the maturation of RGC axons at the target. It is possible, however, that BDNF modulates retinotectal synaptic connectivity by differentially influencing presynaptic RGC axons and postsynaptic tectal cells. Here, we combined single-cell expression of a dominant-negative TrkB-enhanced green fluorescent protein (GFP) fusion protein with confocal microscopy imaging in live Xenopus tadpoles to differentiate between presynaptic and postsynaptic actions of BDNF. Disruption of TrkB signaling in individual RGCs influenced the branching and synaptic maturation of presynaptic axon arbors. Specifically, GFP-TrkB.T1 overexpression increased the proportion of axons with immature, growth cone-like morphology, decreased axon branch stability, and increased axon arbor degeneration. In addition, GFP-TrkB.T1 overexpression reduced the number of red fluorescent protein-synaptobrevin-labeled presynaptic specializations per axon terminal. In contrast, overexpression of GFP-TrkB.T1 in tectal neurons did not alter synaptic number or the morphology or dynamic behavior of their dendritic arbors. Electron microscopy analysis revealed a significant decrease in the number of mature synaptic profiles and in the number of docked synaptic vesicles at retinotectal synapses made by RGC axons expressing GFP-TrkB.T1. Together, our results demonstrate that presynaptic TrkB signaling in RGCs is a key determinant in the establishment of visual connectivity and indicate that changes in tectal neuron synaptic connectivity are secondary to the BDNF-elicited enhanced stability and growth of presynaptic RGCs.

The superficial layers of the rat superior colliculus (sSC) receive innervation from the retina and include nitrergic neurons. We have shown previously that in sSC, eye enucleation reduces NADPH diaphorase staining considerably in all but the most proximal dendrites of nitrergic neurons. We have used immunocytochemistry for neuronal nitric oxide synthase (nNOS) at light and electron microscopic levels and bilateral eye enucleation with varied survival times to determine the regulatory changes imposed by the direct and indirect loss of retinal input on apparent nNOS amount and subcellular distribution. In addition, we have used SDS-PAGE and immunoblotting to test alternatively spliced isoforms in normal and deafferented animals. Our results show that unambiguously identified retinal terminals contact nitrergic neurons. In normal dendrites, nNOS immunoreactivity was distributed almost completely within the cytoplasm of the dendrite and along the postsynaptic membrane at synaptic junctions, in association with endoplasmic reticulum, ribosomes and external mitochondrial membranes. In contrast, nNOS labeling was greatly reduced in sSC deprived of retinal projections, and could only be observed in association with mitochondrial membranes and postsynaptic densities. Immunoblots of the soluble fraction from sSC revealed a surprisingly high proportion of the beta isoform with respect to the alpha counterpart in normal colliculi, suggesting an increase in isoform proportion after enucleation, or at least maintenance of the same proportion. It is suggested that ultrastructural alterations observed in sSC cells of enucleated animals may be consequent to plastic reactions of the sSC cells in response to the removal of retinal afferents., (Copyright 2002 Wiley-Liss, Inc.)

The formation of CNS circuits is characterized by the coordinated development of neuronal structure and synaptic function. The activity-regulated candidate plasticity gene 15 (cpg15) encodes a glycosylphosphatidylinositol (GPI)-linked protein whose in vivo expression increases the dendritic arbor growth rate of Xenopus optic tectal cells. We now demonstrate that tectal cell expression of CPG15 significantly increases the elaboration of presynaptic retinal axons by decreasing rates of branch retractions. Whole-cell recordings from optic tectal neurons indicate that CPG15 expression promotes retinotectal synapse maturation by recruiting functional AMPA receptors to synapses. Expression of truncated CPG15, lacking its GPI anchor, does not promote axon arbor growth and blocks synaptic maturation. These results suggest that CPG15 coordinately increases the growth of pre- and postsynaptic structures and the number and strength of their synaptic contacts.

The present study provides the first electrophysiological evidence for dorsoventral variation in the receptive field properties of tectal cells in pigeons. According to their receptive field organization, visual response properties and laminar locations, 95 tectal neurons recorded in the present study could be categorized into two groups: (1) Fifty-five DL-neurons were recorded in the dorsal, dorso-lateral, lateral and ventro-lateral tectum and characterized by an excitatory receptive field surrounded by an inhibitory receptive field. Most of them almost equally responded to white and black objects, but did not respond to switch-on and -off of a light spot. (2) Forty VC-neurons were recorded in the ventral tectum and characterized by an excitatory receptive field alone. Their responses to switch-on of a light spot and to a white object were significantly stronger than those to light-off and to a black object, respectively. DL-neurons preferred higher velocity, whereas VC-neurons preferred lower velocity. The recording sites of 53 of 95 cells (56%) examined were marked with pontamine sky blue, showing that DL-neurons were located in tectal layers I-IV, predominantly in layer II, whereas VC-neurons were mainly concentrated in sublayer IIc. The receptive fields of VC-neurons were located within the rostroventral visual field possibly corresponding to the red field of the pigeon retina, suggesting that they might be associated with visual food-foraging behaviors., (Copyright 2000 S. Karger AG, Basel.)

