Retinal Ganglion Cell Neurons Research Articles

Apoptosis in human amnion

Retinal ganglion cell (RGC) neurons are believed to die via apoptosis in human primary and secondary open-angle glaucoma. Previous studies have relied solely on the TUNEL (terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate [UTP]-biotin nick end-labeling) method of detecting DNA fragmentation to identify apoptotic nuclei. However, it is now clear that the TUNEL method cannot distinguish between the single- and double-strand DNA breaks that can be a feature of both apoptosis and necrosis. We have developed a double fluorescent labeling method that simultaneously combines in situ end-labeling (ISEL) to detect DNA fragmentation followed by staining with a cyanine dye, YOYO-1, to visualize apoptotic chromatin condensation. This allows for the unequivocal identification of an apoptotic nucleus. Our preliminary data obtained from one case of normal pressure glaucoma suggests that RGC neurons may die via apoptosis when intraocular pressure is not elevated.

Selective disruption of neuregulin-1 function in vertebrate embryos using ribozyme-tRNA transgenes.

The products of the neuregulin-1 gene constitute a set of polypeptide growth factors whose signalling through the ErbB receptors is essential to the growth and differentiation of many cell types in culture. Although studies with neuregulin-1 mutant mice have demonstrated that these growth factors are also essential regulators of cellular differentiation in vivo, the mid-embryonic death of these mutants precludes an analysis of hypothesized neuregulin-1 roles in later aspects of development. To circumvent this early lethality, we have pursued a ribozyme-based strategy for the perturbation of neuregulin-1 function in developing chick embryos. Early administration of a retrovirus carrying neuregulin-1 hammerhead-type ribozymes to blastoderm-stage embryos leads to an embryonic lethal phenotype that results from the failure of ventricular trabeculation in the developing heart, a faithful phenocopy of the mouse neuregulin-1 mutations. Later, more localized delivery of the ribozyme to the developing retina inhibits both the differentiation of retinal ganglion cell neurons and the proliferation of the neuroepithelial cells from which they derive. These results suggest that neuregulin-1 promotes both muscle cell differentiation in the heart and neuronal differentiation in the central nervous system. In addition, they demonstrate the utility of hammerhead ribozymes as a simple, effective and easily adaptable method of conditional gene inactivation in vertebrates.

Elevated Glutamate Levels in the Vitreous Body of Humans and Monkeys With Glaucoma

To explore the possibility that the excitatory amino acid glutamate might be associated with the disease process of glaucoma, which is characterized by the death of retinal ganglion cell neurons and subsequent visual dysfunction. Amino acid analyses were performed on vitreous specimens that were obtained from patients who were undergoing cataract extraction. Samples were collected prospectively from those patients who sustained inadvertent rupture of the posterior capsule between 1988 and 1993. An additional set of specimens, obtained from both eyes of monkeys, was analyzed; in these monkeys, glaucoma had been experimentally induced in one eye only. A twofold elevation in the level of glutamate was detected in the vitreous body of the group of patients with glaucoma when compared with that in a control population of patients with cataracts only. An even greater elevation of the glutamate level was found in the vitreous body of glaucomatous eyes of monkeys when compared with that in control eyes. No statistical differences were detected among other amino acid levels from the vitreous body of glaucomatous and nonglaucomatous eyes in humans or monkeys. The excitatory amino acid glutamate is found in the vitreous body of glaucomatous eyes at concentrations that are potentially toxic to retinal ganglion cells. The increased level of this known neurotoxin is consistent with an "excitotoxic" mechanism for the retinal ganglion cell and optic nerve damage in glaucoma. Therapies to protect neurons against glutamate toxic effects may prove to be useful in the management of this blinding disease.

The HIV-1 envelope protein gp120 induces neuronal apoptosis in hippocampal slices.

The HIV-1 envelope protein gp120 produces neuronal cell damage in primary cultures of a variety of cell types including hippocampal and retinal ganglion cell neurons. The properties of primary cell cultures are, however, often markedly different from those of cells living in their normal environment. We now report that gp120 induces widespread chromatin condensation and lesions in pyramidal granular neurones and in interneurones of rat hippocampal organotypic slice cultures. This damage is clearly of an apoptotic (programmed cell death) type. The use of an in vitro organized structure will enable the molecular and cellular mechanism of action of gp120 to be examined in conditions which are particularly suitable and relevant to the in vivo situation.

