Effects of delayed transplantation of cultured Schwann cells on axonal regeneration from central nervous system cholinergic neurons.

The introduction of transplants consisting of cultured Schwann cells and their associated extracellular matrix (Sc/ECM) into a central nervous system (CNS) lesion cavity facilitates axonal regeneration from injured, adult mammalian neurons with subsequent reinnervation of their appropriate target (Kromer and Cornbrooks: Proceedings of the National Academy of Sciences of the United States of America 82:6330-6334, 1985). In the present study, the effects of a delayed transplantation procedure on the time course of this regenerative response were evaluated. For these experiments, bilateral CNS lesions were created between the septum and hippocampus by removing the fimbria-fornix pathway. Lesion cavities received either no transplants, transplants of collagen, or Sc/ECM transplants at the time the lesion was created or 6 days later. When no transplants or transplants of collagen were used, axonal sprouts extended for very short distances into the lesion cavity. These axons were not preferentially associated with the collagen transplants nor maintained at long post-lesion survival times. In animals that received Sc/ECM transplants, the number of sprouting axons and the progression of axonal growth along the transplants was much more extensive than for the collagen transplants. Although more axons were detected in cavities that received transplants immediately after the fimbria-fornix lesion, axonal regeneration along the transplants was similar regardless of whether there was a delay in transplanting the Schwann cells. By using histochemical techniques to identify acetylcholinesterase (AChE), regenerating AChE-positive axons were first detected in the cavity at 3 days post-transplantation, were associated with the Sc/ECM transplants by 5 days, and crossed the cavity within 8 days post-transplantation. Regenerating, neurofilament-positive axons crossed the CNS-Sc/ECM transplant interfaces in association with laminin-positive, glial fibrillary acidic protein-positive cellular pathways. Upon reaching the caudal end of the Sc/ECM transplant, the cholinergic axons abandoned the transplant and oriented directly toward the adjacent hippocampus. Both the simultaneous and delayed transplantation paradigms demonstrated a similar reinnervation pattern of AChE-positive fibers in the hippocampus, but there was a more rapid penetration and more extensive arborization of fibers in animals receiving the delayed transplants. Cholinergic fibers initially invaded the dentate gyrus molecular layer and hilus between 8 and 14 days post-transplantation. By 45 days post-transplantation, AChE-positive axons were detected throughout the dentate gyrus and regio inferior, but few fibers were present in regio superior of the hippocampus.(ABSTRACT TRUNCATED AT 400 WORDS)

Axotomy enhances the outgrowth of neurites from embryonic rat septal-basal forebrain neurons on a laminin substratum.

Previous studies have suggested that embryonic (nonaxotomized) and regenerating central nervous system neurons differentially respond to the same substrata. In the present study, we have used an in vitro model to test the ability of laminin and type I collagen to promote the outgrowth of neurites from nonaxotomized and axotomized, embryonic septal-basal forebrain (SBF) neurons. Neurons within explants derived from Embryonic Day (E) 15 rats extended neurites that demonstrated similar growth characteristics on a collagen or laminin substratum. E15 neurons could be induced to extend longer neurites on laminin if they were axotomized in vitro and subsequently replated onto a laminin substratum. The carbocyanine dye DiI indicated that neurons which were axotomized could survive and regenerate processes. These regenerating neurites grew 27% longer on laminin than they did on collagen. Similarly, neurons that were axotomized in situ, i.e., E18 SBF neurons, extended neurites that were 29% longer on a laminin substratum. In contrast, E15 explants that were maintained in suspension culture prior to being plated onto a substratum exhibited similar growth on laminin or collagen. The increase in regeneration by E15 neurons on laminin was augmented, by 22%, if nerve growth factor was supplemented to the culture medium. These results demonstrate that laminin is a better substratum, as compared to collagen, for the elongation of neurites from axotomized SBF neurons. Nonaxotomized neurites, on the other hand, do not appear to prefer one substratum over the other. Furthermore, regeneration from embryonic, SBF neurons on laminin is augmented if NGF is used simultaneously.

Proliferation and differentiation of a transfected Schwann cell line is altered by an artificial basement membrane.

