Increased β<Subscript>2</Subscript>-Adrenergic Receptor Activity by Thyroid Hormone Possibly Leads to Differentiation and Maturation of Astrocytes in Culture

Summary (1) Our earlier studies indicate a downsteam regulatory role of the β-adrenergic receptor (β-AR) system in thyroid hormone induced differentiation and maturation of astrocytes. In the present study we have investigated the contributions of the subtypes of β-AR in the above phenomenon. (2) Primary astrocyte cultures were grown under thyroid hormone deficient as well as under euthyroid conditions. [¹²⁵I]Pindolol ([¹²⁵I]PIN) binding studies showed a gradual increase in the specific binding to β₂-AR when observed at 5, 10, 15, and 20 days under both cultural conditions. Thyroid hormone caused an increase in binding of [¹²⁵I]PIN to β₂-AR compared to thyroid hormone deficient controls at all ages of astrocyte culture. (3) Saturation studies using [¹²⁵I]PIN in astrocyte membranes prepared from 20-day-old cultures showed a significant increase in the affinity of the receptors (K _D) in the thyroid hormone treated cells without any change in receptor number (B _max). (4) β₂-AR mRNA levels were measured by real-time PCR during ontogenic development as well as during exposure of 10-day-old hypothyroid cultures to normal levels of thyroid hormone for 2, 6, 12, and 24 h. None of the conditions caused any significant change in the β₂-adrenergic receptor mRNA levels when compared with corresponding hypothyroid controls. (5) Over expression of β₂-AR cDNA in hypothyroid astrocytes caused morphological transformation in spite of the absence of thyroid hormone in the medium. (6) Taken together, results suggest thyroid hormone causes a selective increase in [¹²⁵I]PIN binding to β₂-AR due to increase in receptor affinity, which may lead to maturation of astrocytes.     

A tissue model for the study of cell proliferation in vitro

Summary A procedure for the cultivation of mesentery is described, in which the culture is fully representative of the tissue of origin. The intact mesenteric membrane—exposed to a minimum of trauma—was spread out over a hole in a filter paper strip in fluid medium and was cultivated free-hanging. Specimens from rats and guinea pigs were used. The organ culture model appears especially apt for cytochemical and proliferation studies. Proliferation variables based on Feulgen DNA analysis in individual, morphologically defined cells and on mitotic counting and radiochemical analysis were estimated. The tissue was fully viable in chemically defined growth medium and showed an almost unaltered light microscopical appearance after up to 52 hr in culture. Supported by the Swedish Cancer Society.     

Primary Cultures From Cerebral Cortex and Hippocampus Enriched in Glutamatergic and GABAergic Neurons

The aim was to define a primary culture system enriched in neurons using a defined culture medium, and characterize the model system as to cellular morphology and neuronal phenotypes. We found that these primary neuron enriched cultures from either newborn rat cerebral cortex or hippocampus contain small GABAergic and large glutamatergic neurons as well as astrocytes and microglia. Astrocytes in these cultures are morphologically differentiated with long, slender processes and interact with soluble factors responsible for induction and expression of the glutamate transporter GLT-1. The cultures achieve the highest expression of the vesicular glutamate transporter 1 (VGLUT1) and GLT-1 after 20 days in vitro. Conditioned media from these neuron enriched cultures also induced GLT-1 expression in primary astrocytic cultures, which were free from neurons. The amount of glutamatergic neurons guides the morphological maturation of astrocytes and GLT-1 expression both in the neuron enriched cultures and in the conditioned media supplemented astrocytic cultures. Interestingly, these cultures were not influenced or activated by the inflammatory stimulus lipopolysaccharide. This suggests that soluble factors from neurons protect microglia and astrocytes to become inflammatory reactive. In conclusion we have developed a well characterized culture model system enriched in neurons, taken from newborn rats and cultured in defined media. The neurons express different neuronal phenotypes. Such a model system is valuable when studying interactions between neurons and glial cells.     

Evidence for the presence of thyroid stimulating hormone, thyroglobulin and their receptors in Eisenia fetida: a multilevel hormonal interface between the nervous system and the peripheral tissues

The present study describes the localization and distribution of thyroid-stimulating hormone (TSH), thyroglobulin (TGB) and their receptors in Eisenia fetida (Annelida, Oligochaeta) as revealed by immunohistological methods. Immunopositive neuronal and non-neuronal cells are present in both the central nervous system and some peripheral organs (e.g. foregut and coelomocytes). TSH- and TGB-immunopositive neurons in the various ganglia of the central nervous system are differentailly distributed. Most of the immunoreactive cells are found in the suboesophageal ganglion. The stained cells also differ in their shapes (round, oval, pear-shaped) and sizes (small, 12–25 μm; medium, 20–35 μm; large, 30–50 μm). In all ganglia of the central nervous system, TSH-positive neurons additionally show gamma aminobutyric acid (GABA) immunopositivity. Non-neuronal cells also take part in hormone secretion and transport. Elongated TSH-positive cells have been detected in the capsule of the central ganglia and bear granules or vacuoles in areas lacking neurons. Many of capillaries show immunoreactivity for all four tested antibodies in the entire central nervous system and foregut. Among the coelomocytes, granulocytes and eleocytes stain for TSH and its receptor and for TGB but not for thyroid hormone receptor. Most of the granulocytes are large (25–50 μm) but a population of small cells (10–25 μm) are also immunoreactive. None of the coelomocytes stain for GABA. We therefore suggest that the members of this hormone system can modify both metabolism and immune functions in Eisenia. Coelomocytes might be able to secrete, transport and eliminate hormones in this system.This work was supported by the MTA-PTE Adaptation Biology Research Group and National Research and Developmental Fund (NKP 1/048/2001). M.W. is in receipt of a János Bolyai Scholarship.     