Growth cones are actively influenced by substrate-bound adhesion molecules

As axons advance to appropriate target tissues during development, their growth cones encounter a variety of cell adhesion molecules (CAMs) and extracellular matrix molecules (ECM molecules). Purified CAMs and ECM molecules influence neurite outgrowth in vitro and are thought to have a similar function in vivo. For example, when retinal ganglion cell (RGC) neurons are grown on different CAM and ECM molecule substrates in vitro, their growth cones display distinctive morphologies (Payne et al., 1992). Similarly, RGC growth cones in vivo have distinctive shapes at different points in the pathway from the eye to the tectum, suggesting the presence of localized cues that determine growth cone behaviors such as pathway selection at choice points. In this report, time-lapse video microscopy was utilized to examine dynamic transformations of RGC growth cones as they progressed from L1/8D9, N-cadherin, or laminin onto a different substrate. Contact made by the leading edge of a growth cone with a new substrate resulted in a rapid and dramatic alteration in growth cone morphology. In some cases, the changes encompassed the entire growth cone including those regions not in direct contact with the new substrate. In addition, the growth cones displayed a variety of behavioral responses that were dependent upon the order of substrate contact. These studies demonstrate that growth cones are actively affected by the substrate, and suggest that abrupt changes in the molecular composition of the growth cone environment are influential during axonal pathfinding.

Phosphorylation on carboxyl terminus domains of neurofilament proteins in retinal ganglion cell neurons in vivo: influences on regional neurofilament accumulation, interneurofilament spacing, and axon caliber.

The high molecular weight subunits of neurofilaments, NF-H and NF-M, have distinctively long carboxyl-terminal domains that become highly phosphorylated after newly formed neurofilaments enter the axon. We have investigated the functions of this process in normal, unperturbed retinal ganglion cell neurons of mature mice. Using in vivo pulse labeling with [35S]methionine or [32P]orthophosphate and immunocytochemistry with monoclonal antibodies to phosphorylation-dependent neurofilament epitopes, we showed that NF-H and NF-M subunits of transported neurofilaments begin to attain a mature state of phosphorylation within a discrete, very proximal region along optic axons starting 150 microns from the eye. Ultrastructural morphometry of 1,700-2,500 optic axons at each of seven levels proximal or distal to this transition zone demonstrated a threefold expansion of axon caliber at the 150-microns level, which then remained constant distally. The numbers of neurofilaments nearly doubled between the 100- and 150-microns level and further increased a total of threefold by the 1,200-microns level. Microtubule numbers rose only 30-35%. The minimum spacing between neurofilaments also nearly doubled and the average spacing increased from 30 nm to 55 nm. These results show that carboxyl-terminal phosphorylation expands axon caliber by initiating the local accumulation of neurofilaments within axons as well as by increasing the obligatory lateral spacing between neurofilaments. Myelination, which also began at the 150-microns level, may be an important influence on these events because no local neurofilament accumulation or caliber expansion occurred along unmyelinated optic axons. These findings provide evidence that carboxyl-terminal phosphorylation triggers the radial extension of neurofilament sidearms and is a key regulatory influence on neurofilament transport and on the local formation of a stationary but dynamic axonal cytoskeletal network.

Altered Gene Expression after Optic Nerve Transection: Reduced Neurofilament Expression as a General Response to Axonal Injury

Previous studies have shown that axonal injury (axotomy) in neurons of the mammalian peripheral nervous system (PNS) results in a recapitulation of the developmental program for cytoskeletal gene expression; these changes include the increased expression of a developmentally regulated isotype of β-tubulin (class II) and reduced neurofilament (NF) expression. In the present study we examined the abundance of mRNAs encoding the low-molecular-weight NF protein (NF-L) and class II β-tubulin in RNA purified from the retinae of newborn rats, from the retinae of adult rats at 2, 7, and 14 days after intracranial transection of the ipsilateral optic nerve, and from contralateral control retinae. In order to facilitate comparison with representative PNS neurons, parallel analyses were carried out in axotomized dorsal root ganglion (DRG) sensory neurons. Since NF-L and class II β-tubulin are neuron-specific proteins, axotomy-induced alterations in the levels of retinal mRNAs encoding these proteins largely reflect corresponding changes in expression by retinal ganglion cell neurons. Comparison of retinal RNA from newborn and adult (70-day-old) animals demonstrated a postnatal increase in NF-L and a decline in class II β-tubulin mRNAs comparable to those previously described in DRG neurons, Reductions in NF-L mRNA levels were noted in retina at 2, 7, and 14 days after axotomy and in DRG neurons at 7 and 14 days after axotomy. The abundance of class II β-tubulin mRNAs increased after axotomy in DRG neurons, but not in retina. These observations suggest that reduced NF gene expression represents a general response to axonal injury which is unrelated to the neuron's capacity for either survival or effective axon regeneration.

