Ultrastructure of the crossed isthmotectal projection in Xenopus frogs

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

Topographic projections between the nucleus isthmi and the optic tectum in a teleost,Navodon Modestus

Following horseradish peroxidase injections into the optic tectum of a teleost,Navodon modestus, reciprocal and topographic projections between the nucleus isthmi and the ipsilateral optic tectum were determined. The isthmo-tectal fibers diverge to the optic tectum while maintaining the spatial arrangements of the isthmic cells from which the fibers originate. The tecto-isthmic projections also keep the spatial arrangements in the optic tectum. The tectal fibers converge near the nucleus isthmi and terminate in the non-cellular portion of the nucleus. The reciprocal topography is apparent in the combined results of 9 experiments with one tectal injection in each region. No labeled cells and fibers were found in the contralateral nucleus isthmi.     

A banded distribution of retinal afferents within layer 9A of the normal frog optic tectum

A banded distribution of retinal ganglion cell axons within layer 9A of the superficial tectal neuropil in Rana pipiens was revealed through anterograde labeling with horseradish peroxidase. Layer 9A previously has been demonstrated to mediate binocular vision through a polysynaptic pathway by way of the nucleus isthmi^5,8,9. This nucleus interconnects analogous regions of the two tectal lobes such that isthmic axons retinotopically map the visual world of the ipsilateral eye within tectal layers 9A and 8^9,10. Thus, we have found that a pattern of retinal ganglion cell bands occurs in binocular regions of normal frogs. This pattern is similar, but not identical, to the experimentally produced stripes previously observed in the doubly innervated tecta of 3-eyed and single tecta frogs^2,12–14. Qualitative and quantitative comparisons of these two types of afferent segregation patterns have implicated several structural and functional parameters which might be involved in band formation.     

Two distinct visual pathways through the superficial pretectum in a percomorph teleost

The connections of the superficial pretectum and of nucleus isthmi were examined in a percomorph teleost, Lepomis cyanellus. Horseradish peroxidase was injected either with a pin into the parvicellular nucleus of the superficial pretectum or pressure injected into nucleus isthmi; the isthmal injections retrogradely labelled the neurons of the magnocellular nucleus of the superficial pretectum. Two main visual pathways can be recognized: The first projects from the retina to the parvicellular nucleus, and then to the intermediate nucleus of the superficial pretectum, the inferior raphe nucleus, and the trochlear nucleus. The second projects from the retina via the optic tectum to the magnocellular nucleus of the superficial pretectum, and from there to nucleus isthmi and the lateral thalamic nucleus; nucleus isthmi and the lateral thalamic nucleus project back to the optic tectum, and nucleus isthmi also projects back to the magnocellular nucleus. The two pathways are interconnected to some extent because both nucleus isthmi and the optic tectum project to the parvicellular nucleus; nevertheless, we suggest that they may be functionally and evolutionarily distinct. Compared to percomorphs, the first pathway appears reduced in cyprinid teleosts such as goldfish. Furthermore, the magnocellular nucleus of the second pathway is completely different in cyprinids, both in cellular architecture and in efferent connections. A phylogenetic analysis suggests that cyprinid ancestors went through a period of reduced vision and that the magnocellular nucleus of the superficial pretectum in modern cyprinids has been either extensively modified from the primitive condition or lost entirely and replaced by a superficially similar structure.     

Origins of serotonin-like immunoreactivity in the optic tectum of rana pipiens

We have previously identified a population of serotonin-like immunoreactive (5-HT-ir) retinal ganglion cells in Rana pipiens. In this study, we examined serotonin-like immunoreactivity (5-HTLI) in a probable target of those cells, the optic tectum. We observed both 5-HT-ir fibers and cell bodies in this structure. 5-HT-ir cells were located in the cellular layers of the tectum, layers 2, 4, and 6, and scattered in its superficial layers. 5-HT-ir fibers in the tectum displayed a laminated organization and were located in tectal layers 3, 5, 6, 7, and 9. Retrograde labelling experiments showed that 5-HT-ir retinal ganglion cells projected to the optic tectum. However, these experiments also demonstrated that serotonergic neurons in the midbrain tegmentum, the nucleus isthmi, and the medulla did so as well. 5-HT-ir fibers seen in lamina A of layer 9 were very much reduced in density in animals in which the optic nerve had been lesioned for 3–6 months. Immunoreactive fibers in lamina B of layer 9 were not affected by the lesion. Our results suggest that 5-HT-ir fibers in lamina A of layer 9 are mainly of retinal origin, whereas those in lamina B originate from other brain areas. The 5-HT-ir tectal cells located in the cellular layers probably contribute the 5-HT-ir fibers seen in layers 3, 5, 6, and 7. © 1995 Wiley-Liss, Inc.     