Newly synthesized presynaptic integral membrane proteins in neurons are transported in precursor vesicles from the site of protein biosynthesis in the cell body by fast axonal flow to the presynaptic terminal. We followed the path that presynaptic proteins travel on the way to their central targets of the highly ordered primary visual pathway of the chick and analyzed the developmental changes in the expression of synaptic vesicle protein 2 (SV2), synaptotagmin, and syntaxin. Immunofluorescences revealed that: (1) the onset of protein expression in the retinal ganglion cells occurs in a central to peripheral developmental pattern from embryonic day 4 (E4) onward; (2) the proteins were found first in the inner and later in the outer plexiform layer of the retina; and (3) they were redistributed from the photoreceptor inner segments and cell bodies to the terminals in the outer plexiform layer. From E4 onward, immunopositive axons for SV2, synaptotagmin, and syntaxin were found in the optic nerve, disappearing after E9 for SV2 and synaptotagmin. The optic tract was stained for SV2 and synaptotagmin between E7 and E12, for syntaxin until the posthatching period. Finally, immunoreactivities for the investigated proteins were present at the surface of the tectum from E8 onward, when first retinal axons arrived there. The present study revealed that SV2 and synaptotagmin, but not syntaxin, are, expressed in a transient wave that follows the advancement of optic axons and the proteins towards the optic tectum.

The N-methyl-D-aspartate receptor (NMDAR) is an ionotropic glutamate receptor that is important in neurotransmission as well as in processes of synaptic plasticity in the mammalian superior colliculus (SC). Despite the importance of this receptor in synaptic transmission, there is as yet no evidence that demonstrates directly the synaptic localization of the NMDAR receptor in SC. We have used electron-microscope (EM) immunocytochemistry to localize the NMDAR1 subunit of this receptor protein and its association with sensory afferents in the cat SC. Retinal synaptic terminals were identified by normal morphology and cortical synaptic terminals by degeneration after lesions of areas 17-18 of the visual cortex. At the light-microscope level, label was densest within the superficial gray and upper optic layers, but also present in all other layers. Label was contained within cell bodies, dendrites, and a few putative axons. At the EM level, antibody labeling was found along postsynaptic densifications and internalized within the cytoplasm of a variety of dendrites and some cell bodies. Postsynaptic profiles labeled by NMDAR1 included conventional dendrites and presynaptic dendrites which contained pleomorphic synaptic vesicles and are known to be GABAergic. Many of the labeled postsynaptic densifications of both of these profile types received synaptic inputs from retinal or cortical terminals. Virtually no NMDAR1 immunoreactivity was found on thin dendritic thorns or putative spines, even when these were postsynaptic to retinal or cortical terminals. In summary, these results show that the NMDAR1 subunit is postsynaptic to both retinal and cortical afferents, which are known to be glutamatergic, and are consistent with physiological evidence showing that stimulation of either pathway can activate the NMDA receptor.

The maturation of the neuropil and synapse formation were examined in the retino-receptive layers of the superior colliculus (SCr-r) in the opossum from a period prior to the onset of arborization of retinocollicular fibers (postnatal day 22 - P22), at 44% of the coecal period (CP), to the end of the fast phase of optic fiber myelination and weaning time (P81 - 118% CP). Development of the SCr-r neuropil follows a protracted time course and can be divided into three broad stages, which are characterized by (I) Large extracellular spaces, numerous growth cones that participate rarely in synaptic junctions, vesicles-poor immature synapses (P22-P30), (II) Synapses of varied morphology with abundant synaptic vesicles, and small terminals with dark mitochondria and round synaptic vesicles (RSD terminals) synapsing mostly onto dendritic shafts, flat-vesicles (F) terminals (P40-P56), (III) Sequential appearance of retinal (R) and pleomorphic-vesicles (P) terminals and of RSD terminals synapsing onto spine or spine-like processes, appearance of glomerulus-like synaptic arrays (synaptic islets) (P61-P81). The advancement of synaptogenesis in SCr-r from stage I to II and from stage II to III correlates closely with the differentiation of astrocytes and oligodendrocytes, respectively.

The morphological changes occurring in the visual corticocollicular projection following removal of the contralateral retina (within the first 48 h of postnatal life) were studied using New Zealand rabbits. At 45-50 days after lesion, the corticocollicular terminal field was examined by anterograde transport of Phaseolus vulgaris leucoagglutinin, which was applied iontophoretically in the central region of the contralateral striate cortex. In contrast to normal intact rabbits of the same age, the corticocollicular terminal field was markedly enlarged in experimental animals. In the centre of the field we found abundant oblique fibres which sent out branches. These collateral fibres coursed over long distances, parallel to the pial surface, in the stratum zonale and in the upper part of the stratum griseum superficiale. The presence of these fibres, together with an increased density of synaptic boutons at more superficial levels of the sprouted terminal field, suggest that corticocollicular fibres tended to occupy territories left vacant when retinocollicular axons degenerated after enucleation. The high density and extensive distribution of these corticocollicular fibres may be due to the continued growth of the fibres, which occupy an extensive territory during the early postnatal stages and which, under normal circumstances are retracted during the process of postnatal maturation. Despite the expansion of the field occupied by corticocollicular synapses, its centre coincided topographically with the field centres in normal animals, indicating the existence of intrinsic positional cues that persisted after enucleation and determined the arrangement of visual cortical afferents. This model, which involves substantial changes in terminal field organization, should prove useful in elucidating the cellular and molecular processes underlying regeneration and plasticity in the visual system.

The ultra-structural development of synapses in retino-receptive layers of the opossum superior colliculus was studied by the ethanolic phosphotungstic acid (E-PTA) method. There was a tendency for a slight reduction in the diameter of synaptic disks, a rise and fall of numerical densities and, except for an ephemeral period, a general increase in the proportion of "frown" among curve synapses. The lack of strict synchrony and the occurrence of different patterns of changes suggest that multiple factors contribute to synaptic maturation.

