Contactin1a expression is associated with oligodendrocyte differentiation and axonal regeneration in the central nervous system of zebrafish

Contactin1a (Cntn1a) is a zebrafish homolog of contactin1 (F3/F11/contactin) in mammals, an immunoglobulin superfamily recognition molecule of neurons and oligodendrocytes. We describe conspicuous Cntn1a mRNA expression in oligodendrocytes in the developing optic pathway of zebrafish. In adults, this expression is only retained in glial cells in the intraretinal optic fiber layer, which contains 'loose' myelin. After optic nerve lesion, oligodendrocytes re-express Cntn1a mRNA independently of the presence of regenerating axons and retinal ganglion cells upregulate Cntn1a expression to levels that are significantly higher than those during development. After spinal cord lesion, expression of Cntn1a mRNA is similarly increased in axotomized brainstem neurons and white matter glial cells in the spinal cord. In addition, reduced mRNA expression in the trigeminal/anterior lateral line ganglion in erbb3-deficient mutant larvae implies Cntn1a in Schwann cell differentiation. These complex regulation patterns suggest roles for Cntn1a in myelinating cells and neurons particularly in successful CNS regeneration.     

Embryonic Optic Nerve Tissue Fails to Support Neurite Outgrowth by Central and Peripheral Neurons In Vitro

The failure of axon regeneration in the injured mammalian central nervous system has been ascribed, in part, to the inhibitory effects of myelin proteins. To investigate the influence of myelination on neurite growth and regeneration by both central nervous system and peripheral nervous system neurons, isolated rat neonatal retinal ganglion cells and adult and neonatal dorsal root ganglion neurons were cultured on cryostat sections of both immature unmyelinated and mature fully myelinated adult rat optic nerve. In agreement with earlier studies using neonatal peripheral neurons, the adult optic nerve failed to support neurite outgrowth from any of the neurons tested. A new finding was that tissue sections from unmyelinated optic nerve (aged embryonic days 18 and 20, and postnatal days 1–3), also failed to support the growth of neurites from neonatal retinal ganglion cells and both neonatal and adult dorsal root ganglion neurons. Neonatal retinal ganglion cells also failed to extend neurites on sections of pre-degenerated sciatic nerve, a tissue shown in our previous work to be a good substratum for supporting neurite growth for both neonatal and adult DRG neurons. These results suggest that cells in the immature optic nerve either express widely acting axon growth inhibitory molecules unrelated to previously described myelin proteins, or do not synthesize appropriate axon growth promoting molecules. They also reveal that, for axon regeneration, central nervous system and peripheral sensory neurons require distinct substratum interactions.     

Very Slow Retrograde and Wallerian Degeneration in the CNS of C57BL/Ola Mice

Wallerian degeneration following peripheral nerve transection in C57BL/Ola mice is very slow in comparison to other strains of mice. We show that following optic nerve transection, the axons of retinal ganglion cells in C57BL/Ola mice undergo very slow Wallerian degeneration and that retrograde degeneration of the ganglion cell bodies is much slower than in other strains of mice. The results suggest that the gene product affecting Wallerian degeneration in the peripheral nervous system (PNS) also confers a greater resistance to degeneration on central nervous system (CNS) neurons.     

Tenascin-R inhibits regrowth of optic fibers in vitro and persists in the optic nerve of mice after injury

Tenascin-R, an extracellular matrix constituent expressed by oligodendrocytes and some neuronal cell types, may contribute to the inhibition of axonal regeneration in the adult central nervous system. Here we show that outgrowth of embryonic and adult retinal ganglion cell axons from mouse retinal explants is significantly reduced on homogeneous substrates of tenascin-R or a bacterially expressed tenascin-R fragment comprising the epidermal growth factor-like repeats (EGF-L). When both molecules are presented as a sharp substrate border, regrowing adult axons do not cross into the tenascin-R or EGF-L containing territory. All in vitro experiments were done in the presence of laminin, which strongly promotes growth of embryonic and adult retinal axons, suggesting that tenascin-R and EGF-L actively inhibit axonal growth. Contrary to the disappearance of tenascin-R from the regenerating optic nerve of salamanders (Becker et al., J Neurosci 19:813-827, 1999), the molecule remains present in the lesioned optic nerve of adult mice at levels similar to those in unlesioned control nerves for at least 63 days post-lesion (the latest time point investigated), as shown by immunoblot analysis and immunohistochemistry. In situ hybridization analysis revealed an increase in the number of cells expressing tenascin-R mRNA in the lesioned nerve. We conclude that, regardless of the developmental stage, growth of retinal ganglion cell axons is inhibited by tenascin-R and we suggest that the continued expression of the protein after an optic nerve crush may contribute to the failure of adult retinal ganglion cells to regenerate their axons in vivo.     