Blockade of NMDA receptor-mediated mobilization of intracellular Ca 2+ prevents neurotoxicity

NMDA receptor activation leads to elevated Ca 2+ in cultured rat cortical and retinal ganglion cell neurons. If excessive, this Ca 2+ response in associated with delayed neurotoxicity. We used dantrolene and ionomycin to test if the Ca 2+ response to NMDA was due to mobilization of intracellular Ca 2+ stores rather than merely to Ca 2+ influx. In the presence of EGTA, ionomycin resulted in release and subsequent depletion of intracellular Ca 2+ stores. Henceforth, despite normal extracellular Ca 2+, NMDA elicited only about half of its former Ca 2+ response. Similarly, when dantrolene was used to block Ca 2+ release from intracellular stores, we observed > 50% smaller NMDA-evoked Ca 2+ responses. These results quite surprisingly indicate that at least half the Ca 2+ response to NMDA is due to release of intracellular Ca 2+, a process triggered by influx of extracellular Ca 2+. Dantrolene also protected neurons from NMDA receptor-mediated neurotoxicity. Release of intracellular Ca 2+ may therefore be a necessary step in the cascade leading to neuronal damage induced by excessive NMDA receptor stimulation and may be amenable to pharmacological intervention.

Open-channel block of N-methyl-D-aspartate (NMDA) responses by memantine: therapeutic advantage against NMDA receptor-mediated neurotoxicity

Excessive activation of NMDA receptors is thought to mediate the calcium-dependent neurotoxicity associated with hypoxic-ischemic brain injury, trauma, epilepsy, and several neurodegenerative diseases. For this reason, various NMDA antagonists have been investigated for their therapeutic potential in these diseases, but heretofore none have proven to be both effective and safe. In the present study, memantine, an adamantane derivative similar to the antiviral drug amantadine, is shown to block the channels activated by NMDA receptor stimulation. From whole-cell and single-channel recording experiments, the mechanism of action of memantine is deduced to be open-channel block, similar to MK-801; however, unlike MK-801, memantine is well tolerated clinically. Compared to MK-801, memantine's safety may be related to its faster kinetics of action with rapid blocking and unblocking rates at low micromolar concentrations. Furthermore, at these levels memantine is an uncompetitive antagonist and should theoretically allow near-normal physiological NMDA activity throughout the brain even in the face of pathologically high focal concentrations of glutamate. These pharmacological properties confer upon memantine a therapeutic advantage against NMDA receptor-mediated neurotoxicity with few side effects compared with other organic NMDA open-channel blockers. Moreover, memantine is increasingly effective against escalating levels of glutamate, such as those observed during a stroke. Low micromolar concentrations of memantine, levels known to be tolerated by patients receiving the drug for the treatment of Parkinson's disease, prevent NMDA receptor-mediated neurotoxicity in cultures of rat cortical and retinal ganglion cell neurons; memantine also appears to be both safe and effective in a rat stroke model. These results suggest that memantine has considerable therapeutic potential for the myriad of clinical entities associated with NMDA receptor-mediated neurotoxicity.

Requirement for macrophages in neuronal injury induced by HIV envelope protein gp120.

HIV-1-related neuronal injury may involve a complex web of viral proteins and cytokines, but neurons themselves are not infected. The HIV envelope protein gp120 has been shown to engender an early increase in neuronal free calcium followed by delayed excitotoxic-like damage, which is prevented by N-methyl-D-aspartate (NMDA) antagonists. In the present study, we found that the injurious effects of gp120 on retinal ganglion cell neurons require the presence of macrophages in mixed neuronal glial cultures of postnatal retina. Within 24 hours of incubation, 20 pM gp120 injured nearly 40% of retinal ganglion cells in cultures containing macrophages and other glial cells, whereas no deleterious effects of gp120 were noted on retinal ganglion cells in cultures depleted of macrophages. Thus, the toxic effect of gp120 on neurons appears to be an indirect one, mediated by activation of macrophages and perhaps other glial cells.