During development, many CNS projection neurons establish topographically ordered maps in their target regions. Myelin-associated inhibitors of neurite growth contribute to the confinement of fiber tracts during development and limit plastic changes after CNS projections have been formed. Neutralization of myelin-associated growth inhibitors leads to an expansion of the retinal innervation of the superior colliculus (SC). In the lesioned adult mammalian CNS, these long projection neurons are usually unable to regrow axons over long distances after lesion due to myelin-associated inhibitors, which interfere with axonal growth in vivo and in vitro. Application of a specific antibody directed against myelin-inhibitors (IN-1) promotes regrowth of corticospinal tract or retinal ganglion cell axons. In the present study, we asked whether application of an antibody to myelin-associated growth inhibitors would lead to disturbances of target-specific axon guidance. To examine this issue, we used an in vitro model, the "stripe assay", to examine the behavior of rat retinal ganglion cell axons on membranes from embryonic and deafferented adult rat SC. On membrane preparations from embryonic rat SC, retinal fibers avoid posterior tectal membranes, possibly due to the presence of a repulsive factor. Nasal retinal axons show a random growth pattern. On membranes prepared from the deafferented adult rat SC, temporal and nasal axons prefer to grow on membranes prepared from their specific target region, which suggests the involvement of target-derived attractive guidance components. The results of the present study show that retinal axons grow significantly faster in the presence of IN-1 antibody that neutralizes myelin-associated growth inhibitors present in the membrane preparations from the adult rat SC. IN-1 antibody, however, does not interfere with specific axonal guidance. This suggests that axonal guidance and specific target finding are independently regulated in retinal axons.

The early development of the optic tract in hamsters was studied by labeling retinal axons with Dil applied to the eye, and then examining the labeled axons in flatmount preparations of the rostral brain stem. This technique permits a panoramic view of the entire retinal projection, from the chiasm to the caudal end of the superior colliculus. In the E11 embryo, retinal axons have reached the chiasm. They defasciculate as they emerge from the nerve, prior to reaching the ventral midline of the diencephalon, then converge again as they pass over to the opposite side. At the midline, many axonal trajectories crisscross, implying some shuffling of relative positions. Retinal axons are tightly bundled within the optic tract. Upon reaching the ventral border of the lateral geniculate body (LGB), they splay out over the nucleus, revealing a wavefront of pioneer axons individually distributed across the rostro-caudal extent of the LGB. Later-emerging retinal axons course over the surface of the thalamus in waves; subsequent waves of axons interdigitate between the lead fibers without fasciculating along them. Past the LGB, the axons undergo a second change in relative positions as the ribbon of fibers swerves caudally, prior to entering the superior colliculus. Retinal axons are tipped with growth cones of varying morphologies. No strong correlation is evident between the structural complexity of the growth cone and its position within the tract. In the majority of cases, ipsilaterally and contralaterally directed axons follow a similar developmental course along the optic tract, without any indication of a temporal lag in the ipsilateral projection as claimed in earlier reports. Understanding the changes in spatial distribution of embryonic retinal axons as they navigate along the optic tract provides a further step towards elucidating how point-to-point projections form in developing sensory systems.

We have investigated the pattern of glutamate-like immunoreactivity in the superficial layers of the rat superior colliculus by means of postembedding immunocytochemical methods for light and electron microscopy. At the light microscopic level, labelling was faintly to moderately intense in most perikarya of the stratum zonale, stratum griseum superficiale and stratum opticum. Furthermore, strong glutamate-immunoreactive terminal-like elements were accumulated most densely in stratum zonale, stratum griseum superficiale and stratum opticum. At the electron microscopic level, a postembedding immunogold method revealed that the vast majority of those labelled elements corresponded to retinal and visual cortical terminals. These profiles were about twice as heavily labelled as their postsynaptic partners. To determine the contribution of retinal and cortical afferents to the pattern of glutamate-like immunoreactivity, rats were subjected to right retinal ablation, left cortical ablation or combined right retinal and left cortical ablations. After retinal ablation, strongly labelled perikarya were observed in the retinorecipient layers. Furthermore, a prominent loss of glutamate-immunoreactive terminal-like elements occurred in stratum zonale and stratum griseum superficiale. Ipsilateral superior colliculus to cortical ablation exhibited subtle changes characterized by a moderate increase in perikaryal immunostaining in stratum zonale, stratum griseum superficiale and stratum opticum and by an apparent discrete reduction of labelled dots in stratum griseum superficiale and stratum opticum. In cases with combined lesions, strongly immunoreactive cell bodies and dendrites were accompanied by a massive disappearance of labelled terminal-like elements in stratum zonale, stratum griseum superficiale and stratum opticum. The effect of retinal and visual cortical ablations on the pattern of glutamate-like immunoreactivity suggests that these afferents are the major sources for glutamate-immunoreactive terminals in the rat superior colliculus. In addition, these findings provide further evidence for glutamate as neurotransmitter in the visual pathways studied.

Prior morphological studies of individual retinal X and Y axon arbors based on intraaxonal labeling with horseradish peroxidase have been limited by restricted diffusion or transport of the label. We used biocytin instead as the intraaxonal label, and this completely delineated each of our six X and 14 Y axons, including both thalamic and midbrain arbors. Arbors in the lateral geniculate nucleus appeared generally as has been well documented previously. Interestingly, all of the labeled axons projected a branch beyond thalamus to the midbrain. Each X axon formed a terminal arbor in the pretectum, but none continued to the superior colliculus. In contrast, 11 of 14 Y axons innervated both the pretectum and the superior colliculus, one innervated only the pretectum, and two innervated only the superior colliculus. Two of the Y axons were quite unusual in that their receptive fields were located well into the hemifield ipsilateral with respect to the hemisphere into which they were injected. These axons exhibited remarkable arbors in the lateral geniculate nucleus, diffusely innervating the C-laminae and medial interlaminar nucleus, but, unlike all other X and Y arbors, they did not innervate the A-laminae at all. In addition to these qualitative observations, we analyzed a number of quantitative features of these axons in terms of numbers and distributions of terminal boutons. We found that Y arbors contained more boutons than did X arbors in both thalamus and midbrain. Also, for axons with receptive fields in the contralateral hemifield (all X and all but two Y axons), 90-95% of their boutons terminated in the lateral geniculate nucleus; the other two Y axons had more of their arbors located in midbrain.