Fetal tectal transplants in the cortex of adult rats become innervated both by retinal ganglion cell axons regenerating through peripheral nerve grafts and by cortical neurons

The present work elucidates the connectivity of adult retinal ganglion cell axons regenerating through grafted peripheral nerve segments with co-grafted immature brain target cells. The optic nerve of rats was transected intraorbitally and its segment distal to the transection was replaced by a 3 cm length of peroneus communis graft, that is known to permit regeneration of a certain proportion of the severed axonal population. Five weeks after optic nerve transection and peripheral nerve transplantation the regenerating optic tract axons were guided into rat fetal mesencephalic co-grafts (E14-16) placed in superficial cavities prepared in the occipital cortex. The rationale of the experimental setup was based on the fact that regrowth of retinal axons started at the 6th day after transection, whereas the fastest-growing axons reached the distal end of the transplanted peripheral nerve 4 weeks later growing with a velocity of about 1.33 mm/day. Therefore, grafting the fetal superior colliculus at the time axons arrive distally resulted in ingrowth of several hundreds of retinal axons into this immature, retinoreceptive brain tissue. Retinal axons which penetrated the fetal grafts contacted tectal neurons and GFAP-immunoreactive glia and formed typical retinocollicular axonal arbors as detected by anterograde labeling with RITC from the retina. In addition, sprouting fibers from the adjacent adult cortical neurons penetrated frequently the fetal transplants. By 'bridging' lesions with peripheral nerve pieces and providing immature neurons as targets for growing neurites, this transplantation model is suitable for investigations on whether regenerating adult neurites are capable of reforming connections. The co-transplantation technique may serve as a tool for understanding whether interrupted circuitries in the central nervous system can be functionally restored over long distances by the use of peripheral nerve grafts and immature nervous system tissue.     

Effects of low level laser treatment on the survival of axotomized retinal ganglion cells in adult Hamsters

Injury to axons close to the neuronal bodies in the mammalian central nervous system causes a large proportion of parenting neurons to degenerate. It is known that optic nerve transection close to the eye in rodents leads to a loss of about half of retinal ganglion cells in 1 week and about 90% in 2 weeks. Using low level laser treatment in the present study, we demonstrated that treatment with helium-neon(660 nm) laser with 15 m W power could delay retinal ganglion cell death after optic nerve axotomy in adult hamsters. The effect was most apparent in the first week with a short period of treatment time(5 minutes) in which 65–66% of retinal ganglion cells survived the optic nerve axotomy whereas 45–47% of retinal ganglion cells did so in optic nerve axotomy controls. We also found that single dose and early commencement of laser irradiation were important in protecting retinal ganglion cells following optic nerve axotomy. These findings thus convincingly show that appropriate laser treatment may be neuroprotective to retinal ganglion cells.     

Up-regulation of cadherin-2 and cadherin-4 in regenerating visual structures of adult zebrafish

Cadherins are homophilic cell adhesion molecules that control development of a variety of tissues and maintenance of adult structures. In this study, we examined expression of zebrafish cadherin-2 (Cdh2, N-cadherin) and cadherin-4 (Cdh4, R-cadherin) in the visual system of adult zebrafish after eye or optic nerve lesions using immunocytochemistry and immunoblotting. Both Cdh2 and Cdh4 immunoreactivities were specifically up-regulated in regenerating retina and/or the optic pathway. Furthermore, temporal expression patterns of these two cadherins were distinct during the regeneration of the injured tissues. Cadherins have been shown to regulate axonal outgrowth in the developing nervous system, but this is the first report, to our knowledge, of increased cadherin expression associated with axonal regeneration in the vertebrate central nervous system. Our results suggest that both Cdh2 and Cdh4 may be important for regeneration of injured retinal ganglion cell axons.     

