Identification and characterization of Escherichia coli DNA helicase II mutants that exhibit increased unwinding efficiency

Ganglion cells with intraretinal axon collaterals have been described in monkey (Usai et al., 1991), cat (Dacey, 1985), and turtle (Gardiner & Dacey, 1988) retina. Using intracellular injection of horseradish peroxidase and Neurobiotin in in vitro whole-mount preparations of human retina, we filled over 1000 ganglion cells, 19 of which had intraretinal axon collaterals and wide-field, spiny dendritic trees stratifying in the inner half of the inner plexiform layer. The axons were smooth and thin (approximately 2 microm) and gave off thin (<1 microm), bouton-studded terminal collaterals that extended vertically to terminate in the outer half of the inner plexiform layer. Terminal collaterals were typically 3-300 microm in length, though sometimes as long as 700 microm, and were present in clusters, or as single branched or unbranched varicose processes with round or somewhat flattened lobular terminal boutons 1-2 microm in diameter. Some cells had a single axon whereas other cells had a primary axon that gave rise to 2-4 axon branches. Axons were located either in the optic fiber layer or just beneath it in the ganglion cell layer, or near the border of the ganglion cell layer and the inner plexiform layer. This study shows that in the human retina, intraretinal axon collaterals are associated with a morphologically distinct ganglion cell type. The synaptic connections and functional role of these cells are not yet known. Since distinct ganglion cell types with intraretinal axon collaterals have also been found in monkey, cat, and turtle, this cell type may be common to all vertebrate retinas.     

Randomized retinal ganglion cell axon routing at the optic chiasm of GAP-43-deficient mice: association with midline recrossing and lack of normal ipsilateral axon turning

During mammalian development, retinal ganglion cell (RGC) axons from nasal retina cross the optic chiasm midline, whereas temporal retina axons do not and grow ipsilaterally, resulting in a projection of part of the visual world onto one side of the brain while the remaining part is represented on the opposite side. Previous studies have shown that RGC axons in GAP-43-deficient mice initially fail to grow from the optic chiasm to form optic tracts and are delayed temporarily in the midline region. Here we show that this delayed RGC axon exit from the chiasm is characterized by abnormal randomized axon routing into the ipsilateral and contralateral optic tracts, leading to duplicated representations of the visual world in both sides of the brain. Within the chiasm, individual contralaterally projecting axons grow in unusual semicircular trajectories, and the normal ipsilateral turning of ventral temporal axons is absent. These effects on both axon populations suggest that GAP-43 does not mediate pathfinding specifically for one or the other axon population but is more consistent with a model in which the initial pathfinding defect at the chiasm/tract transition zone leads to axons backing up into the chiasm, resulting in circular trajectories and eventual random axon exit into one or the other optic tract. Unusual RGC axon trajectories include chiasm midline recrossing similar to abnormal CNS midline recrossing in invertebrate "roundabout" mutants and Drosophila with altered calmodulin function. This resemblance and the fact that GAP-43 also has been proposed to regulate calmodulin availability raise the possibility that calmodulin function is involved in CNS midline axon guidance in both vertebrates and invertebrates.     