Rapidly dividing transfected Schwann cells were grown on Matrigel, a reconstituted basement membrane gel. Matrigel decreased the proliferation of the cells by 75% when compared to sister cultures that were grown on an untreated plastic substrate. Some transfected cells plated onto a Matrigel substrate formed colonies similar to that observed when the cells were plated on a plastic substrate. Additionally, many cells on Matrigel assembled themselves into fascicles projecting away from the colonies. These fascicles were composed of transfected Schwann cells that had assumed a bipolar appearance reminiscent of quiescent secondary Schwann cells in culture. Transfected cells grown on Matrigel contained approximately 10-fold less glial fibrillary acidic protein when compared to sister cultures grown on an untreated plastic substrate. By indirect immunofluorescence laminin immunoreactivity appeared as globules within the cytoplasm of the cells which were cultured on a plastic substrate. However, cells that were grown on the Matrigel substrate appear to organize laminin in a linear array around themselves. These results demonstrate that the presence of an artificial basement membrane alters the morphology, rate of proliferation, and state of differentiation of a transfected Schwann cell line.

Transient modulation of Schwann cell antigens after peripheral nerve transection and subsequent regeneration.

Schwann cells within the distal portion of a transected nerve undergo a series of poorly understood events in response to injury and loss of axonal contact. These events may influence the regeneration of PNS neurons. In this study we examined the alteration of antigens located in the basal lamina, plasma membrane and cytoplasm of Schwann cells within the distal nerve stump: (a) after a complete transection of the sciatic nerve, and (b) subsequent to reestablished contact between regenerating axons and dedifferentated Schwann cells separated from contact with neurons. Visualization of laminin and heparan sulphate proteoglycan molecules at various intervals after nerve transection always revealed intact basal lamina channels. In response to loss of axonal contact, vimentin expression by Schwann cells within the distal nerve stump increased, becoming a predominant intermediate filament protein of the cytoskeleton while glial fibrillary acid protein (GFAP) expression decreased. This reversal in the prominence of intermediate filament protein was maintained until the onset of axonal reinnervation, at which point expression of GFAP increased and vimentin decreased. Expression of the Schwann cell plasma membrane associated protein, C4, closely mimicked GFAP expression during axon degeneration and subsequent reinnervation. In the normal uninjured nerve, tissue plasminogen activator (tPA) and S-100 were localized in the region near the Schwann cell-axon interface and the outer Schwann cell plasma membrane. In response to loss of axonal contact, the S-100 and tPA immunoreactivity associated with the Schwann cell-axon interface was lost while that localized around the outer Schwann cell plasma membrane remained unchanged. The results of this study demonstrate that Schwann cells modulate a portion of their antigenic repertoire in response to a loss of axonal contact and after contact with regenerating axons.

Effect of nerve growth factor on the elongation of neurites from axotomized rat embryonic septal-basal forebrain neurons: an in vitro analysis.

The administration of nerve growth factor (NGF) into the brain of a fornix-fimbria lesioned rat can rescue many cholinergic, septal-basal forebrain (SBF) neurons from imminent cell death. Unfortunately, it is unclear if NGF can stimulate regenerative growth from axotomized, SBF neurons. In the present study, we used an in vitro model system to determine if NGF could affect neurite outgrowth from nonaxotomized and/or axotomized, embryonic SBF neurons. Axotomized neurons were obtained by severing the neuritic fields surrounding embryonic day (E) 15 SBF explants maintained in primary culture. Acetylcholinesterase (AChE) histochemistry was used to assess the effects of NGF on cholinergic neurites. We report that 1) neurite outgrowth on type I collagen from E15 SBF neurons in primary culture (nonaxotomized neurons) was not affected by NGF. 2) NGF enhanced the outgrowth (regeneration) of axotomized, SBF neurons on a collagen substratum; however, neurons had to be treated with NGF both before and after axotomy to stimulate regeneration effectively. Application of NGF either before or after axotomy did not enhance regenerative neurite outgrowth. 3) SBF neurons had to be axotomized for NGF to facilitate neurite outgrowth. This is supported by the observation that SBF explants, initially maintained in NGF-supplemented medium in suspension culture, did not demonstrate enhanced neurite outgrowth in the presence of NGF when plated onto a substratum. 4) The regenerative growth of AChE-negative, as well as AChE-positive, neurites was facilitated by NGF treatment. In addition to data concerning neurite outgrowth, we also found that the NGF receptor, as recognized by the antibody 192-IgG, expands its distribution as time in culture progresses; i.e., staining, originally confined to cell bodies and proximal processes within the explant, later included neurites that emanated from the explant. Thus, our results demonstrate that NGF can stimulate regenerative growth from axotomized, but not nonaxotomized, embryonic SBF neurons. We hypothesize that, given the appropriate substratum for axon elongation in vivo, NGF can stimulate the regeneration of SBF neurons in the injured adult brain.