Combining visual information from the two eyes: the relationship between isthmotectal cells that project to ipsilateral and to contralateral optic tectum using fluorescent retrograde labels in the frog, Rana pipiens

The frog nucleus isthmi (homolog of the mammalian parabigeminal nucleus) is a visually responsive tegmental structure that is reciprocally connected with the ipsilateral optic tectum; cells in nucleus isthmi also project to the contralateral optic tectum. We investigated the location of the isthmotectal cells that project ipsilaterally and contralaterally using three retrograde fluorescent label solutions: Alexa Fluor 488 10,000 mw dextran conjugate; Rhodamine B isothiocyanate; and Nuclear Yellow. Dye solutions were pressure-injected into separate sites in the superficial optic tectum. Following a 6-day survival, brains were fixed, sectioned, and then photographed. Injection of the different labels at separate, discrete locations in the optic tectum result in retrograde filling of singly labeled clusters of cells in both the ipsilateral and contralateral nucleus isthmi. Generally, ipsilaterally projecting cells are dorsal to the contralaterally projecting cells, but there is a slight overlap between the two sets of cells. Nonetheless, when different retrograde labels are injected into opposite tecta, there is no indication that individual cells project to both tecta. The set of cells that project to the ipsilateral tectum and the set of cells that project to the contralateral tectum form a visuotopic map in a roughly vertical, transverse slab. Our results suggest that nucleus isthmi can be separated into two regions with cells in the dorsolateral portion projecting primarily to the ipsilateral optic tectum and cells in the ventrolateral nucleus isthmi projecting primarily to the contralateral optic tectum.     

Laminar segregation of GABAergic neurons in the avian nucleus isthmi pars magnocellularis: A retrograde tracer and comparative study

The isthmic complex is part of a visual midbrain circuit thought to be involved in stimulus selection and spatial attention. In birds, this circuit is composed of the nuclei isthmi pars magnocellularis (Imc), pars parvocellularis (Ipc), and pars semilunaris (SLu), all of them reciprocally connected to the ipsilateral optic tectum (TeO). The Imc conveys heterotopic inhibition to the TeO, Ipc, and SLu via widespread γ‐aminobutyric acid (GABA)ergic axons that allow global competitive interactions among simultaneous sensory inputs. Anatomical studies in the chick have described a cytoarchitectonically uniform Imc nucleus containing two intermingled cell types: one projecting to the Ipc and SLu and the other to the TeO. Here we report that in passerine species, the Imc is segregated into an internal division displaying larger, sparsely distributed cells, and an external division displaying smaller, more densely packed cells. In vivo and in vitro injections of neural tracers in the TeO and the Ipc of the zebra finch demonstrated that neurons from the external and internal subdivisions project to the Ipc and the TeO, respectively, indicating that each Imc subdivision contains one of the two cell types hodologically defined in the chick. In an extensive survey across avian orders, we found that, in addition to passerines, only species of Piciformes and Rallidae exhibited a segregated Imc, whereas all other groups exhibited a uniform Imc. These results offer a comparative basis to investigate the functional role played by each Imc neural type in the competitive interactions mediated by this nucleus. J. Comp. Neurol. 521:1727–1742, 2013. © 2012 Wiley Periodicals, Inc.     

Early development of the retinal line of decussation in normal and albino ferrets.

This work describes the retinal origin of the crossed and uncrossed projections in newborn, 9-day-old and adult normally pigmented and albino ferrets. Horseradish peroxidase (HRP) was injected unilaterally into the thalamic and midbrain visual centers of ferrets to label retinal ganglion cells retrogradely. In normally pigmented adults, the retinal line of decussation was sharp and passed through the area centralis. Ganglion cells with uncrossed axons occupied the entire temporal retina. In albino adults, ganglion cells with uncrossed axons were distributed in the periphery of the temporal retina away from the area centralis. In the normally pigmented adults, about 11% of the retinal ganglion cells had uncrossed axons compared to about 4% in the albinos. At birth, normally pigmented ferrets had a sharp line of decussation with most (about 98%) uncrossed ganglion cells found in the temporal retina. In the newborn albinos, most uncrossed ganglion cells were in the temporal retina (about 89%), but there were many fewer than in the normal neonates and, as in the albino adults, the uncrossed ganglion cells were distributed along the temporal most margin of the retina. In the normal neonates, about 11% of the ganglion cells had uncrossed axons, compared to about 3% in the albino neonates. The area centralis and visual streak were not evident until 9 days after birth. From these results we conclude that the retinal line of decussation is essentially mature by birth in the ferret, and the degree of the albino's abnormality is as extreme in neonates as in adults. The retinal decussation is virtually mature at a stage of development when the crossed and uncrossed retinal afferent axons are completely intermingled in their target nuclei and prior to the onset of significant retinal ganglion cell loss.     