In the chick, retinal axons enter the optic tectum through a superficial lamina, then branch into distinct deeper retinorecipient laminae, where they arborize and form synapses. To study factors that guide this laminar selectivity, we devised an organotypic culture system in which a transverse tectal section is overlaid with a retinal explant large enough to allow unimpeded access to all tectal laminae. Outgrowth, branching, and arborization patterns of retinal axons on tectal slices were lamina-selective, indicating the existence of localized cues that guide retinal axons. Further studies suggested that some of these cues are: (1) associated with cell membranes or extracellular matrix (because axons grew selectively on chemically fixed tectal sections); (2) intrinsic to the tectum (because axons grew selectively on tectal sections prepared from enucleated embryos); (3) distinct from topographic cues (because axons from nasal and temporal retina behaved similarly on anterior tectal slices); and (4) selective for retinal axons (because axons growing from other explants exhibited different laminar preferences).

The electron microscopic autoradiographic tracing method has been used to examine the morphology and postsynaptic relationships of five projections (retina, cortical area 17, superior colliculus (tectal), parabigeminal nucleus, and pretectum) to the dorsal lateral geniculate nucleus of the greater bush baby Galago crassicaudatus. Retinal terminals have been examined in the contralaterally innervated layer of each of the three matched pairs [parvi- (X-cell), magno- (Y-cell), and koniocellular (small, W-cell)] of geniculate layers. These terminals are large and contain pale mitochondria and round vesicles (RLPs). RLPs are presynaptic to juxtasomatic regions of parvi- and magnocellular neurons. In contrast, RLPs innervate more distal regions of koniocellular neurons. Labeled cortical, tectal, and parabigeminal terminals are relatively small and contain round vesicles and dark mitochondria. Cortical terminals in each of the three representative layers are presynaptic to small diameter dendrites. No convergence of cortical and retinal terminals has been seen in any layer. Labeled tectal and parabigeminal terminals are found primarily in the koniocellular layers, but the latter are also seen in all other layers. Tectal and parabigeminal terminals have been observed converging with retinal terminals on dendrites of some koniocellular neurons. Labeled pretectogeniculate terminals contain densely packed pleomorphic vesicles, dark mitochondria, and a dark cytoplasmic matrix. These terminals, which are present in each of the representative layers, are presynaptic to conventional dendrites and profiles containing loosely dispersed pleomorphic vesicles and a pale cytoplasmic matrix.

Dynamic rearrangements of axon terminal arbors may be critical for establishing appropriate connections in the developing nervous system. Here, the changes in complex retinal axon arbors in the tecta of live Xenopus larvae were followed during the formation of the topographic retinotectal projection. Three-dimensional reconstructions of terminal arbors made with a confocal microscope at hourly intervals revealed rapid remodeling of arbor extensions. Shorter branches were extended and retracted very rapidly, suggesting that they probe the environment for the optimal sites to form stable branches. About 27% of longer branches were present throughout the entire observation period and may be sites of stabilized synaptic contacts. Treatment of the animals to block postsynaptic activity resulted in increased rates of arbor rearrangements, which may coincide with decreased synapse stability. These studies reveal the dynamic behavior of nerve arbors and provide estimates for the lifetimes of retinotectal branches.

The large glycoprotein tenascin is one of the extracellular matrix proteins that is abundant in the developing nervous system. To determine its distribution and possible role in the ontogenty of the avian retinotectal system, the distribution of the protein, the expression of its mRNA, and the effect of the protein on growing retinal neurites in vitro was investigated. Immunocytochemistry demonstrated that relatively little tenascin was present in the optic fiber layer of the retina, the optic nerve and tract. Tenascin, however, was abundant in the stratum opticum of the tectum, the target of retinal axons in the brain. Whereas tenascin protein is found only in discrete portions and layers of the brain, in situ hybridization studies showed that tenascin mRNA was expressed throughout development by radial glial cells at the ventricular surfaces of the brain, distant from the tissue localization of the protein. Injection of antitenascin antiserum into the tectal ventricle disturbed the distribution of the protein in the tectum by binding tenascin closer to its origin at the ventricular border. This suggests that the localization of tenascin in the brain is not restricted to its site of synthesis, but is mediated by tenascin-binding proteins that are expressed in defined areas and layers in the brain. In vitro studies showed that tenascin did not promote neurite outgrowth of retinal axons. On the contrary, the addition of tenascin to retinal explants growing on collagen or on L1 slowed the growth rate of optic axons by as much as 50%. The inhibitory function in vitro suggests that this protein has a modulatory role in axonal growth in vivo. The absence of tenascin from the optic fiber layer of retina, optic nerve, and optic tract and its abundance in the tectum suggest that tenascin may function to slow the rapidly growing optic nerve fibers once they arrive at the tectum. The slowing of optic fiber outgrowth may then facilitate terminal arborization and synapse formation within the tectum. The abundant tenascin in synaptic layers may also serve to stabilize synapses once they have formed.