Age-dependent patterns and rates of neurite outgrowth from CNS neurons on Schwann cell-derived basal lamina and laminin substrata.

In the present study, we have examined the growth characteristics of CNS neurons on type I collagen, detergent-treated collagen (dColl), Schwann cell-derived basal lamina (SC-BL), and purified laminin substrata. Neurons from the cerebral cortex, septal basal forebrain, and lumbosacral spinal cord were obtained from embryonic age (E) 15 and E18 rats and grown in vitro as explants on the test substrata. Neurons from either embryonic age displayed radial neurite outgrowth on collagen and dColl substrata. However, pretreatment of collagen with detergents slightly diminished its ability to support neurite outgrowth, as evidence by the 20-40% decrease in the rate of neurite growth on dColl versus the rate calculated for neurons on collagen. In contrast to the similar growth characteristics of E15 and E18 neurons on collagen and dColl, the pattern of neurite outgrowth for CNS neurons on SC-BL and laminin substrata was age dependent. Most E15 neurons grown on SC-BL extended neurites that grew identically to those observed on dColl; these 'non-orienting' neurites maintained a radial orientation to their outgrowth despite encountering interposing channels of SC-BL and grew at rates equal to that calculated for neurons on dColl. E15 neurons placed on laminin substrata showed similar growth patterns and rates equal to that calculated for neurons on dColl. E15 neurons placed on laminin substrata showed similar growth patterns and rates to neurons on collagen. In contrast, neurons from E18 rats exhibited neurites that preferentially grew in intimate association with SC-BL channels once contact with the channels was established. These 'orienting' neurites faithfully elongated within the SC-BL and demonstrated a 1.4- to 2.0-fold increase in growth rate compared with the sister cultures of neurons grown on dColl. Furthermore, E18 neurons exhibited a 1.4-fold increase in growth on laminin compared with E18 neurons grown on collagen. A minor population of neurites exhibiting similar characteristics to orienting neurites was also observed in E15 cultures. It is hypothesized that orienting and non-orienting neurites reflect the outgrowth of 'regenerating' and 'developing' neurons, respectively, and may indicate an inherent difference in the ability of regenerating and developing neurons to recognize and respond to the same guidance signals.

Identification of trophic factors and transplanted cellular environments that promote CNS axonal regeneration.

As indicated in this review, we have begun to elucidate cellular environments and trophic factors that promote the regeneration of adult mammalian CNS neurons. In the present paradigm, bilateral aspiration lesions of the fornix-fimbria are used to axotomize septal neurons and transect the septal cholinergic projection to the dorsal hippocampus in order to evaluate the influence of trophic factors, such as NGF, on neuronal survival and the ability of cellular transplants of PNS tissue to promote axonal regeneration in vivo. Initial results demonstrate that NGF is a potent trophic molecule that prevents retrograde degeneration of septal cholinergic neurons. Observations from transplantation studies demonstrate that viable Schwann cells obtained from PNS nerve grafts or Schwann cell-ECM cultures provide a favorable cellular milieu for CNS regeneration. These cellular transplants induce a remarkable sprouting response from septal cholinergic neurons and promote the rapid elongation of septal axons that reinnervate the denervated hippocampus. In stark contrast to the Schwann cell-laden transplants, transplants including only ECM channels synthesized by cultured Schwann cells do not promote axonal regeneration within the time periods that we have examined. Therefore, we hypothesize that viable Schwann cells are crucial for the process of regeneration because they contribute both trophic and tropic factors to the injured CNS neurons. The significant early sprouting phenomenon associated with transplants containing Schwann cells strongly suggests that soluble Schwann cell-synthesized factors induce axon elongation and possibly enhance the survival of injured septal neurons. The trophic factors probably function in a manner similar, if not identical, to the action of NGF on axotomized septal neurons. Moreover, Schwann cells appear to provide tropic signals, such as LAM or a LAM-NGF complex, that can act, when in the proper stereoconfiguration, to promote the elongation and orientation of regenerating axons. Thus, our current data indicate that in order to promote optimal axonal regeneration from injured CNS neurons, both trophic and tropic factors must be supplied from exogenous sources.

Variation in content and function of non-neuronal cells in the outgrowth of sympathetic ganglia from embryos of differing age.