Position of axons in the cat's optic tract in relation to their retinal origin and chiasmatic pathway.

The positions of the crossed and uncrossed optic axons of distinct diameter classes has been examined in the optic tract of the adult cat. In addition, the retinal origin of axons occupying different positions within the tract has been studied. Since the position of a fibre within the optic tract reflects its time of arrival during development, we have used axonal position as an indicator of age and have related this to the chiasmatic pathway choice of the axons. Cats were either monocularly enucleated, to reveal the position and diameter of surviving crossed and uncrossed optic axons in semithin and thin sections, or implants of horseradish peroxidase (HRP) were placed so as to retrogradely label the ganglion cells giving rise to axons within the deep (early arriving), or superficial (later arriving) parts of the tract selectively. This was accomplished by either 1) surgically implanting HRP into the superficial portion of the optic tract, via a transbuccal approach, or 2) making such a transbuccal transection of the superficial fibres, followed by intracerebral injections of HRP to retrogradely label the surviving, deeper, optic axons from their target nuclei. The deep parts of the optic tract contain fine and medium, crossed and uncrossed axons arising from mainly medium sized cells in the contralateral nasal and the ipsilateral temporal retina; there is a clear line of decussation. In contrast, the superficial parts of the tract contain mainly fine diameter axons arising from small cells in the whole contralateral retina, and a small proportion of large diameter axons arising from large, alpha cells in the whole contralateral retina and in the ipsilateral temporal retina. The likelihood that axons from the temporal retina will project contralaterally therefore increases as development proceeds, since these axons are found in the superficial parts of the tract only. This suggests that a time-dependent signal that weakens with age is responsible for directing early arriving optic axons from the temporal retina to take an exclusively uncrossed course.     

Change in the neurochemical signature and morphological development of the parvocellular isthmic projection to the avian tectum

Neurons can change their classical neurotransmitters during ontogeny, sometimes going through stages of dual release. Here, we explored the development of the neurotransmitter identity of neurons of the avian nucleus isthmi parvocellularis (Ipc), whose axon terminals are retinotopically arranged in the optic tectum (TeO) and exert a focal gating effect upon the ascending transmission of retinal inputs. Although cholinergic and glutamatergic markers are both found in Ipc neurons and terminals of adult pigeons and chicks, the mRNA expression of the vesicular acetylcholine transporter, VAChT, is weak or absent. To explore how the Ipc neurotransmitter identity is established during ontogeny, we analyzed the expression of mRNAs coding for cholinergic (ChAT, VAChT, and CHT) and glutamatergic (VGluT2 and VGluT3) markers in chick embryos at different developmental stages. We found that between E12 and E18, Ipc neurons expressed all cholinergic mRNAs and also VGluT2 mRNA; however, from E16 through posthatch stages, VAChT mRNA expression was specifically diminished. Our ex vivo deposits of tracer crystals and intracellular filling experiments revealed that Ipc axons exhibit a mature paintbrush morphology late in development, experiencing marked morphological transformations during the period of presumptive dual vesicular transmitter release. Additionally, although ChAT protein immunoassays increasingly label the growing Ipc axon, this labeling was consistently restricted to sparse portions of the terminal branches. Combined, these results suggest that the synthesis of glutamate and acetylcholine, and their vesicular release, is complexly linked to the developmental processes of branching, growing and remodeling of these unique axons.     