The retinal projection to the superior colliculus can be made abnormally dense by inducing a "compressed" retinal projection into a subnormal tectal volume, or abnormally sparse by monocular enucleation early in development. Any or all of the features of cell number, axonal arbor, dendritic arbor, and synaptic density could potentially be adjusted to compensate for such variations in the convergence of one cell population on another. We have examined the consequences of neonatal partial tectal ablation or monocular enucleation for synaptic length, density, and relative numbers of synapse classes in the superficial gray layer of the hamster superior colliculus. Monocular enucleation resulted in a reduction of synaptic density in the superficial gray layer of the colliculus ipsilateral to the remaining eye. This decrease in density was entirely accounted for by a reduction of the number of synapses with round vesicles, large asymmetric terminal specializations, and pale mitochondria characteristic of retinocollicular terminals (RLP synapses). There was no compensatory increase in any other synaptic class. RLP synapses were larger in monocular enucleates. Partial tectal ablation had no effect on synaptic density, nor on the relative proportions of different synaptic types. Synapses of the RLP class were slightly smaller than normal. These results suggest that synaptic density is normally at a maximum that cannot be altered by increases in potential input. However, density may be reduced by decreasing the number of inputs. Terminal classes do not appear to compete with each other within the collicular volume, suggesting that postsynaptic cells controls both the classes and numbers of their potential inputs.

The characteristics and distribution pattern of retinal afferent terminals making synaptic contacts on narrow field vertical neurons in the stratum griseum superficiale of the rabbit superior colliculus were studied using the Golgi-gold substitution technique in combination with either autoradiographic or degenerative methods. At the level of light microscopy, identified gold-toned vertical neurons showed similar features to those previously described by others. Although their axons were frequently seen rising from the basal dendritic tuft, they could also emerge from an apical dendrite, but rarely from the cell body. The electron microscopic study revealed that these neurons received radiolabelled or degenerated profiles with typical features of retinal terminals, while more proximal parts seemed to receive fewer terminals but of larger size. Axo-somatic synaptic junctions were very rare and, interestingly, retinal terminals were not seen forming synapses on the basal dendritic arborization. The present results indicate that the narrow field vertical cells are targets for retinotectal projection.

In a variety of species, developing retinal axons branch initially more widely in their visual target centers and only gradually restrict their terminal arbors to smaller and defined territories. Retinotectal axons in fish, however, appeared to grow in a directed manner and to arborize only at their retinotopic target sites. To visualize the dynamics of retinal axon growth and arbor formation in fish, time-lapse recordings were made of individual retinal ganglion cell axons in the tectum in live zebrafish embryos. Axons were labeled with the fluorescent carbocyanine dyes Dil or DiO inserted as crystals into defined regions of the retina, viewed with 40x and 100x objectives with an SIT camera, and recorded, with exposure times of 200 msec at 30 or 60 sec intervals, over time periods of up to 13 hr. (1) Growth cones advanced rapidly, but the advance was punctuated by periods of rest. During the rest periods, the growth cones broadened and developed filopodia, but during extension they were more streamlined. (2) Growth cones traveled unerringly into the direction of their retinotopic targets without branching en route. At their target and only there, the axons began to form terminal arborizations, a process that involved the emission and retraction of numerous short side branches. The area that was permanently occupied or touched by transient branches of the terminal arbor--"the exploration field"--was small and almost circular and covered not more than 5.3% of the entire tectal surface area, but represented up to six times the size of the arbor at any one time. These findings are consistent with the idea that retinal axons are guided to their retinotopic target sites by sets of positional markers, with a graded distribution over the axes of the tectum.

After early postnatal ablation of one superior colliculus together with the ipsilateral eye in Syrian hamsters, retinofugal fibers abnormally cross the tectal midline and innervate the remaining superior colliculus. The fibers of this aberrant decussation are confined mainly to the superficial gray layer, with little ingrowth or termination in the deeper stratum opticum; laterally, most termination is in the superficial part of the superficial gray. Establishment of this abnormal pattern is temporally correlated with the appearance of oligodendrocytes at progressively more superficial locations in the colliculus. Oligodendrocytes express, on their surface, molecules that are inhibitory to neurite growth. This raises the possibility that their differential distribution in the superior colliculus during growth of retinal fibers is causally involved in the generation of the observed termination pattern. We tested this hypothesis by applying the monoclonal antibody IN-1, which neutralizes this inhibitory activity, during the time of postnatal fiber growth and terminal arbor formation. We found that in the presence of IN-1, but not a control antibody, recrossing retinofugal fibers, observed at postnatal day 12, traverse the stratum opticum as well as the superficial gray, with greater depth of termination in superficial gray and stratum opticum. This pattern resembles that of the normal contralateral retinotectal projection. The results indicate that neurite growth inhibitors expressed by oligodendrocytes are responsible for restricting the innervation of a target area in postnatal plasticity.

The morphology and synaptic relationships of anterogradely labeled retinal, visual cortical (area 17), and parabigeminal terminals have been analyzed within the superficial gray (stratum griseum superficiale) of Galago crassicaudatus. Our data regarding the retinocollicular projection reveal two populations of terminals based upon size. The population of smaller terminals are found in clusters, while the larger occur in isolation. Both populations of retinocollicular terminals form synapses primarily with dendritic spines, but synapses upon pale vesicle filled (PVF) profiles and dendritic shafts also occur. Corticotectal terminals contain round vesicles and make asymmetrical synapses, primarily onto dendritic spines; few form synapses with PVF profiles. Our findings suggest the possibility that there are two populations of corticotectal terminals based upon differences in size and morphology. Parabigeminotectal profiles contain densely packed round vesicles and make asymmetrical synapses. These terminals, which are exclusively cholinergic in Galago, are presynaptic to dendrites of various sizes. Convergence of retinal and cortical terminals has been observed. This convergence occurs on distinctly separate regions of the postsynaptic membrane. In contrast, convergence of retinal and parabigeminal terminals occurs on the same region of the postsynaptic cell(s).

Spatial gradients of axon guiding molecules have long been suspected to provide positional and directional cues for retinal ganglion cell axons growing within the optic tectum. With the identification of a guiding activity from tectal cell membranes, it has become possible to investigate the potential physiological significance of molecular gradients for retinal growth cone behavior in vitro. A subset of retinal growth cones, those from the temporal half, were highly sensitive to small concentration changes of the guiding component. The degree of response was correlated with the strength of the gradient. These findings demonstrate that the neural growth cone can read gradients of surface-associated information.