Effect of different optic nerve lesions on retinal ganglion cell death in the frog Rana pipiens

Following optic nerve crush in various species of frog, a proportion of the retinal ganglion cells re-establishes functional contact with the optic tectum. However, as much as 50% of the retinal ganglion cells die during this process. The determinants of an individual ganglion cell's fate have not been established. In this study of Rana pipiens, cell survival after optic nerve crush was compared with that after nerve cut followed by stump separation, a procedure that considerably delayed entry of optic axons to the brain. It was also ascertained, in the case of delayed ingrowth, whether application of nerve growth factor immediately after lesion influenced the cell death process. This study confirmed that retinal ganglion cell death is a relatively late event in regeneration, because in several animals where anterograde HRP labeling demonstrated regenerating axons within the tectum, no cell death had occurred. There was no statistically significant difference in cell death at 75 days after lesion between animals receiving nerve crush and those receiving nerve cut with stump separation, even though most crush animals had regenerated a complete visual projection, whereas most nerve cut animals had not. The application of NGF did not influence the level of cell death at 75 days after lesion. These results suggest that contact of optic axons with the optic tract or tectum is not necessary for retinal ganglion cell death to occur. However, this does not necessarily mean that contact with the brain is not involved with cell death during regeneration following nerve crush because it is possible that the mechanisms of cell death are different when axons are prevented from regenerating. Further investigations are therefore required to establish the reasons for this cell death.     

Slow transport rates of cytoskeletal proteins change during regeneration of axotomized retinal neurons in adult rats

To investigate cytoskeletal changes associated with axonal regrowth from damaged nerve cells in the mammalian CNS, we examined the slow transport of axonal proteins during the regeneration of adult rat retinal ganglion cell (RGC) axons. Although normally such RGC axons do not regrow after injury in the CNS, they can extend several centimeters when their nonneuronal environment is changed by replacing the optic nerve (ON) with a grafted segment of peripheral nerve (PN). Proteins transported in axons of RGCs from intact control and PN-grafted animals were labeled by an intraocular injection of 35S-methionine and examined 4-60 days later by SDS PAGE. During RGC regeneration into PN grafts, the transport rate of tubulin and neurofilament increased twofold, whereas that of actin decreased to nearly one third of its normal rate. Thus, in these regenerating RGC axons, all three major cytoskeletal proteins were largely transported within a single rate component rather than in the two separate components (SCa and SCb) normally observed in the intact ON. Furthermore, the 200 kDa neurofilament protein (NF-H) was persistently detected in Western blots during periods of active regeneration, a finding that contrasts with the late appearance of the NF-H during the developmental growth of retinal axons. The changes in slow transport observed during RGC regeneration in adult rats may reflect growth-associated responses of mature CNS neurons during periods of active axonal extension.     

Autoimmune T cells retard the loss of function in injured rat optic nerves

We recently demonstrated that autoimmune T cells protect neurons from secondary degeneration after central nervous system (CNS) axotomy in rats. Here we show, using both morphological and electrophysiological analyses, that the neuroprotection is long-lasting and is manifested functionally. After partial crush injury of the rat optic nerve, systemic injection of autoimmune T cells specific to myelin basic protein significantly diminished the loss of retinal ganglion cells and conducting axons, and significantly retarded the loss of the visual response evoked by light stimulation. These results support our challenge to the traditional concept of autoimmunity as always harmful, and suggest that in certain situations T cell autoimmunity may actually be beneficial. It might be possible to employ T cell intervention to slow down functional loss in the injured CNS.     

Presence of growth inhibitors in fish optic nerve myelin: Postinjury changes

This study shows that the fish optic nerve, which is able to regenerate after injury, contains myelin-associated growth inhibitors similar to the growth inhibitors present in mammalian central nervous system (CNS) myelin. The ability of nerves to regenerate was previously correlated with the ability of sections from these nerves to support neuronal attachment to and axonal growth in vitro. Thus neuroblastoma cells or embryonic neurons became attached to and grew axons on sections of rat sciatic nerve or fish optic nerve, which are spontaneously regenerating systems, but not on sections of rat optic nerve, a nongenerating system. Failure of the latter to support axonal growth has been attributed, at least in part, to growth inhibitors. Recently it was shown that adult neurons, which differ in their growth requirement from embryonic neurons, are unable to extend neurites on sections of normal sciatic nerve but are able to extend neurites on sections of sciatic nerve that was injured prior to its excision. We found a similar in the fish optic nerve, i.e. that the nerve is normally not permissive to growth of adult retinal axons but becomes growth permissive after injury. The nonpermissiveness of the normal fish optic nerve was found to correlate with the presence of myelin-associated growth-inhibitory molecules. This inhibitory activity of fish myelin was neutralized by IN-1 antibodies, known to neutralize rat myelin growth inhibitors apparently similar or even identical to those of rat, but possibly present in lower amounts than in the rat. Results are discussed with respect to the possibility that fish optic nerve, like the rat sciatic regeneration of adult neurons. Such changes might include elimination or neutralization of growth inhibitors. © 1994 Wiley-Liss, Inc.     