Studies on cellular interactions in the developing nervous system are greatly facilitated by the availability of tissue culture preparations that contain single or combined populations of neurons and non-neuronal cells (NNCs). Using superior cervical ganglia (SCG) from early E15 rats on air-dried collagen, we were able to prepare cultures containing neurons along with Schwann cells (SCs) as the only NNC type present without the use of antimitotic treatment and cultures containing only neurons, following brief antimitotic treatment. Light-microscopic observation of E15 outgrowth showed a uniform population of flattened cells, unlike that of E20 cultures, which contained a mixture of spindle-shaped and flattened cells. Autoradiograms following [3H]thymidine administration to E15 cultures revealed a striking gradient of nuclear labeling: Only a few cells were labeled near the explant and nearly all cells were labeled at the growth front. This was in marked contrast to E20 cultures, in which nuclei were labeled throughout the outgrowth. The conclusion that the labeling gradient is explained by the presence of SCs without other NNC types in E15 cultures was confirmed by immunocytochemical studies. Anti-laminin antibodies stain only those extracellular matrix components related to the SC surface, whereas anti-fibronectin antibodies stain fibroblast-related components (Cornbrooks et al., 1983a). Anti-laminin antibodies stained cell surfaces in both E15 and E20 outgrowth. E15 outgrowth did not stain with anti-fibronectin antibodies although marked staining was obtained in E20 cultures. Electron microscopy confirmed the presence of only SCs in E15, and of both SCs and fibroblasts in E20 outgrowth. Thus, it appears that there is a narrow developmental window in which the ganglia contain neurons and SCs but relatively few fibroblast components; cultures prepared from ganglia at this stage form outgrowth containing only neurites and SCs without antimitotic treatment. Surprisingly, neither SC ensheathment nor SC basal lamina formation was normal in E15 and E20 outgrowth. When either E15 or E20 SCG SCs were transplanted onto dorsal root ganglion neurons free of endogenous SCs, however, the sensory neurites were typically ensheathed or myelinated and basal lamina appeared 9 d later, identifying the SCG NNCs as functionally competent SCs.(ABSTRACT TRUNCATED AT 400 WORDS)

Basal lamina-associated heparan sulphate proteoglycan in the rat PNS: characterization and localization using monoclonal antibodies.

Cultured rat Schwann cells produce a basal lamina (BL)-associated heparan sulphate proteoglycan (HSPG). The HSPG has an apparent molecular weight of greater than 450 kD, is sensitive to both heparinase and heparitinase and contains a core protein of approximately 400 kD. Two independently derived monoclonal antibodies, B3 and C17, recognize this HSPG. Using B3 and C17, we found that this HSPG, or immunologically related material, is present in BLs throughout the body and in a small number of connective tissue sites without a formed BL. In the PNS it is present in BLs of Schwann cell-axon units, in synaptic and extrasynaptic portions of muscle fibre BL, and in the BLs of satellite cells that ensheath neurons in sympathetic and sensory ganglia. This HSPG is not detectable in the neuropil of the brain and spinal cord. Neurons, Schwann cells and fibroblasts cultured alone do not assemble a BL or accumulate immunocytochemically detectable amounts of this HSPG, but it is present in BLs assembled in myotube and in Schwann cell-neuron cultures. Thus, this HSPG is a component of most, if not all, BLs in the PNS.

Transplants of Schwann cell cultures promote axonal regeneration in the adult mammalian brain.