Nasal and temporal retinal neurites were confronted in culture with glial cells from the rostral and caudal parts of the optic tectum and with glial cells from the diencephalon. Twenty of each of the six classes of encounter between individual growth cones and isolated glial cells were analysed by time-lapse videorecording. The results show that growth cones from the temporal retina collapse when they contact glial cells from the caudal tectum, but do not collapse when they contact glia from other areas. Growth cones of nasal retinal fibres do not collapse on contact with any of the glial types examined. This suggests that the inhibitory phenomena described by others are in part due to the cell surface characteristics of glial cells, and that there are differences between glia from the front and back of the optic tectum.

Leu-enkephalin-like immunoreactive (ENK-I) elements appearing in the superficial layers of the rat superior colliculus (SC) after eye-enucleation were examined by means of immunoelectronmicroscopy. ENK-I somata were of a single type and formed symmetric and asymmetric synapses with non-immunoreactive axon terminals. Some degenerating retinal terminals made synaptic contacts only with small ENK-I dendrites, suggesting that deafferentation-induced ENK-I neurons in the rat SC receive retinal input onto the distal portions of their dendrites.

The ultrastructure of the pigeon and quail ventral lateral geniculate nucleus was analyzed with standard electron microscopy and horseradish peroxidase tracing of its retinal and tectal afferents. Six types of neurons were distinguished: two large, two medium-sized, and two small types. The latter do not project to the optic tectum and appear to be interneurons. Large and medium-sized neurons project to the optic tectum and are thus relay neurons. Profiles with round, large synaptic vesicles were identified as retinal axon terminal afferents and those with pleomorphic, loosely grouped synaptic vesicles as tectal afferents. Gap junctions were seen between perikarya of small neurons and also with unidentified profiles.

The fine structure of substance P (SP) and adenosine deaminase (ADA) immunoreactive structures in synaptic contacts localized to the superficial layers of the superior colliculus of the rat was investigated by means of immunoelectron microscopy. We also examined the possibility of retinal innervation of SP- and ADA- containing neurons by immunohistochemistry after degeneration of retinal terminals caused by enucleation. SP-like immunoreactive presynaptic terminals of the stratum griseum superficiale (SGS) formed both asymmetric and symmetric synaptic contacts. Presynaptic dendritelike structures were also observed. SP immunoreactive postsynaptic elements made contacts with terminals showing diverse features. ADA-like immunoreactive structures were seen only as postsynaptic elements to different kinds of nonimmunoreactive terminals and were mostly localized in the ventral third of the SGS and the dorsalmost stratum opticum (SO). After enucleation, degenerating retinal terminals were found to form synaptic contacts with SP and ADA immunoreactive structures. The highest number of such degenerating terminals on ADA immunoreactive structures was observed 2 days after retinal denervation, very few being seen after 5 days. These degenerating terminals were restricted to the ventral SGS and dorsal SO. SP immunoreactive structures postsynaptic to degenerating retinal terminals were most numerous 5 days after enucleation and mainly localized in the dorsal SGS. Occasionally, SP immunoreactive dendritelike processes forming synapses with degenerating retinal terminals were simultaneously presynaptic to other nonimmunoreactive profiles, defining, therefore, serial synapses. The present results suggest that SP-I and ADA-I collicular neurons may be part of distinct channels carrying visual information to the lateral posterior and lateral geniculate nuclei of the thalamus, respectively.

The immunocytochemical analysis of the viper optic tectum was carried out with an anti-gamma-aminobutyric acid (anti-GABA) antiserum following retinal deafferentation for survival times ranging from 10 to 90 days. The ultrastructure of the SGFS neuropil revealed that among the two types of axon terminals, namely pleiomorphic (P) and spherical (S) boutons, a subtype of the latter (S2), corresponding to the retinal terminals, was found to degenerate at varying rates. In most cases, this resulted in the glial engulfment of the presynaptic partner, leaving the postsynaptic differentiation free (FPSD). Beyond the two first months, the asymmetrical freed postsynaptic differentiations (FPSDs) were progressively and partly reafferented by GABA-positive P axon terminals through a sliding process. Three months postoperative, the number of GABA-positive P axon terminals which, in normal animals, establish asymmetrical contacts (1-2%), was found to increase to approximately 10%. The postsynaptic differentiation may represent a mismatched receptor area for the new competing presynaptic partners. The functional implications of such 'axonal terminal sprouting' are discussed.

Intracellular recording and HRP injection were used to characterize retinocollicular axons in the hamster. Eighteen retinotectal axons were filled with HRP. The type of axon recovered most often (N = 10) had a receptive field with center-surround organization, gave sustained responses to flashed spots, had linear spatial summation, and responded only to more slowly moving stimuli. These fibers had a mean axonal diameter of 0.58 microns (SD = 0.21) and an average conduction latency of 3.4 msec (SD = 1.2) to optic chiasm (OX) stimulation. They gave rise to circular or vertically elongated arbors with an average cross-sectional area of 33,238 microns2 (SD = 12,763) and were almost completely restricted to the stratum griseum superficiale (SGS). We recovered 6 fibers with very different structural and functional properties. The receptive fields of these axons also had center-surround organization, but gave phasic responses to flashed stimuli, had nonlinear spatial summation, and responded to rapidly moving stimuli. These axons had an average diameter of 0.84 micron (SD = 0.09) and an average latency of 2.1 msec (SD = 0.40) to OX stimulation. They had elongated arbors with an average cross-sectional area of 78,045 microns2 (SD = 14,252) and innervated the SGS, the stratum opticum (SO), and upper stratum griseum intermediale (SGI). We also recovered 2 fibers that had thin (0.3 and 0.6 micron) axons and latencies of 4.2 and 3.6 msec to OX stimulation. They both gave rise to horizontally oriented arbors in the stratum zonale (SZ) and upper SGS.