A quantitative analysis of cells in the ganglion cell layer of the chick retina

This study investigated the organization of cells in the ganglion cell layer (GCL) using Nissl staining, retrograde cell degeneration with axotomy of the optic nerve, and retrograde cell labeling by injections of horseradish peroxidase (HRP) into the optic nerve of chicks (posthatching day 1 and 8, P-1 and P-8). The total number of cells in the GCL was 6.1 x 10(6) (P-1) and 4.9 x 10(6) (P-8), and the cell density was 14,300 cells/mm2 (P-1) and 10,400 cells/ mm2 (P-8) on average. Two high-density areas, the central area (CA) and the dorsal area (DA), were observed in the central and dorsal retinas in both P-1 (22,000 cells/mm2 in CA, 19,000 cells/mm2 in DA) and P-8 chicks (19,000 cells/mm2 in CA, 12,800 cells/mm2 in DA). The cell densities in the temporal periphery (TP) and the nasal (NP) peripheral retinas were 7,800 cells/mm2 and 12,500 cells/mm2, respectively, in P-1 and 5,000 cells/ mm2 and 8,000 cells/mm2, respectively, in P-8 chicks. The cell density in the temporal periphery was 35% (P-8) lower than in the nasal periphery in both P-1 and P-8 chicks. Thirty percent (1.9 x 10(6) cells in P-1) of the total cells in the GCL were resistant to axotomy of the optic nerve. The distribution of the axotomy-resistant cells showed two high-density areas in the central and dorsal retinas, corresponding to the CA (5,800 cells/mm2) and the DA (3,200 cells/mm2). These cells also exhibited a center-peripheral increase (2,200 cells/mm2 in the TP) in P-1 chicks, but the high-density area was not found in the dorsal retina of P-8 chicks. From these data and the HRP study, the number of presumptive ganglion cells in P-8 chicks was estimated to be 4 x 10(6) (8,600 cells/mm2 on average), and the density in each area was 13,500 (CA), 10,200 (DA), and 4,300 (TP) cells/mm2. The peripheral/ center ratios of the density of ganglion cells were significantly different along the nasotemporal and dorsoventral axes. The density of ganglion cells decreased more rapidly toward the temporal periphery (TP/CA ratio: 0.47 in P-1 and 0.32 in P-8) than toward the nasal periphery (NP/CA ratio: 0.67 in P-1 and 0.52 in P-8). In contrast, there was no significant difference in the peripheral/center ratios between the dorsal retina (DP/CA ratio: 0.6 in P-1 and 0.56 in P-8) and ventral retina (VP/CA ratio: 0.58 in P-1 and 0.51 in P-8). A small peak in the density of the presumptive ganglion cells was detected in the dorsal retina of both P-1 chicks (10,800 cells/mm2) and P-8 chicks (10,200 cells/mm2). The HRP-labeled cells were small in the CA (M +/- SD: 35.7 +/- 9.1 microm2) and DA (40.0 +/- 11.3 microm2), and their sizes increased toward the periphery (63.4 +/- 29.7 microm2 in the TP) accompanied by a decrease in the cell density. However, the axotomy-resistant cells did not significantly increase in size toward the peripheral retina (12.2 +/- 2.2 microm2 in the CA, 15.2 +/- 3.2 microm2 in the DA, 15.1 +/- 3.8 microm2 in the TP). The characteristic distribution of ganglion cells could be related to visual behavior based upon the specialization of avian visual fields.     

Histomorphometry of the optic nerves of normal dogs and dogs with hereditary glaucoma

The beagle dog with hereditary primary open-angle glaucoma, unlike other animal models of human glaucoma, possesses a slowly progressive, sustained elevation of intraocular pressure. The effects of this insidious elevation in intraocular pressure on the axons of the optic nerves of three beagles at early stages of glaucoma and two beagles with advanced signs of glaucoma were compared to the optic nerves of four age-matched normal dogs. Plastic embedded optic nerve cross-sections (1 micron) 1 mm posterior to the lamina cribrosa were osmicated and stained with Toluidine Blue. Axons from 0.2 to > 2.0 microns in diameter were counted and measured in 16 cross-sectional regions of equal size within the whole optic nerve using a computerized image analysis system. The mean optic nerve axon diameters in the normal, early glaucomatous, and advanced glaucomatous dogs were 1.53, 1.25 and 1.13 microns respectively. The average total optic nerve axon count in the normal dogs was 148,303. Approximately 16% of the total axonal fibers were counted in each nerve. The counts of optic nerve axons 2.0 microns or greater in diameter were reduced by up to 60% in the central regions of the optic nerves of affected beagles. The large diameter axons of the peripheral optic nerve of the beagle dogs with glaucoma were more resistant to the elevated intraocular pressure. The counts of axons > 0.6 to 0.8 micron in diameter were significantly increased in glaucomatous beagles.     

Early axon outgrowth of retinal ganglion cells in the fetal rhesus macaque

Employing retinal explants and retrograde transport techniques, we studied the formation of the arcuate fascicles by examining the growth of the central retina, the emergence of the adult fiber layer pattern, and the projections of retinal ganglion cells in the central and peripheral retina. Sixty days prior to foveal pit formation, the distance from the incipient fovea to the optic disk was equal to the adult, even though the retinal area was only 8% of the adult. Arcuate fibers, at this age, were observed to avoid the incipient fovea, with no fascicles and few axons projecting over this region. A small population of 15.2% of the ganglion cells located within 2 mm of the incipient fovea possessed an axon with an aberrant trajectory that wound around and projected 50 to several hundred microns away from the optic disk, compared to only 3% at other retinal locations. The incidence of disorder decreased with increasing fetal age, establishing mature values in late fetal periods. These findings suggest that the area of the central retina does not increase after embryonic day 60 and that guidance factors are present that allow outgrowing ganglion cell axons to distinguish and avoid that portion of the retina that will become the fovea.     