Terminations of individual optic tract fibers in the lateral geniculate nuclei of Galago crassicaudatus and Tupaia belangeri

The morphology and laminar distribution of individual optic fibers projecting to the lateral geniculate nucleus (GL) of Galago and Tupaia were studied following iontophoretic injections of horseradish peroxidase (HRP) into the optic tract. In Galago the GL is composed of three functionally matched pairs of layers, each characterized by cells of a given size, one large, one medium-sized, and one small. The results show that there is a close correspondence between the size of the afferent fibers and the size of the neurons in the target layer: large axons project to the magnocellular layers, medium-sized axons project to the parvicellular layers, and small fibers project to the intercalated layers. In Tupaia the GL is composed of two functionally matched pairs and two unmatched layers. Optic fibers that project to the medial matched pair (1 and 2) are only slightly larger than those that project to the lateral matched pair (4 and 5), but both are larger than those that project to the unmatched layers (3 and 6). In both species terminal arbors and the distribution of terminal boutons within layers corresponded closely with the organization of dendritic processes of cells in the target layer. This correspondence was particularly evident in the parvicellular layers in Galago and in layer 6 in Tupaia: parvicellular terminal arbors, like the dendrites of parvicellular cells, are organized in narrow columns oriented along lines of projection, whereas layer 6 terminal arbors, like the dendrites of layer 6 cells, are oriented in elongated strips perpendicular to lines of projection. In both species there was evidence for sublaminar terminations in some layers. These were restricted to the parvicellular layers in Galago and layers 4 and 5 in Tupaia. With the exception of a small number of fine fibers in the intercalated layers in Galago, optic fibers in both species terminated in one and only one layer in a set. The significance of this result depends on the relation between ganglion cell classes and what is being segregated in different GL layers. Lateral geniculate lamination varies even in closely related species and has evolved independently in such distantly related lines as carnivores and primates. It is not surprising, therefore, that what is being segregated varies from species to species.     

Direct retinal projections to the lateroposterior and pulvinar nuclear complex (LP-Pul) in the cat, as revealed by the anterograde HRP method.

Retinal projections to the lateroposterior and pulvinar nuclear complex (LP-Pul) in the cat were studied using anterograde horseradish peroxidase (HRP) tracing. After injecting HRP into the vitreous cavity of one eye, HRP-labeled presumed axon terminals were found in the LP-Pul bilaterally, with a contralateral predominance. The areas of distribution of these terminals were seen as a thin sheet at the lateral extreme of the LP-Pul, and as a few small spots within the LP-Pul, especially in the regions along the dorsomedial border of the caudal part of the LP-Pul.     

Development of the tecto–thalamic projection neurons and the differential expressions ofcalcium-binding proteins in the rat

We studied expression of calbindin-D 28 K and parvalbumin in tecto–thalamic projection neurons and during the formation of their tecto–thalamic projections using a double-labeling with Fluoro–Gold. To discern the completion of these projections, Fluoro–Gold, an opalescent fluorescent dye, was injected into the dorsal lateral geniculate and/or the lateral posterior nucleus in rats of various ages from neonates to adults. After one day’s survival, the brains were removed and sections of the brain were immunohistochemically processed using Cy3, a red fluorescent dye, as a marker for calbindin-D 28 K or parvalbumin. The three types of tecto–thalamic neurons, which have been described previously in the adult rats, were identified in the present study. The results revealed that in developing rats: 1) A population of medium-sized neurons (the presumed pyriform cells) express calbindin-D 28 K as early as the day of birth prior to the formation of their tecto–thalamic projection that occured on postnatal day 4. Most (over 90%) of them project to the dorsal lateral geniculate nucleus; 2) A population of large neurons (the presumed wide-field vertical cells) express calbindin-D 28 K on postnatal day 7, and most of them (over 90%) project to the lateral posterior nucleus; 3) Another population of medium-sized neurons (the presumed narrow-field cells) express parvalbumin on post-natal day 17, but only a half (45%) of them project to the dorsal lateral geniculate nucleus. In the developing nervous system, calcium ions play important roles in the biological and molecular events underlying neural development. Changes in the free intracellular calcium ion level, indicating neuronal activity has been reported to be correlated with onset of calbindin-D 28 K or parvalbumin-immunoreactivity that participate in the regulation of intracellular calcium homeostasis in neurons. Therefore, the present findings may reflect distinct developmental events in the different classes of tectal relay neurons that form parallel visual pathways, but which have such different functions as the detection of luminance, discrimination of direction, and the detection of fast movements.     