Redox modulatory site of the NMDA receptor-channel complex: regulation by oxidized glutathione.

We monitored increases in both intracellular calcium concentration [( Ca2+]i) and whole-cell current responses induced by N-methyl-D-aspartate (NMDA), applied with co-agonist glycine, using fura-2 digital imaging and patch-clamp recording techniques. Extracellular application of oxidized glutathione (GSSG), but not reduced glutathione (GSH), inhibited responses mediated by activation of the NMDA subtype of glutamate receptor in cultures of rat cortical and retinal ganglion cell neurons. The NMDA responses were persistently inhibited by GSSG (500 microM to 10 mM) until introduction of a selective sulfhydryl reducing agent, dithiothreitol, which resulted in complete recovery of the responses. Exposure of the neurons to 5,5-dithio-bis-2-nitrobenzoic acid (DTNB), an efficacious oxidizing agent, also resulted in persistently smaller responses to NMDA. The addition of GSSG following exposure to DTNB, however, did not result in a further decrement in NMDA responses in our experimental paradigm. These findings suggest that a predominant action of GSSG is oxidation of vicinal thiol groups to form a peptide disulfide bond(s) comprising the redox modulatory site of the NMDA receptor-channel complex. Evidence for such regulatory sulfhydryl centers associated with the NMDA receptor has been presented previously. Moreover, the fact that DTNB produced little if any additional attenuation of the NMDA [Ca2+]i response when administered after GSSG implies that GSSG is also an efficacious oxidant at this site. GSSG displayed little or no effect on [Ca2+]i responses elicited by high extracellular K+ or by kainate, suggesting that, at least under the conditions of the present experiments, GSSG was somewhat selective for the NMDA redox modulatory site. Based on these observations, we suggest that GSSG exerts its NMDA-specific redox effects in a novel extracellular manner.

Neuronal injury due to HIV‐1 envelope protein is blocked by anti‐gp120 antibodies but not by anti‐CD4 antibodies

Picomolar concentrations of native or recombinant coat protein gp120, from the human immunodeficiency virus type 1 (HIV-1), injured rat retinal ganglion cell neurons in culture. This form of neurotoxicity could be completely abrogated by anti-gp120 but not by control preimmune serum, suggesting that the lethal effects of the purified preparations of the envelope protein were due to gp120 and not to a contaminant. Entry of HIV-1 is mediated by gp120 binding to a surface protein, designated "CD4," which is located, for example, on T lymphocytes. However, in the present study, specific anti-CD4 antibodies, at concentrations known to block effects mediated by high-affinity binding to CD4 on the surface of rat T cells, did not prevent neuronal injury induced by gp120. These findings suggest that injury of central neurons engendered by gp120 may be responsible, at least in part, for the neurologic manifestations observed in as many as 2/3 of the patients with acquired immunodeficiency syndrome, such as dementia, myelopathy, and visual loss, even in the absence of superinfection. In contrast with previous studies, however, this report suggests that the deleterious effects of gp120 on neurons may not be mediated via binding to the CD4 molecule.

VIP-mediated increase in cAMP prevents tetrodotoxin-induced retinal ganglion cell death in vitro

Afferent influences on natural cell death were modeled in retinal cultures derived from neonatal rats. Tetrodotoxin (TTX) blockade of electrical activity produced a significant reduction in surviving retinal ganglion cell (RGC) neurons during a critical period of development, similar in magnitude to the reduction observed during natural cell death in the intact retina at a similar developmental stage. The addition of vasoactive intestinal peptide (VIP) protected the RGCs from the lethal action of TTX. This effect was specific, since the related peptides PHI-27 and secretin produced no significant increase in RGC survival. Radioimmunoassay of cyclic nucleotides showed that TTX decreased culture levels of cAMP and that this trend was reversed by VIP Decreases in RGC survival associated with TTX electrical blockade were prevented by 8-bromo:cAMP or forskolin. Furthermore, VIP 10–28, the C-terminal fragment that inhibits VIP stimulation of adenylate cyclase, reduced the number of surviving RGCs. Thus, our results suggest that VIP, acting by increasing cAMP, has a neurotrophic effect on electrically blocked RGCs and may be an endogenous factor modulating normal cell death in the retina.