Transplantation of embryonic brain tissue or mature peripheral nerves into the adult mammalian central nervous system promotes axonal regrowth from axotomized central nervous system neurons; however, the cellular origin and molecular nature of the factors promoting axonal growth in vivo are unknown. To further characterize cellular environments that facilitate regeneration of central nervous system axons, we developed a methodology whereby cultured cell preparations can be transplanted into the brain of mature mammals. For this procedure, lesions are produced in the septal-hippocampal system of adult rats, and selected regions from collagen-supported Schwann cell/neuron cultures (consisting of Schwann cells, extracellular matrix, and degenerating neuronal processes and myelin but devoid of neuronal perikarya and fibroblasts) are positioned within the intracephalic cavity so that they bridge the lesion gap (approximately 3 mm) separating the septum and hippocampus. At various time up to 3 weeks after transplantation, specimens were prepared for acetylcholinesterase histochemistry and the immunocytochemical localization of laminin (an extracellular matrix protein) and C-4 (a Schwann cell membrane antigen). All specimens (from uninjured controls and from animals with either acellular collagen or mature Schwann cell/extracellular matrix transplants) contained laminin immunoreactivity associated with the meninges, choroid plexus, ependyma, and cerebral blood vessels. All animals with transplants showed prominent laminin staining on astrocytic processes along the intracephalic cavity, but only the Schwann cell/extracellular matrix transplants exhibited dense laminin and C-4 immunoreactivity within the cellular portion of the transplants. Regeneration of acetylcholinesterase-positive septal fibers occurred only in animals containing Schwann cell/extracellular matrix transplants. By 6 days after transplantation, acetylcholinesterase-positive fibers were observed both on laminin-positive cellular tissue strands connecting the septum and the Schwann cell/extracellular matrix transplants and on the initial portions of the transplants. By day 14, acetylcholinesterase-positive fibers traversed the entire lesion cavity in intimate association with the laminin- and C-4-positive cellular layer of the transplants and reinnervated the host hippocampus. However, cholinergic fibers were not associated with all laminin-containing processes along the lesion cavity nor did they grow along acellular collagen transplants. These results indicate the presence of factors in transplants of cultured Schwann cells and their associated extracellular matrix that promote rapid regeneration of central nervous system cholinergic axons in vivo.

Synthesis by Schwann cells of basal lamina and membrane-associated heparan sulfate proteoglycans.

Primary cultures that contain only Schwann cells and sensory nerve cells synthesize basal lamina. The assembly of this basal lamina appears to be essential for normal Schwann cell development. In this study, we demonstrate that Schwann cells synthesize two major heparan sulfate-containing proteoglycans. Both proteoglycans band in dissociative CsCl gradients at densities less than 1.4 g/ml, and therefore, presumably, have relatively low carbohydrate-to-protein ratios. The larger of these proteoglycans elutes from Sepharose CL-4B in 4 M guanidine hydrochloride (GuHCl) at a Kav of 0.21 and contains heparan sulfate and chondroitin sulfate chains of Mr 21,000 in a ratio of approximately 3:1. This proteoglycan is extracted from cultures by 4 M GuHCl but not Triton X-100 and accumulates only when Schwann cells are actively synthesizing basal lamina. The smaller proteoglycan elutes from Sepharose CL-4B at a Kav of 0.44 and contains heparan sulfate and chondroitin sulfate chains of Mr 18,000 in a ratio of approximately 4:1. This proteoglycan is extracted by 4 M GuHCl or by Triton X-100. The accumulation of this proteoglycan is independent of basal lamina production.

Biosynthesis of type IV collagen by cultured rat Schwann cells.

We have obtained evidence that rat Schwann cells synthesize and secrete type IV procollagen. Metabolic labeling of primary cultures of Schwann cells plus neurons and analysis by SDS PAGE revealed the presence of a closely spaced pair of polypeptides in the medium of these cultures that (a) were susceptible to digestion by purified bacterial collagenase, (b) co-migrated with type IV procollagen secreted by rat parietal endoderm cells, and (c) were specifically immunoprecipitated by antibodies against mouse type IV collagen. Limited pepsin digestion of metabolically labeled medium or cell layers produced a pepsin-resistant fragment characteristic of pro-alpha 1(IV) chains. Removal of neuronal cell bodies from the cultures immediately before labeling did not reduce the amount of type IV procollagen detected in the medium. This indicated that Schwann cells, not neurons, were responsible for synthesis of type IV procollagen. We believe type IV procollagen is a major constituent of the Schwann-cell extracellular matrix based upon (a) its presence in a detergent-insoluble matrix preparation, (b) its presence in the cell layer of the cultures in a state in which it can be removed by brief treatment with bacterial collagenase or trypsin, and (c) positive immunofluorescence of Schwann cell-neuron cultures with anti-type-IV collagen antibodies. Secretion of type IV procollagen was substantially reduced when Schwann cells were maintained in the absence of neurons. This observation may account for the previously reported finding that Schwann cells assemble a basal lamina only when co-cultured with neurons (Bunge, M. B., A. K. Williams, and P. M. Wood, 1982, Dev. Biol., 92:449).

In vivo and in vitro observations on laminin production by Schwann cells.