Maturational gradients in the retina of the ferret

In the present set of studies, we have examined the site for the initiation of retinal maturation in the ferret. A variety of maturational features across the developing inner and outer retina were examined by using standard immunohistochemical, carbocyanine dye labelling, and Nissl-staining techniques, including 1) two indices of early differentiation of the first-born retinal ganglion cells, the presence of beta-tubulin and of neuron-specific enolase; 2) the receding distribution of chondroitin sulfate proteoglycans within the inner retina; 3) the distribution of the first ganglion cells to grow axons along the optic nerve; 4) the emergence of the inner plexiform layer; 5) the emergence of the outer plexiform layer and 6) the onset of synaptophysin immunoreactivity within it; 7) the differentiation of calbindin-immunoreactive horizontal cells; and 8) the cessation of proliferative activity at the ventricular surface. Although we were able to define distinct maturational gradients that are associated with many of these features of inner and outer retinal development (each considered in detail in this report), with dorsal retina maturing before ventral retina, and with peripheral retina maturing last, none showed a clear initiation in the region of the developing area centralis. Rather, maturation began in the peripapillary retina dorsal to the optic nerve head, which is consistent with previous studies on the topography of ganglion cell genesis in the ferret. These results make clear that the order of retinal maturation and the formation of the area centralis are not linked, at least not in the ferret.     

Fine structure and development of dorsal root ganglion neurons and Schwann cells in the newborn opossum Monodelphis domestica

The aim of these experiments was to determine the state of maturity of dorsal root ganglia and axons in opossums (Monodelphis domestica) at birth and to assess quantitatively changes that occur in early life. Counts made of dorsal root ganglion cells at cervical levels showed that the numbers were similar in newborn and adult animals, approximately 1,600 per ganglion. In cervical dorsal root ganglia of newborn animals, division of neuronal precursors cells had ceased. The number of axons in cervical dorsal roots was similar in newborn and adult animals (about 4,500). For each ganglion cell body, approximately three axons were counted in the dorsal root. At birth, dorsal roots contained several bundles about 30 microns in diameter consisting of small axons (0.05-2 microns in diameter). A few non-neural cells were identified as Schwann cell perikarya, each enclosing a number of neurites. Later, marked changes occurred in Schwann cells and in their relationship to axons in the roots. Thus, at 12 days, an increase occurred in the number of Schwann cells and fibroblasts, and the bundles had enlarged to about 80 microns with little increase in axon diameter (0.1-2 microns). By 18 days, the bundles were larger, and myelination had already started. At 23 days, the dorsal root contained more than 500 myelinated axons that could reach 5 microns in diameter. The adult dorsal root enclosed about 900 myelinated axons. Throughout this time, the relationship between the Schwann cells and axons changed. Together, these results indicate that the number of axons and cell bodies of sensory dorsal root ganglia in opossum do not show major changes after birth. In addition, these results set the stage for quantitative studies of regeneration of dorsal column fibers in injured neonatal opossum nervous system.     

Reevaluation of the growth-permissive substrate properties of goldfish optic nerve myelin and myelin proteins

To determine whether optic nerve myelin of goldfish carries mammalian-like neurite growth inhibitory proteins which can be neutralized by the antibody IN-1, myelin fractions of fish optic nerves were used as substrates for fish retinal ganglion cell axons and rat dorsal root ganglia (DRG). Axonal growth was monitored and compared with that of IN-1 treated preparations. Growth of fish retinal axons and rat DRG neurites was substantial on goldfish optic nerve myelin and no improvement was observed with IN-1. In contrast, rat CNS myelin allowed only poor growth, and number of axons and length of DRG neurites increased significantly with IN-1. In addition, proteins of fish optic nerve myelin and bovine CNS myelin were extracted, reconstituted in liposomes and applied to growth cones. When goldfish myelin proteins in liposomes were seeded onto growth cones, 77% of fish and 89% of rat DRG growth cones continued to elongate, and the proportion of elongating fish growth cones (80%) did not significantly change when liposomes were pretreated with IN-1. But 73% of fish and 93% of rat growth cones collapsed with liposomes containing proteins from bovine CNS myelin. Upon IN-1 treatment, only 24% of fish growth cones collapsed. Thus, axon growth in vitro indicates that goldfish optic nerves, which permit successful axon regeneration in vivo, lack mammalian-like neurite growth inhibitors which are neutralized by IN-1.     