A polyclonal antibody raised to a protein from goldfish optic tectum recognises, immunohistochemically, axons throughout normal goldfish visual pathway. In goldfish with injured optic nerve, this antibody recognises degenerating neuronal debris as well as regenerating fibres. On immunoblot, the antibody recognises, primarily, a neuronal intermediate filament protein in the region of 145 kDa. Such an antibody should prove useful in studies pertaining to goldfish visual pathway.

There are several different groups of ganglion cells in the retina of the frog. Although their axons are thought to terminate in different layers of the optic tectum, little is known about the morphology of their terminal arbors or their synaptic targets. The present paper reports the results of a layer-by-layer study of horseradish peroxidase labeled retinal axons in the optic tectum of Rana pipiens. Light and electron microscopy was used to study the axon's laminar distribution, patterns of arborization, and synaptic contacts. Labeled retinal axons were found in all of the superficial layers of the tectum (A-G). From layer to layer, the retinal axons differed markedly in the diameter of their parent axons (0.2-3.0 microns) and in the morphology and horizontal extent of their terminal arbors. Five classes of synaptic terminals could be distinguished in the tectum. The retinal terminals belonged to a class characterized by round, medium-sized synaptic vesicles. They made synaptic contact with dendrites and other axon terminals in each of the layers. They were always the presynaptic component. The postsynaptic dendrites were often the vertically oriented processes of cells located in the deeper layers. The postsynaptic terminals belonged to a class distinguished by their flat, medium-sized vesicles. These terminals in turn contacted what appeared to be dendrites. In layer eight, the retinal axons were often large, spoon-shaped boutons that ended in apposition with the somata of the layer.

The ultrastructural organization of the pretecto-olivary projection neurons within the nucleus of the optic tract and dorsal terminal accessory optic nucleus of rabbits was studied by using anti-GABA immunolabelling and retrograde transport of WGA-HRP. GABA-like immunoreactivity was determined with a postembedding colloidal gold technique. WGA-HRP was injected in the dorsal cap of the inferior olive. The WGA-HRP-labelled neurons were incubated with gold-substituted silver peroxidase. Neurons projecting to the inferior olive had large to medium-sized cell bodies and were GABA negative. In the nucleus of the optic tract, projection neurons are found in the rostral parts, while the majority of the local GABAergic interneurons are mainly found in the caudal parts. In the dorsal terminal nucleus both types of neurons are intermingled. The projection neurons were frequently in synaptic contact by GABAergic terminals. These neurons also receive retinal afferents indicating the existence of a two-step synaptic connection from the retina to the inferior olive. It is suggested that this class of projection neurons forms the "direction-selective" neurons that can be antidromically stimulated from the inferior olive. The GABAergic terminals on the identified projection neurons are of axonal origin (F-terminals), whereas presynaptic dendrites of interneurons (P-terminals) were seldom observed to be in synaptic contact with retrogradely labelled profiles. The strong input of GABA on direction-selective neurons indicates that GABA is directly involved in modulating retinal signals to the inferior olive.

Changes in synaptic structure were examined in junctions of the retino-recipient layers of the opossum superior colliculus (SC) at critical developmental stages (30, 40 and 61 day-old pouch young and adult controls). Criteria of classification were the presence and direction of curvature (smile, flat, frown and irregular) and the degree of aggregation of paramembranous components (immature, intermediate and mature). The results favor the aggregation of dense components vis-a-vis curvature in the assessment of maturational changes.

Embryonic and regenerating retinal axons in fish are able to seek out their retinotopic target sites in the tectum. Neither a specific preordering of axons in the retinotectal pathway nor activity-dependent axon-target interactions are required for appropriate axonal targeting. Axon-target recognition appears to be predominantly mediated by positional cell surface markers. The discrimination of position-dependent differences by retinal axons in a special in vitro assay is consistent with this concept. To understand retinal axonal regeneration we have analyzed the glial cells of the fish optic nerve and the expression of growth-associated cell surface molecules on the regenerating axons. The surfaces of the glial cells identified as oligodendrocytes are excellent substrates for the elongation of regenerating axons. Raising monoclonal antibodies we have found 3 cell surface proteins specific for growing axons. In the normal adult goldfish optic nerve, these proteins are only expressed by the few new axons from the newborn ganglion cells at the retinal margin. They are re-expressed on all axons during regeneration. A known cell surface molecule, NCAM, is expressed in a similar, specific spatiotemporal pattern on the fish retinal axons and may--in normal nerves--contribute to the establishment of the age-related fiber association. Whether the re-expression of NCAM and the antigens detected by the novel monoclonal antibodies are functionally involved in axonal growth and regeneration remains to be investigated.

The ultrastructure of the optic tectum in the squirrel fish, Holocentrus rufus, has been studied and the normal synaptic organization is described. Synaptic terminals were classified into eight types (S1-S6, F1, F2) by their morphology and synaptic relations. The distribution pattern for each type of terminal was determined by counting the relative number of terminals in each layer. Most S1 terminals are localized in Stratum marginale (SM), whereas S2 terminals are most common in Stratum fibrtosum et griseum superficiale (SFGS). S3, S4, S5, F1, and F2 terminals are limited mainly to SFGS and Stratum griseum centrale (SGC). S6 terminals are most frequently seen in SGC and Stratum griseum periventriculare (SPV). In order to determine the origins of the various types of synaptic terminals in the optic tectum, the telencephalon, eye, and optic tectum were removed unilaterally and areas of resultant degeneration examined. Electron microscopic observations show that nearly all S2 terminals in SFGS of the contralateral optic tectum degenerate after eye enucleation, whereas some S4 terminals in SFGS and SGC exhibit degenerative changes after removal of the ipsilateral telencephalon. Unilateral ablation of the optic tectum was associated with degenerative changes in occasional S5 terminals within SGC of contralateral optic tectum. All experiments resulted in some increased electron density of S3 and F terminals, some of which were identified as F2 terminals. The possible origins of S1 and S6 terminals, which were not altered in the present experiments, are discussed.