Recent reports on the dystrophic mouse mutant suggest that the prominent extracellular matrix component of peripheral nerve tissues plays an important role in peripheral nerve development. We have examined the disposition of two prominent extracellular matrix components, fibronectin and laminin, both in mature peripheral nerve in vivo and in an in vitro system that allows study of Schwann cells in various functional states. In frozen sections of whole nerve, staining with antibodies to fibronectin and laminin shows that fibronectin stains throughout the endoneurium while laminin staining is restricted to regions known to contain basal lamina, particularly the basal lamina of each ensheathing Schwann cell. Tissue culture studies indicate that fibronectin staining at the light microscopic level is a reliable marker for fibroblasts (and not Schwann cells) in culture; conversely, antibodies to laminin stain components related to the Schwann cell surface but not components related to fibroblasts. Unexpectedly, Schwann cells in culture produce laminin at all stages in development, whether in contact with axons or not. As Schwann cells in culture begin to ensheathe axons, punctate regions of laminin on their surfaces become confluent. After ensheathment is completed, a continuous line of staining is found in the region of the Schwann cell basal lamina. It has been established that Schwann cells produce a basal lamina only when in contact with axons. Therefore, the production of laminin appears to be necessary but not sufficient for basal lamina formation. The constancy of laminin production by Schwann cells provides a reliable basis for distinguishing between Schwann cells and fibroblasts in tissue culture.

Factors affecting schwann cell basal lamina formation in cultures of dorsal root ganglia from mice with muscular dystrophy.

Pure populations of sensory neurons (N), Schwann cells (S) and fibroblasts (Fb) were established in culture from normal and dystrophic (dy) mice in order to investigate the cellular origin(s) of the peripheral nervous system abnormalities present in murine muscular dystrophy. These cell types were placed together in various combinations and their subsequent interactions were monitored with the light and electron microscope. The formation of the basal lamina (BL) which in normal tissue, completely surrounds the external aspect of the Schwann cell (when in contact with axons) was documented by morphometric analysis of electron micrographs. Defects in Schwann cell BL formation, observed throughout the PNS of the dy mouse in vivo, were used as a marker for the expression of the dystrophic abnormality in culture. Initially mature cultures of dy tissues containing only S and N (SN) without Fb were examined and found to contain an incomplete BL that surrounded only 82.8 +/- 12.2% of the externally directed plasmalemma of axon-related Schwann cells. The following recombination cultures were established: (1) normal S were placed on dystrophic N; (2) dystrophic S were placed on dystrophic N; (3) dystrophic S were placed on normal N; and (4) normal Fb were added to a dystrophic SN culture. After a 5-week period, the BL formed by normal S in direct contact with dystrophic N was thick and continuous (97.7 +/- 2.2 coverage). On the other hand, in culture situations (without Fb) containing dystrophic S in contact with either dystrophic or normal neurites, the BL coverage was considerably less (58.5 +/- 14.8% and 55.4 +/- 13.2%, respectively). The addition of normal Fb obtained from sciatic nerve explants to dystrophic SN cultures in time resulted in the formation of a morphologically complete BL (98.9 +/- 1.4% coverage). We conclude that neuronal signal(s) are adequate to induce complete BL formation by Schwann cells in the dystrophic tissue but that dystrophic Schwann cells are incapable of forming a complete BL. Furthermore, this deficiency of dy Schwann cells is apparently corrected by the presence of normal Fb by an unknown mechanism.

Expression of the trembler mouse mutation in organotypic cultures of dorsal root ganglia.

Organotypic dorsal root ganglion (DRG) cultures were established from all the embryos of two trembler (Tr/+) female mice mated to normal (+/+) males to determine if the trembler mutation would be expressed in nerve tissue culture. Dorsal root ganglia from normal mice maintained in our culture system exhibit substantial myelination after 6 weeks of growth. This normal pattern was observed in approximately one half of the cultures in the present series. The remaining half of the explants had marked PNS myelin abnormalities readily detectable at the light microscopic level in living cultures; furthermore, the ultrastructural appearance of these Tr/+ cultures was similar to that of adult trembler sciatic nerve. An analysis of unmyelinated nerve fibers in Tr/+ cultures revealed that the number of neurites resident within each non-myelinating Tr/+ Schwann cell was significantly less than the number observed in +/+ cultures. There are distinctive PNS myelin abnormalities which: (1) develop in DRG cultures established from embryos at risk for the trembler mutation; (2) are highly reliable and readily detectable markers for the trembler genotype; and (3) are similar to the trembler PNS defects detectable in vivo.