Retinal ganglion cell axon progression from the optic chiasm to initiate optic tract development requires cell autonomous function of GAP-43

Pathfinding mechanisms underlying retinal ganglion cell (RGC) axon growth from the optic chiasm into the optic tract are unknown. Previous work has shown that mouse embryos deficient in GAP-43 have an enlarged optic chiasm within which RGC axons were reportedly stalled. Here we have found that the enlarged chiasm of GAP-43 null mouse embryos appears subsequent to a failure of the earliest RGC axons to progress laterally through the chiasm-tract transition zone to form the optic tract. Previous work has shown that ventral diencephalon CD44/stage-specific embryonic antigen (SSEA) neurons provide guidance information for RGC axons during chiasm formation. Here we found that in the chiasm-tract transition zone, axons of CD44/SSEA neurons precede RGC axons into the lateral diencephalic wall and like RGC axons also express GAP-43. However unlike RGC axons, CD44/SSEA axon trajectories are unaffected in GAP-43 null embryos, indicating that GAP-43-dependent guidance at this site is RGC axon specific or occurs only at specific developmental times. To determine whether the phenotype results from loss of GAP-43 in RGCs or in diencephalon components such as CD44/SSEA axons, wild-type, heterozygous, or homozygous GAP-43 null donor retinal tissues were grafted onto host diencephalons of all three genotypes, and graft axon growth into the optic tract region was assessed. Results show that optic tract development requires cell autonomous GAP-43 function in RGC axons and not in cellular elements of the ventral diencephalon or transition zone.     

Different cell surface areas of polarized radial glia having opposite effects on axonal outgrowth

During neuronal development neurites are likely to be specifically guided to their targets. Within the chicken retina, ganglion cell axons are extended exclusively into the optic fibre layer, but not into the outer retina. We investigated, whether radial glial cells having endfeet at the optic fibre layer and somata in the outer retina, might be involved in neurite guidance. In order to analyse distinct cell surface areas, endfeet and somata of these glial cells were purified. Glial endfeet were isolated from flat mounted retina by a specific detachment procedure. Glial somata were purified by negative selection using a monoclonal antibody/complement mediated cytolysis of all non-glial cells. Retinal tissue strips were explanted either onto pure glial endfeet or onto glial somata. As revealed by scanning and fluorescence microscopy, essentially no ganglion cell axons were evident on glial somata, whereas axonal outgrowth was abundant on glial endfeet. However, when glial somata were heat treated and employed thereafter as the substratum, axon extension was significantly increased. Time-lapse video recording studies indicated that purified cell membranes of glial somata but not of endfeet induced collapse of growth cones. Collapsing activity was destroyed by heat treatment of glial membranes. The collapsing activity of retinal glia was found to be specific for retinal ganglion cell neurites, because growth cones from dorsal root ganglia remained unaffected. Employing four different kinase inhibitors revealed that the investigated protein kinase types were unlikely to be involved in the collapse reaction. The data show for the first time that radial glial cells are functionally polarized having permissive endfeet and inhibitory somata with regard to outgrowing axons. This finding underscores the pivotal role of radial glia in structuring developing nervous systems.     

Neurotrophins and their receptors in the tench retina during optic nerve regeneration