Chiasmatic Specificity in the Regenerating Mammalian Optic Nerve

The mammalian central nervous system is capable of regenerating; however, there is no evidence that the regenerating axons can navigate along their normal pathways and reestablish topographically organized projections: essential for functional return of vision. Here retinal ganglion cells in the opossumMonodelphiswere birthdated with tritiated thymidine on the sixth postnatal day (P6), before being lesioned in the temporal retina at P8. Retrograde tracing with horseradish peroxidase injected into the ipsilateral optic tract at P24 showed that the temporal crescent had reformed behind the retinal lesion. By comparisons of cell and thymidine counts from lesioned and control regions of retina, it was estimated that about 40% of the normal number of ganglion cells are able to regenerate into the ipsilateral optic tract following a lesion in the temporal retina at P8. A clear line of decussation (separation of ipsilateral and contralateral projections) reformed in the lesioned temporal retina and regenerating ganglion cells labeled with DiI were turned at appropriate points on passing through the optic chiasm. This is evidence of chiasmatic specificity with regard to lesioned retinal ganglion cells regenerating into the ipsilateral optic tract.     

PTEN knockdown with the Y444F mutant AAV2 vector promotes axonal regeneration in the adult optic nerve

The lack of axonal regeneration is the major cause of vision loss after optic nerve injury in adult mammals. Activating the PI3 K/AKT/m TOR signaling pathway has been shown to enhance the intrinsic growth capacity of neurons and to facilitate axonal regeneration in the central nervous system after injury. The deletion of the m TOR negative regulator phosphatase and tensin homolog(PTEN) enhances regeneration of adult corticospinal neurons and ganglion cells. In the present study, we used a tyrosine-mutated(Y444 F) AAV2 vector to efficiently express a short hairpin RNA(sh RNA) for silencing PTEN expression in retinal ganglion cells. We evaluated cell survival and axonal regeneration in a rat model of optic nerve axotomy. The rats received an intravitreal injection of wildtype AAV2 or Y444 F mutant AAV2(both carrying sh RNA to PTEN) 4 weeks before optic nerve axotomy. Compared with the wildtype AAV2 vector, the Y444 F mutant AAV2 vector enhanced retinal ganglia cell survival and stimulated axonal regeneration to a greater extent 6 weeks after axotomy. Moreover, post-axotomy injection of the Y444 F AAV2 vector expressing the sh RNA to PTEN rescued ~19% of retinal ganglion cells and induced axons to regenerate near to the optic chiasm. Taken together, our results demonstrate that PTEN knockdown with the Y444 F AAV2 vector promotes retinal ganglion cell survival and stimulates long-distance axonal regeneration after optic nerve axotomy. Therefore, the Y444 F AAV2 vector might be a promising gene therapy tool for treating optic nerve injury.     

RNA content of normal and axotomized retinal ganglion cells of rat and goldfish

The responses of rat and goldfish retinal ganglion cells to axotomy were examined by a quantitative cytochemical method for RNA and by morphometric measurement 1-60 (rat) and 3-90 (goldfish) days after interruption of one optic nerve or tract intracranially. Unoperated control animals were studied also. The RNA content of axotomized neurons of rat fell 7-60 days postoperatively. Additionally, atrophy of the axotomized somas occurred. Over time, neuronal atrophy approximately paralleled the loss of RNA, and mean cell area and RNA content were reduced by about 25% 60 days after axotomy. Incorporation of 3H-uridine by axotomized neurons declined also. Axotomized retinal ganglion cells of goldfish behaved differently from those of the rat and showed increases in RNA content, most conspicuously 14-60 days postoperatively. Enlargement of axotomized fish neurons occurred but was less proportionately than concomitant increases in RNA content. The nonaxotomized ganglion cells of goldfish displayed statistically significant increases in size and RNA content 14-49 days after unilateral optic nerve or tract lesions. In contrast, alterations in rat retinal ganglion cells contralateral to interruption of one optic nerve were of limited and questionable significance. The contrasting reactions to axotomy by the retinal ganglion cells of these two vertebrates, one of which regenerates optic axons and one of which does not, may support the proposition that the somal response to axon injury has an important bearing upon the success or failure of CNS regeneration.(ABSTRACT TRUNCATED AT 250 WORDS)     