Comparison of delayed administration of competitive and uncompetitive antagonists in preventing NMDA receptor‐mediated neuronal death

Activation of N-methyl-D-aspartate (NMDA) receptors is thought to mediate toxic damage to central neurons due to hypoxia-ischemia, hypoglycemia, and trauma. We studied identified rat retinal ganglion cell neurons in vitro, a useful system for the study of excitotoxicity, and compared the protective effects of delayed administration of a competitive antagonist, 2-amino-5-phosphonovalerate (APV), and of an uncompetitive antagonist, MK-801, after glutamate-induced injury. We used maximally protective doses of the 2 antagonists. Under these conditions, both antagonists were able to prevent neuronal cell death if they were present within minutes of exposure to an endogenous glutamate-related toxin. In contrast, MK-801, but not APV, protected significantly against NMDA-mediated neurotoxicity when administered 1 to 4 hours after the initial insult. Thus, at least under certain conditions, an uncompetitive NMDA antagonist may offer a distinct advantage over a competitive antagonist when given several hours after a neurologic injury.

Postnatal development of the spatial contrast sensitivity of X- and Y- cells in the kitten retinogeniculate pathway

The sensitivity to spatial contrast patterns of single retinal ganglion cell axons and neurons in the A-layers of the dorsal lateral geniculate nucleus (LGNd) was measured in 4 1/2- and 6 1/2-week-old kittens and adult cats. Drifting sinusoidal grating stimuli were presented at 6-12 spatial frequencies to obtain spatial contrast sensitivity functions (SCSFs). The SCSFs were normalized for the postnatal growth of the kitten eye and were interpreted using a difference of Gaussians model of the receptive field (RF). The average optimal spatial frequency, spatial frequency bandwidth, and the proportion of cells that were selective for spatial frequency did not differ significantly between the kittens and adults for ganglion cells belonging to the cluster 1 (X-) or cluster 2 (Y-) functional types. The spatial resolution of kitten ganglion cells was also adultlike, except for that of Y-ganglion cells with peripheral RFs, which was significantly higher than in the adult. The spatial resolution of X-LGNd neurons with peripheral RFs was significantly poorer at 4 1/2 weeks than in the older animals. The proportion of X-LGNd neurons that were selective for spatial frequency increased between 4 1/2 and 6 1/2 weeks postnatally, but the spatial frequency bandwidth of selective cells did not change. The increased proportion of spatially selective LGNd neurons is probably due to the maturation of intrageniculate inhibitory circuits. Developmental changes in spatial resolution were interpreted as resulting from an increase (Y-retinal ganglion cells) or decrease (X-LGNd neurons) in RF center size. A model of retinogeniculate development is presented that attributes postnatal expansion of Y-retinal ganglion cell RF centers to increased functional convergence from more distal retinal neurons and reduction in LGNdX-cell RF center size to decreased convergence from X-retinal ganglion cells.

Phosphorylation of neurofilament proteins by protein kinase C

The low molecular mass (70 kDa) subunit of neurofilaments (NF-L) contains at least three phosphorylation sites in vivo and is phosphorylated by multiple kinases in a site-specific manner [(1987) J. Neurochem. 48, S101; Sihag, R.K. and Nixon, R.A. submitted]. In this study, we observed that the three subunits of neurofilament proteins from retinal ganglion cell neurons are substrates for purified mouse brain protein kinase C. Two-dimensional α-chymotryptic phosphopeptide map analyses of the NF-L subunit demonstrated that protein kinase C phosphorylates four polypeptide sites, two of which incorporate phosphate when retinal ganglion cells are pulse-radiolabeled with [ 32P]orthophosphate in vivo.

Signal-to-noise comparisons for X and Y cells in the retina and lateral geniculate nucleus of the cat.