Development of retinopetal projections in the cichlid fish, Herotilapia multispinosa

This paper reports a study of the development of cells that project to the retina from the telencephalic nucleus olfactoretinalis and the diencephalon. Stell et al. (Proc. Natl. Acad. Sci. USA 81:940-944, '84) have shown that the FMRFamide-immunoreactive (FMRFamide-ir) cells in the nucleus olfactoretinalis project to the retina. Therefore, we used immunocytochemistry to study the development of these cells in the nucleus olfactoretinalis. Twenty hours after fry hatched, FMRFamide-ir cells were unambiguously seen in the nucleus olfactoretinalis. At this time the axons of these cells could be traced into the optic nerve. A few hours later the axons were visible in the retina and soon attained their adult position in the inner plexiform layer near the amacrine cells. In older frey, tracers were used to fill retinopetal cells in both the nucleus olfactoretinalis and the diencephalon. Counts of these cells demonstrated that over one-third of the adult number of retinopetal cells in the nucleus olfactoretinalis are present and have axons in the retina when the fry is 9 days old, and the percentage grows to one-half by the time the fry is 1 month old. Development of the retinopetal cells in the diencephalon lags behind that of the cells in the nucleus olfactoretinalis. However, about one-third of the adult number extend their axons into the optic nerve by 1 month of age. These results support our suggestion that the retinopetal cells have axons in the old part of the optic nerve because these cells were born and extended axons early in the life of the fish.     

Retinal projection to the pretectal nucleus lentiformis mesencephali in pigeons (Columba livia)

In birds, the nucleus of the basal optic root (nBOR) and the nucleus lentiformis mesencephali (LM) are retinal‐recipient nuclei involved in the analysis of optic flow and the generation of the optokinetic response. The nBOR receives retinal input from displaced ganglion cells (DGCs), which are found at the margin of the inner nuclear and inner plexiform layers, rather than the ganglion cell layer. The LM receives afferents from retinal ganglion cells, but whether DGCs also project to LM remains unclear. To resolve this issue, we made small injections of retrograde tracer into LM and examined horizontal sections through the retina. For comparison, we also had cases with injections in nBOR, the optic tectum, and the anterior dorsolateral thalamus (the equivalent to the mammalian lateral geniculate nucleus). From all LM injections both retinal ganglion cells and DGCs were labeled. The percentage of DGCs, as a proportion of all labeled cells, varied from 2–28%, and these were not different in morphology or size compared to those labeled from nBOR, in which the proportion of DGCs was much higher (84–93%). DGCs were also labeled after injections into the anterior dorsolateral thalamus. The proportion was small (2–3%), and these DGCs were smaller in size than those projecting to the nBOR and LM. No DGCs were labeled from an injection in the optic tectum. Based on an analysis of size, we suggest that different populations of retinal ganglion cells are involved in the projections to LM, nBOR, the optic tectum, and the anterior dorsolateral thalamus. J. Comp. Neurol. 522:3928–3942, 2014. © 2014 Wiley Periodicals, Inc.     

Mesencephalic and diencephalic afferent connections to the thalamic nucleus rotundus in the lizard,Psammodromus algirus

The present work is an analysis of the afferent projections to the thalamic nucleus rotundus in a lizard, both at the light- and electron-microscopic level, using biotinylated dextran amine (BDA) as a neuroanatomical tracer. This study has confirmed previously reported afferent projections to nucleus rotundus in reptiles and has also identified a number of new cellular aggregates projecting to this dorsal thalamic nucleus. After BDA injections into nucleus rotundus, retrogradely labelled neurons were observed consistently within the following neuronal groups in the midbrain and the diencephalon: (i) the stratum griseum centrale of the optic tectum; (ii) the nucleus subpretectalis in the pretectum; (iii) the nucleus ansa lenticularis posterior, the posterior nucleus of the ventral supraoptic commissure, and the posteroventral nucleus, in the dorsal thalamus and (iv) the lateral suprachiasmatic nucleus and part of the reticular complex in the ventral thalamus. Tectal axons entering nucleus rotundus were fine and varicose and formed exclusively asymmetric synaptic contacts, mainly on small dendritic profiles. Rotundal neurons had symmetric synapses made by large boutons probably of nontectal origin. After comparing our results with those in other reptiles, birds and mammals, we propose that the sauropsidian nucleus rotundus forms part of a visual tectofugal pathway that conveys mesencephalic visual information to the striatum and dorsal ventricular ridge, and is similar to the mammalian colliculo-posterior/intralaminar-striatoamygdaloid pathway, the function of which may be to participate in visually guided behaviour.     