Molecular characterization of E587 antigen: An axonal recognition molecule expressed in the goldfish central nervous system

The E587 antigen (Ag) is a 200-Kd membrane glycoprotein originally identified by a monoclonal antibody on new and regenerating retinal ganglion cell axons in the adult goldfish. We report the isolation of cDNAs encoding the E587 Ag and identify it as a member of the L1 family of cell adhesion molecules (CAMs). The predicted amino acid sequence of E587 Ag shows an approximately equal identity (40%) to mouse L1, chick neuron-glia CAM, and chick neuron-glia-related CAM. Although the overall similarity is low, there is a high conservation of structural domains and specific sequence motifs. Wholemount in situ hybridizations were performed on goldfish between 34 hours and 3 days postfertilization (pf). A dramatic increase in E587 Ag mRNA was observed between 34 and 48 hours pf. The expression of E587 Ag mRNA in neurons shortly precedes axonogenesis. A marked decrease in expression occurs by 3 days pf, when the axonal scaffold has already been established. Wholemount immunohistochemistry on embryos demonstrates expression of E587 Ag on all major tracts. E587 Ag is absent from mature retinal ganglion cell axons, but its expression is induced by optic nerve transection. A corresponding induction of E587 Ag mRNA in retinal ganglion cells is shown by in situ hybridization. Furthermore, E587 Ag mRNA was detected in the optic nerve, which suggests that nonneuronal cells also express this molecule. E587 Ag was previously shown to promote retinal axon fasciculation and outgrowth in young fish and to mediate axon-glial interactions in vitro. The expression pattern and developmental regulation of E587 Ag in the central nervous system, its reexpression in retinal ganglion cells following optic nerve transection, and its relation to the L1 family indicate that E587 Ag functions as a cell recognition molecule important during axonal growth and regeneration. J. Comp. Neurol. 377:286–297, 1997. © 1997 Wiley-Liss, Inc.     

Nogo-A expression in the intact and injured nervous system

The expression of Nogo-A mRNA and protein in the nervous system of adult rats and cultured neurons was studied by in situ hybridisation and immunohistochemistry. Nogo-A mRNA was expressed by many cells in unoperated animals, including spinal motor, DRG, and sympathetic neurons, retinal ganglion cells, and neocortical, hippocampal, and Purkinje neurons. Nogo-A protein was strongly expressed by presumptive oligodendrocytes, but not by NG2+glia and was abundant in motor, DRG, and sympathetic neurons, retinal ganglion cells, and many Purkinje cells, but was difficult to detect in dentate gyrus neurons and some neocortical neurons. Cultured fetal mouse neocortical neurons and adult rat DRG neurons strongly expressed Nogo-A in their perikarya, growth cones, and axonal varicosities. All axons in the intact sciatic nerve contained Nogo-A and many but not all regenerating axons were strongly Nogo-A immunopositive after sciatic nerve transection. Ectopic muscle fibres that developed among the regenerating axons were also Nogo-A immunopositive. Following injury to the spinal cord, Nogo-A mRNA was upregulated around the lesion and Nogo-A protein was strongly expressed in injured dorsal column fibres and their sprouts which entered the lesion site. Following optic nerve crush, Nogo-A accumulated in the proximal and distal stumps bordering the lesions.     

Neuroanatomy of the visual afferents in the horseshoe crab (Limulus polyphemus)

The central nervous system of Limulus consists of a circumesophageal ring of fused ganglia and a paired ventral nerve cord. The anterior portion, the protocerebrum, receives sensory inputs including visual information. Three optic nerves, one each from the lateral eye, median ocellus, and ventral eye enter each side of the protocerebrum. The central connections of each optic nerve were determined by staining cut nerve trunks with cobalt chloride. The lateral optic nerve innervates the lamina, medulla, optic tract, ventral central body, and ocellar ganglion. The branching patterns of single axons, probably those of eccentric cells in the lateral eye retina, were observed. Single, large-diameter axons in the lateral optic nerve ramify at seven loci including sites in each of the structures innervated by the lateral optic nerve as a whole. The median optic nerve innervates the ocellar ganglion, central body, optic tract, and medulla. Three types of branching patterns were observed for single, large-diameter fibers in the median optic nerve. One type bypasses the ocellar ganglion and innervates the central body. A second type passes through the ocellar ganglion and optic tract without branching and innervates the posterior medulla. A third type innervates the ocellar ganglion, ventral central body, optic tract, and medulla. The ventral optic nerve is composed of large-diameter axons of ventral photoreceptors. Each axon enters the ganglion cell layer of the medulla and branches over a planar area less than 150 μm in diameter. We also observed that axons from mechanoreceptors on the anterior carapace innervate the posterior neuropil of the medulla, and that about 5% of the neurons in the medullar ganglion cell layer send axons to the ipsilateral circumesophageal connective.     