Neural connections between the eye and optic tectum in Xenopus laevis were anatomically traced by observing the tectal location of Wallerian degeneration after discrete retinal lesion. These retinotectal connections were mapped in postmetamorphic frogs and tadpoles at stage 51, the stage at which retinal axons have grown into about the rostral one-half of the tectum. The course of the experimental degeneration was the same in frogs and tadpoles, but degeneration proceeded faster in the younger animals. In the frogs, connections were ordered, with nasal retina mapping to the caudal part of the tectum and temporal retina mapping to the rostral tectum. In the tadpoles, within the innervated area at the rostral tectum, the retino-tectal connections were generally organized as in the adults, with the temporal retina mapping to the rostral part of the innervated tectum and nasal retina mapping primarily to the caudal part. But a portion of the nasal fibers consistently mapped to the far rostral tectum as well. Electron microscopic observations showed degenerating synaptic terminals at both rostral and caudal portions of the innervated tectum after lesion of just the nasal retina. Degeneration was not seen in control animals. These results indicate that some fibers (particularly from nasal retina) may shift their terminals caudally on the tectum to match tectal growth and produce the adult pattern of connections. If there is such connection readjustment, the 'aberrant' connections from nasal retina in tadpoles may be an indication of this process.

The optic tecta of 55 Rutilus rutilus were examined at intervals varying from 2 days to 4 years after unilateral retinal ablation. Qualitative ultrastructural examination of the retinal target lamina of the optic tectum (stratum fibrosum et griseum superficiale, SFGS) revealed that an initial period of degeneration and glial reaction, each of which could take one of a variety of forms and which lasted for 1-3 months after ablation, was followed by the temporary formation of heterologous synapses which persisted for a further 1-12 months. This in turn was followed by the degeneration of these synapses during the second year after ablation. Quantitative analysis at the level of the light microscope revealed a shrinkage of the SFGS throughout the level of the light microscope revealed a shrinkage of the SFGS throughout the first 14 postoperative months with no further reduction taking place thereafter. Analysis at the ultrastructural level revealed that this shrinkage was due to the disappearance, and not to the reduction in size, of pre- and postsynaptic profiles accompanied by glial reaction. No signs of collateral sprouting were seen throughout the survival period. Thus, partial deafferentation of the SFGS leads in the long run to a marked impoverishment of its neuronal network, without any apparent compensation.

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

Retinal and cortical afferents to the superior colliculus of the rabbit were labelled simultaneously by injecting 3H-leucine into the right eye and HRP into the left visual cortex. It could be shown that there is some convergence of retinal and cortical input onto common postsynaptic elements in the superficial grey, but these cases were found to be rather rare indicating that most afferents from the retina and the visual cortex terminate either on different postsynaptic cells or on different parts of common postsynaptic cells.

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

The growth of regenerating retinal axons into the central portion of the optic tectum of adult goldfish was examined with the light and electron microscopes. Optic tracts were cut and, two days to five months later, the animals were perfused and the tecta prepared for microscopy. Regenerating axons first reached central regions of the tectum seven to ten days postoperatively. Regenerating axons appear in very large numbers and travel in fascicles in the stratum opticum (SO) and in the adjacent neuropil, the stratum fibrosum et griseum superficiale (SFGS). In the SO, the fascicles are bordered by glial cells and degenerating debris. Within the SFGS, however, the fascicles do not seem to be similarly associated with glial cells and degenerating debris. The youngest regenerating axons are very slender processes, containing microtubules but few or no neurofilaments or dense granular material. By 10 to 14 days postoperatively, neurofilaments can be seen and, in addition, large numbers of vesicles with dense cores appear. The vesicles with dense cores increase in numbers until about 28 days postoperatively and then become quite rare. That vesicles with dense cores were seen in regenerating axons in both SO and SFGS during the period of growth into the tectum but were not seen in axon terminals at any time, suggests that they may be concerned with axon elongation. During the period one month to five months postoperatively, the regenerating axons gradually increase in diameter but do not reach preoperative sizes, suggesting that the regenerative changes may still be occurring. Remyelination is delayed and proceeds slowly. Many axons remain unmyelinated for as long as five months postoperatively.

Optic and nonoptic fibers and synapses were counted in the primary optic innervation layer (S-SO-SFGS) in anteromedial tectum in normal goldfish and in fish 30, 60, and 240 d after the optic nerve was crushed. A newly developed "cold-fill" HRP-labeling protocol was used to label optic afferents for electron microscopy, and counts were then made on EM photomontages of columns through the HRP-labeled S-SO-SFGS. Normal numbers of retinotectal synapses were present at 30 d regeneration, at a time when activity-dependent refinement of the optic projection is incomplete. Normal numbers were also found at 60 and 240 d, when refinement is largely completed. In contrast to this constancy in optic synapse numbers, there was nearly 10 times the normal number of optic fibers in the SFGS at 30 d, and these were reduced by 50% at 60 d, remaining over 4 times normal at 240 d. These findings imply extensive rearrangement of optic synapses during map refinement. They also indicate that synapse rearrangement is associated with the elimination of optic collaterals.