The spike trains of X and Y retinal ganglion cell axons and neurons in the lateral geniculate nucleus (LGN) of cats were compared to determine if the visual signal could be better discriminated from the maintained activity in the LGN relative to the retina. Curves for relative or receiver operating characteristics (ROC) were derived from the interspike interval data of the spike trains using maintained activity as "noise" and visually-driven activity as the "signal". Analyses were also made using spike densities and more restricted time intervals. Although it was expected that neurons in the LGN might better distinguish the signal from the noise, the results of the ROC curve and spike density analyses did not bear out this expectation; that is, neither the X or Y cells in the LGN provided better discrimination of the visually-driven activities from the maintained activities compared to the incoming retinal information. Thus, at least in the anesthetized preparation, the LGN does not play a role in increasing the signal-to-noise ratio.

Nicotinic antagonists enhance process outgrowth by rat retinal ganglion cells in culture.

Functional nicotinic cholinergic receptors are found on mammalian retinal ganglion cell neurons in culture. The neurotransmitter acetylcholine (ACh) can be detected in the medium of many of these retinal cultures, after release presumably from the choline acetyltransferase-positive amacrine cells. The postsynaptic effect of endogenous or applied ACh on the ganglion cells can be blocked with specific nicotinic antagonists. Here it is shown that within 24 hours of producing such a pharmacologic blockade, the retinal ganglion cells begin to sprout or regenerate neuronal processes. Thus, the growth-enhancing effect of nicotinic antagonists may be due to the removal of inhibition to growth by tonic levels of ACh present in the culture medium. Since there is a spontaneous leak of ACh in the intact retina, the effects of nicotinic cholinergic drugs on process outgrowth in culture may reflect a normal control mechanism for growth or regeneration of retinal ganglion cell processes that is exerted by ACh in vivo.

Posttranslational modification of neurofilament proteins by phosphate during axoplasmic transport in retinal ganglion cell neurons

The progressive modification of newly synthesized neurofilament proteins (NFPs) during axoplasmic transport in mouse retinal ganglion cell (RGC) neurons was studied after RGC perikarya were pulse-labeled with 32P-orthophosphate or radiolabeled amino acids. The 3 NFP subunits, H(igh), M(iddle), and L(ow), were among a group of axonally transported proteins that incorporated high levels of 32P. Covalent addition of phosphate slowed the electrophoretic mobility of H and M on SDS polyacrylamide gels and shifted the charge of all 3 subunits toward more acidic pH values, thereby providing an index of the phosphorylation state of this radiolabeled population of NFPs. NFPs were extensively phosphorylated before they entered axons at the optic nerve level, and continued to be modified during transport along RGC axons at the optic nerve and tract level. H and M exhibited charge shifts of 0.2-0.6 units toward a more acidic pH during axoplasmic transport. The charge modifications became more prominent when NFPs reached distal axonal levels, which may indicate regional differences in the activity of this modification process along axons. By contrast, the L subunit became more basic in charge, consistent with decreases in the phosphorylation state during transport. Additional observations (Nixon and Lewis, 1986) that a considerable proportion of phosphate groups initially added to L and M were later removed as neurofilaments advanced along RGC axons support the notion that the changing phosphorylation state of NFP subunits during axoplasmic transport reflects a dynamic equilibrium between phosphorylation and dephosphorylation events. Topographical remodeling of phosphate groups on NFPs during axoplasmic transport is proposed as a possible mechanism for coordinating interactions between neurofilaments and other constituents, as these elements are transported and integrated into the axonal cytoskeleton.

Phosphorylation and dephosphorylation of neurofilament proteins in retinal ganglion cell neurons in vivo.

The cytoskeleton of most higher eukaryotic cells is composed of three filamentous systems, which serve varying dynamic and structural roles in cellular function. Two of these systems, the 80 A microfilaments and the 230 A microtubules, are composed of subunit proteins that are phylogenetically highly conserved. By contrast, the 100 A intermediate filaments are encoded by a large multigene family, the members of which are differentially expressed in different tissues (Fuchs and Hanukoglu, 1983). On the basis of biochemical and immunological criteria, five major classes of intermediate filaments have been defined (Lazarides, 1980). These include keratin filaments, found in cells of epithelial origin; desmin filaments, predominantly found in smooth, skeletal and cardiac muscle cells; vimentin filaments, present in cells of mesenchymal origin; glial filaments, constituents of certain glial cell types; and neurofilaments, present in many differentiated neurons of vertebrates and invertebrates. This classification emphasizes the tissue specificity of intermediate filaments, although it is now known that subunits from more than one class may coexist in some tissues at certain developmental stages (Osborn et al., 1980; Drager, 1983).