Distribution of the phosphorylated form of microtubule associated protein 1B in the fish visual system during optic nerve regeneration

Microtubule associated proteins are a heterogeneous group of proteins that have been implicated in regulating microtubule stability. They play an important role in the organisation of the neuronal cytoskeleton during neurite outgrowth, plasticity and regeneration. The fish visual system presents a considerable degree of plasticity. Thus, the retina grows continually throughout life and the optic nerve regenerates after crush. In the present study, we compared the distribution of the microtubule associated protein 1B in its phosphorylated form (MAP1B-phos) in the normal adult fish visual system with that observed during optic nerve regeneration after adult optic nerve crush using a specific monoclonal antibody mAb-150. Expression of MAP1B-phos was observed in some ganglion cell somata and in developing, growing axons within the control optic nerve. Few immunoreactive terminals were seen in the control optic tectum. After optic nerve crush, we found additional MAP1B-phos expression in regenerating axons throughout the visual system. Our results demonstrate that MAP1B-phos is present in growing and regenerating axons of fish retinal ganglion cells, which suggests that the phosphorylated form of MAP1B may play an important role in developmental and regeneration processes within the fish central nervous system.     

FGF-2 modulates expression and distribution of GAP-43 in frog retinal ganglion cells after optic nerve injury

Basic fibroblast growth factor (bFGF or FGF-2) has been implicated as a trophic factor that promotes survival and neurite outgrowth of neurons. We found previously that application of FGF-2 to the proximal stump of the injured axon increases retinal ganglion cell (RGC) survival. We determine here the effect of FGF-2 on expression of the axonal growth-associated phosphoprotein (GAP)-43 in retinal ganglion cells and tectum of Rana pipiens during regeneration of the optic nerve. In control retinas, GAP-43 protein was found in the optic fiber layer and in optic nerve; mRNA levels were low. After axotomy, mRNA levels increased sevenfold and GAP-43 protein was significantly increased. GAP-43 was localized in retinal axons and in a subset of RGC cell bodies and dendrites. This upregulation of GAP-43 was sustained through the period in which retinal axons reconnect with their target in the tectum. FGF-2 application to the injured nerve, but not to the eyeball, increased GAP-43 mRNA in the retina but decreased GAP-43 protein levels and decreased the number of immunopositive cell bodies. In the tectum, no treatment affected GAP-43 mRNA but FGF-2 application to the axotomized optic nerve increased GAP-43 protein in regenerating retinal projections. We conclude that FGF-2 upregulates the synthesis and alters the distribution of the axonal growth-promoting protein GAP-43, suggesting that it may enhance axonal regrowth.     

Distribution and morphology of retinal ganglion cells in the Japanese quail

A ganglion cell density map was produced from the Nissl-stained retinal whole mount of the Japanese quail. Ganglion cell density diminished nearly concentrically from the central area toward the retinal periphery. The mean soma area of ganglion cells in isodensity zones increased as the cell density decreased. The histograms of soma areas in each zone indicated that a population of small-sized ganglion cells persists into the peripheral retina. The total number of ganglion cells was estimated at about 2.0 million. Electron microscopic examination of the optic nerve revealed thin unmyelinated axons to comprise 69% of the total fiber count (about 2.0 million). Since there was no discrepancy between both the total numbers of neurons in the ganglion cell layer and optic nerve fibers, it is inferred that displaced amacrine cells are few, if any. The spectrum in optic nerve fiber diameter showed a unimodal skewed distribution quite similar to the histogram of soma areas of ganglion cells in the whole retina. This suggests a close correlation between soma areas and axon diameters. Retinal ganglion cells filled from the optic nerve with horseradish peroxidase were classified into 7 types according to such morphological characteristics as size, shape and location of the soma, as well as dendritic arborization pattern. Taking into account areal ranges of somata of each cell type, it can be assumed that most of the ganglion cells in the whole retinal ganglion cell layer are composed of type I, II and III cells, and that the population of uniformly small-sized ganglion cells corresponds to type I cells and is an origin of unmyelinated axons in the optic nerve.