Thalamocortical and thalamo-amygdaloid projections from the parvicellular division of the posteromedial ventral nucleus in the cat

Projections from the parvicellular division of the posteromedial ventral thalamic nucleus (VPMpc) of the cat were examined. After injection of horseradish peroxidase conjugated with wheat germ agglutinin (WGA-HRP) into the VPMpc, both anterogradely labeled axon terminals and retrogradely labeled neuronal cell bodies were found ipsilaterally in three discrete regions of the cerebral cortex, i.e., in the orbital cortex, caudoventral part of the infralimbic cortex, and medial part of the fundus of the posterior rhinal sulcus (perirhinal area); in the subcortical regions, anterogradely labeled axon terminals were seen ipsilaterally in the rostrodorsal part of the lateral amygdaloid nucleus. Neuronal connections between these VPMpc-recipient regions were further verified by injecting WGA-HRP into each of the three cortical and the lateral amygdaloid regions. After injection of WGA-HRP into each of the three cortical regions, labeled neuronal cell bodies and axon terminals were seen ipsilaterally in the VPMpc, especially in its medial part, and in the other two of the three VPMpc-recipient cortical regions. In the rostrodorsal part of the lateral amygdaloid nucleus, both axon terminals and neuronal cell bodies were labeled after WGA-HRP injection into the perirhinal area, and only axon terminals were labeled after WGA-HRP injection into the orbital cortex, but no labeling was observed after WGA-HRP injection into the infralimbic cortex. After injection of WGA-HRP into the rostrodorsal portion of the lateral amygdaloid nucleus, both axon terminals and neuronal cell bodies were labeled ipsilaterally in the perirhinal area and the ectorhinal area, and only neuronal cell bodies were labeled ipsilaterally in the VPMpc (especially in its medial part) and orbital cortical region; no labeling was observed in the infralimbic cortex. The present results indicate that the VPMpc of the cat is connected reciprocally with the orbital, infralimbic, and perirhinal cortical regions on the ipsilateral side, that the three VPMpc-recipient cortical regions are reciprocally connected with each other, that the VPMpc sends fibers ipsilaterally to the rostrodorsal part of the lateral amygdaloid nucleus, which may relay information from the VPMpc to the perirhinal cortical area, and that the VPMpc-recipient area in the lateral amygdaloid nucleus receives cortical fibers from the orbital and perirhinal cortical regions.     

Nucleus isthmi provides most tectal choline acetyltransferase in the frogRana pipiens

Up to 9 weeks following the removal of unilateral retinal input, choline acetyltransferase (ChAT) activity in the de-afferented tectal lobe is not significantly different from the intact tectal lobe. At 14 weeks, there is a 29% increase in the de-afferented side compared to the intact side. Following unilateral lesion of nucleus isthmi, ChAT activity in the tectal lobe ipsilateral to the lesion is approximately 30% of that measured in the contralateral lobe. Following bilateral n. isthmi lesion, ChAT activity in each tectal lobe is reduced by approximately 94% from intact tectal lobe controls. Thus, nucleus isthmi is the principal source of cholinergic input to the tectum.     

R-cadherin expression in the developing and adult zebrafish visual system.

Cell adhesion molecules in the cadherin family have been implicated in histogenesis and maintenance of cellular structure and function in several organs. Zebrafish have emerged as an important new developmental model, but only three zebrafish cadherin molecules have been identified to date (N-cadherin, paraxial protocadherin, and VN-cadherin). We began a systematic study to identify other zebrafish cadherins by screening zebrafish cDNA libraries using an antibody raised to the cytoplasmic domain of mouse E-cadherin. Here, we report a partial cDNA with extensive sequence homology to R-cadherin. Spatial and temporal expression of this putative zebrafish R-cadherin was examined in embryos and adults by Northern analysis, RNase protection, and in situ hybridization. R-cadherin message increased during embryogenesis up to 80 hours postfertilization (hpf) and persisted in adults. In the embryonic brain, R-cadherin was first expressed in groups of cells in the diencephalon and pretectum. In adult zebrafish brain, R-cadherin continued to be expressed in several specific regions including primary visual targets. In the retina, R-cadherin was first detected at about 33 hours postfertilization in the retinal ganglion cell layer and the inner part of the inner nuclear layer. Expression levels were highest during periods of axon outgrowth and synaptogenesis. Retrograde labeling of the optic nerve with 1,1'-dioctadecyl-3,3,3',3', tetramethylindocarbocyanine perchlorate (DiI) followed by in situ hybridization confirmed that a subset of retinal ganglion cells in the embryo expressed R-cadherin message. In the adult, R-cadherin expression continued in a subpopulation of retinal ganglion cells. These results suggest that R-cadherin-mediated adhesion plays a role in development and maintenance of neuronal connections in zebrafish visual system.