To understand the role of neurotrophins in the visual system, we investigated the distribution of both neurotrophins and their receptors within the retina of a fish that has the capacity to spontaneously regenerate its optic nerve axons after lesion. Intact retinas and retinas from tench, whose optic nerve had been crushed, were analyzed by immunohistochemistry and in situ hybridization. Trk receptors were mainly immunolocalized in cells of the inner nuclear and ganglion cell layers, a distribution coincident with that of their mRNAs. Nerve growth factor (NGF) immunoreactivity was detected exclusively in Müller cell processes, and brain-derived neurotrophic factor (BDNF) was found in both neuronal bodies and Müller cell processes. Neurotrophin-3 (NT-3) was detected in most of the cell nuclei, and neurotrophin-4/5 (NT-4/5) was localized in fibers and in a few cells in the inner retina. An increase in both TrkA protein and mRNA was detected during axonal regeneration within the retinal ganglion cell layer, reaching a maximum 30 days postcrush and returning to normal levels by day 90, when optic nerve regeneration is almost completed in this fish. None of the other neurotrophins and receptors showed appreciable changes. The heterogeneous distribution patterns of neurotrophins and their receptors in fish retina, their differences from the distribution observed in other species, and the TrkA changes after optic nerve crush suggest an important role for these molecules in the normal physiology of the fish retina and during the regeneration process.     

Inhibition of protein tyrosine kinases impairs axon extension in the embryonic optic tract

The role of protein tyrosine kinase (PTK) activity in the development of the retinal projection was examined in vivo by applying inhibitors of cytoplasmic PTKs, herbimycin A and lavendustin A, to intact brain preparations of Xenopus embryos. The inhibitors were present during the period when retinal ganglion cell axons first navigate through the optic tract to reach their target, the optic tectum. A majority of inhibitor-treated retinal axons stalled at the beginning of the optic tract, leading to an 80% reduction in projection length at the highest doses. All inhibitor-treated axons that did extend into the optic tract exhibited normal pathfinding behavior. Tyrosine kinase assays of inhibitor-treated brains demonstrated that at doses at which retinal axon extension was severely impaired, PTK activity, including that of src family proteins, was reduced by 50-60%. Consistent with the in vivo findings, PTK inhibitors reduced neurite outgrowth from cultured retinal neurons by 70-80%. This contrasts with the strong enhancement of outgrowth induced by the same inhibitors in cultured chick ciliary ganglion neurons and suggests that the mediation of outgrowth by PTK activity may vary in different neuronal types. Inhibitor-treated growth cones cultured on laminin were larger than normal, suggesting that tyrosine phosphorylation can modulate growth cone-substrate adhesive interactions. Our results in vivo and in vitro provide complementary evidence that retinal axon outgrowth is inhibited by pharmacological blockers of PTK activity and indicate that inhibitor-sensitive PTKs normally play a role in promoting retinal neurite extension.     

Seven retinal specializations in the tubular eye of the deep-sea pearleye, Scopelarchus michaelsarsi: a case study in visual optimization

The deep-sea pearleye, Scopelarchus michaelsarsi (Scopelarchidae) is a mesopelagic teleost with asymmetric or tubular eyes. The main retina subtends a large dorsal binocular field, while the accessory retina subtends a restricted monocular field of lateral visual space. Ocular specializations to increase the lateral visual field include an oblique pupil and a corneal lens pad. A detailed morphological and topographic study of the photoreceptors and retinal ganglion cells reveals seven specializations: a centronasal region of the main retina with ungrouped rod-like photoreceptors overlying a retinal tapetum; a region of high ganglion cell density (area centralis of 56.1 x 10(3) cells per mm2) in the centrolateral region of the main retina; a centrotemporal region of the main retina with grouped rod-like photoreceptors; a region (area giganto cellularis) of large (32.2+/-5.6 microm2), alpha-like ganglion cells arranged in a regular array (nearest neighbour distance 53.5+/-9.3 microm with a conformity ratio of 5.8) in the temporal main retina; an accessory retina with grouped rod-like photoreceptors; a nasotemporal band of a mixture of rod- and cone-like photoreceptors restricted to the ventral accessory retina; and a retinal diverticulum comprised of a ventral region of differentiated accessory retina located medial to the optic nerve head. Retrograde labelling from the optic nerve with DiI shows that approximately 14% of the cells in the ganglion cell layer of the main retina are displaced amacrine cells at 1.5 mm eccentricity. Cryosectioning of the tubular eye confirms Matthiessen's ratio (2.59), and calculations of the spatial resolving power suggests that the function of the area centralis (7.4 cycles per degree/8.1 minutes of arc) and the cohort of temporal alpha-like ganglion cells (0.85 cycles per degree/70.6 minutes of arc) in the main retina may be different. Low summation ratios in these various retinal zones suggests that each zone may mediate distinct visual tasks in a certain region of the visual field by optimizing sensitivity and/or resolving power.     

Effects of sciatic nerve crush on the L7 spinal roots and dorsal root ganglia in kittens

The number and size distribution of axons and neurons were examined in the L7 spinal roots and ganglia of kittens 14 to 220 days after early postnatal sciatic nerve crush. The results show that motoraxons in the ventral root as well as axons and perikarya of sensory neurons in the dorsal root remained growth-retarded throughout the examined period. This was most evident in the dorsal root. Both ventral and dorsal roots showed some loss of myelinated axons, but this was only half that previously observed after sciatic nerve resection. Whereas in the dorsal roots and dorsal root ganglia the loss seemed to be nonselective with respect to size, axons in the gamma range were primarily affected in the ventral roots. In the dorsal roots the proportion of unmyelinated axons was comparable with controls but in the ventral roots it was somewhat elevated. In most cases the loss of dorsal root ganglion neurons was relatively greater than the decrease of dorsal root axons.     

Intramembrane structure of the sensory axon terminals in bullfrog muscle spindles

Much physiologic and morphologic research has been done into the sensory mechanism of the frog muscle spindle. However, no freeze-fracture study has described in detail the shape and intramembrane structure of the nonmyelinated sensory axon terminals of the frog muscle spindle. In this study, muscle spindles were isolated from the red part of bullfrog semitendinous muscles. Chemically fixed spindles were subjected to freeze fracturing. The sensory axon endings were reconstructed, and the size and density of intramembrane particles (IMPs) were measured along the sensory nerve endings. The axon terminals had four distinctive parts: parent trunks (>0.5 microm in diameter), primary branches (0.15-0.5 microm), terminal branches (<0.1 pm), and varicosities (0.02-0.5 microm). IMPs ranged from 5 nm to 21 nm in diameter and were present in the intramembrane space of the plasma membrane all throughout the nonmyelinated sensory nerve endings. Mean IMP sizes in the protoplasmic face (PF) and the external face (EF), respectively, were 8.1 nm and 8.4 nm in the parent trunks, 8.8 nm and 8.8 nm in the primary branches, 9.4 nm and 9.0 nm in the varicosities, and 8.7 nm and 8.7 nm in the terminal branches. Mean IMP size in the PF was smallest in the parent trunk and largest in the varicosity. Mean IMP densities (numbers of IMPs per microm2) in the PF and the EF, respectively, were 2,500 and 700 in the parent trunks, 2,200 and 500 in the primary branches, 1,700 and 400 in the varicosities, and 1,000 and 300 in the terminal branches. Density decreased with the tapering of the axon terminal, with IMPs distributed evenly in the PF and the EF. The characteristic intramembrane structure of sensory nerve endings is discussed.     

Leech photoreceptors project their galectin-containing processes into the optic neuropils where they contact AP cells

We characterized a subset of leech sensory afferents, the photoreceptors, in terms of their molecular composition, anatomical distribution, and candidate postsynaptic partners. For reagents, we used an antiserum generated against purified LL35, a 35 kD leech lactose-binding protein (galectin); monoclonal antibody (mAb) Lan3-2, which is specific for a mannose-containing epitope common to the full set of sensory afferents; and dye injections. Photoreceptors differ from other types of sensory afferents by their abundant expression of galectin. However, photoreceptors share in common with other sensory modalities the mannose-containing epitope recognized by mAb Lan3-2. Photoreceptors from a given segment project their axons directly into the CNS ganglion innervating the same segment. They assemble in a target region, the optic neuropil, which is separate from the target regions of other sensory modalities. They also extend their axons as an optic tract into the connective to innervate optic neuropils of other CNS ganglia, thereby providing extensive intersegmental innervation for the 33 CNS ganglia comprising the leech nerve cord. Because of its intimate contact with the optic neuropil, a central neuron, the AP effector cell, is a strong candidate second order visual neuron. In confocal images, the AP cell projects its primary axon for about 100 microns alongside the optic neuropil. In electron micrographs, spines emanating from the axon of the AP cell make contact with vesicle laden nerve terminals of photoreceptors. Leech photoreceptors and their second order visual neurons represent a simple visual system for studying the mechanisms of axonal targeting.     

Amyloid precursor protein is synthesized by retinal ganglion cells, rapidly transported to the optic nerve plasma membrane and nerve terminals, and metabolized

We have investigated the synthesis, axonal transport, and processing of the beta-amyloid precursor protein (APP) in in vivo rabbit retinal ganglion cells. These CNS neurons connect the retina to the brain via axons that comprise the optic nerve. APP is synthesized in retinal ganglion cells and is rapidly transported into the optic nerve in small transport vesicles. It is then transferred to the axonal plasma membrane, as well as to the nerve terminals and metabolized with a t1/2 of less than 5 h. A significant accumulation of C-terminal amyloidogenic or nonamyloidogenic fragments is seen in the optic nerve 5 h after [35S]-methionine, [35S]cysteine injection, which disappears by 24 h. The major molecular mass species of APP in the optic nerve is approximately 110 kDa, and is an APP isoform that does not contain a Kunitz protease inhibitor domain. Higher molecular mass species containing this sequence are seen mostly in the retina. A protease(s) that can potentially cleave APP to generate an amyloidogenic fragment is present in the same optic nerve membrane compartment as APP.     

Glial cells are increased proportionally in transgenic optic nerves with increased numbers of axons

To study how an increase in axon number influences the number of glial cells in the mammalian optic nerve, we have analyzed a previously described transgenic mouse that expresses the human bcl-2 gene from a neuron-specific enolase promoter. In these mice, the normal postnatal loss of retinal ganglion cell axons is greatly decreased and, as a consequence, the number of axons in the optic nerve is increased by approximately 80% compared with wild-type mice. Remarkably, the numbers of oligodendrocytes, astrocytes, and microglial cells are all increased proportionally in the transgenic optic nerve. The increase in oligodendrocytes apparently results from both a decrease in normal oligodendrocyte death and an increase in oligodendrocyte precursor cell proliferation, whereas the increase in astrocytes apparently results from an increase in the proliferation of astrocyte lineage cells. Unexpectedly, the transgene is expressed in oligodendrocytes and astrocytes, but this does not seem to be responsible for the increased numbers of these cells. These findings indicate that developing neurons and glial cells can interact to adjust glial cell numbers appropriately when neuronal numbers are increased. We also show that the expression of the bcl-2 transgene in retinal ganglion cells protects the cell body from programmed cell death when the axon is cut, but it does not protect the isolated axon from Wallerian degeneration, even though the transgene-encoded protein is present in the axon.     

Increased axon number in the anterior commissure of mice lacking a corpus callosum

Relatively few behavioral deficits are apparent in subjects with hereditary absence of the corpus callosum (CC). The anterior commissure (AC) has been suggested to provide an extracallosal route for the transfer of interhemispheric information in subjects with this congenital defect. Anterior commissure size, axon number, axon diameter, and neuronal distribution were compared between normal mice and those with complete CC absence. No difference in midsagittal AC area was found between normals and acallosals, nor were differences found in the numbers or diameters of myelinated axons. However, axon counts indicated an 17% increase or about 70,000 more unmyelinated axons in the AC of acallosal mice, and the mean diameter of unmyelinated axons was slightly less than in normal mice (0.24 vs 0.26 microm). This decrease in axon diameter enabled more axons to pass through the AC without increasing its midsagittal area. The topographical distribution of neurons sending axons through the AC, assessed with lipophilic dyes, was qualitatively similar for almost all the known regions of origin of the anterior commissure in normal and acallosal mice. There was a pronounced deficit of AC cells in the anterior piriform cortex of BALB/c mice, but this occurred whether or not the mouse suffered absent CC. Although the increase in AC axon number is far smaller than the number of CC axons that fail to reach the opposite hemisphere, the higher number of axons present in the AC of acallosal mice may contribute to the functional compensation for the loss of the CC.     

Expression of vimentin and GFAP and development of the retina in the trout

The glial cell development was studied during the edification of the retina and the optic tract, in a teleost, the rainbow trout. The intermediate filament proteins, vimentin and glial fibrillary acidic protein (GFAP) were visualized by an indirect immunohistochemical method. Results show that both vimentin and GFAP are early expressed in the developing retina and, particularly in the Müller cells, a coexpression of vimentin and GFAP is observed from embryonic to adult stages. The ganglion cell layer and the optic fiber layer both exhibit GFAP-positive structures. The deep staining for GFAP is also seen in the optic nerve and induces us to credit astrocyte-like cells with a leading role in the pattern formation of this tract.