Rapid chondrocyte maturation by serum-free culture with BMP-2 and ascorbic acid

In serum-containing medium, ascorbic acid induces maturation of prehypertrophic chick embryo sternal chondrocytes. Recently, cultured chondrocytes have also been reported to undergo maturation in the presence of bone morphogenetic proteins or in serum-free medium supplemented with thyroxine. In the present study, we have examined the combined effect of ascorbic acid, BMP-2, and serum-free conditions on the induction of alkaline phosphatase and type X collagen in chick sternal chondrocytes. Addition of either ascorbate or rhBMP-2 to nonconfluent cephalic sternal chondrocytes produced elevated alkaline phosphatase levels within 24–72 h, and simultaneous exposure to both ascorbate and BMP yielded enzyme levels at least threefold those of either inducer alone. The effects of ascorbate and BMP were markedly potentiated by culture in serum-free medium, and alkaline phosphatase levels of preconfluent serum-free cultures treated for 48 h with BMP + ascorbate were equivalent to those reached in serum-containing medium only after confluence. While ascorbate addition was required for maximal alkaline phosphatase activity, it did not induce a rapid increase in type X collagen mRNA. In contrast, BMP added to serum-free medium induced a three- to fourfold increase in type X collagen mRNA within 24 h even in the presence of cyclohexamide, indicating that new protein synthesis was not required. Addition of thyroid hormone to serum-free medium was required for maximal ascorbate effects but not for BMP stimulation. Neither ascorbate nor BMP induced alkaline phosphatase activity in caudal sternal chondrocytes, which do not undergo hypertrophy during embryonic development. These results indicate that ascorbate + BMP in serum-free culture induces rapid chondrocyte maturation of prehypertrophic chondrocytes. The mechanisms for ascorbate and BMP action appear to be distinct, while BMP and thyroid hormone may share a similar mechanism for induction. J. Cell. Biochem. 66:394–403, 1997. © 1997 Wiley-Liss, Inc.     

Clonal growth and culture life span of bovine adrenocortical cells in a serum-free medium

Summary The life span and growth from clonal density of bovine adrenocortical cell cultures were studied in serum-supplemented medium and a serum-free defined medium, which supported sustained cell proliferation and steroid production. The total culture life span was 79 population doublings in serum-supplemented medium with fibroblast growth factor (FGF) and 36 population doublings in the defined medium without serum. Older passage cell cultures grown in the defined medium progressively lost the ability to produce 11β- and 21-hydroxylated steroids, which was observed previously for cultures in serum-supplemented medium, and also had a decline of 17α-hydroxylated steroid production. The cloning efficiency in the defined medium was 12.2% as compared to 24% in serum-supplemented medium with FGF. Five isolated clonal cell lines grown in the defined medium were characterized for steroid function in response to steroidogenic agents. All five clonal cell lines had stimulated steroid production with 8-bromo-cAMP, but only four of the clonal lines were stimulated also by adrenocorticotropin. None of the clonal cell lines produced 11β-, 21- or 17α-hydroxylated steroids in response to treatment with either steroidogenic agent, results that were similar to data obtained from older mass cultures. The apparent deficiency of the defined medium as compared to serum-supplemented medium for maximum support of the culture life span and cloning efficiency may be useful in studies of cellular aging and its relation to differentiated function for this cell culture system. This study was supported by the Iowa Diabetes and Endocrinology Research Center (grant AM25295 from the National Institutes of Health, Bethesda, MD). D.A.F. was supported by a National Research Service Award from the National Institutes of Health (grant HL07485).     

THE EXPRESSION OF DIFFERENTIATION BY CHICK EMBRYO THYROID IN CELL CULTURE : II. Modification of Phenotype in Monolayer Culture by Different Media

Previous results with thyroid secretory cells in monolayer culture seem contradictory with respect to phenotypic stability of this cell type. On the one hand, in "minimal" medium the cells lose structural and functional specializations which can be returned only by three-dimensional growth in organ culture upon addition of fibroblasts derived from the thyroid capsule. On the other hand, in "rich" medium used for cloning, cytoarchitecture and function remain unaltered in either mass or clonal cultures. The apparent discrepancy has been resolved by plating cell suspensions in both media and changing to the alternate medium once the cells have become established. It has been shown that a number of characteristics, including hormone levels, are reversed each time such a change in medium is made. These modulations are discussed in terms of the normal variations in structure and function of the gland in vivo.     

Changes in the properties of fetal rat liver during organ culture

Summary Explants of fetal rat liver maintained in organ culture lost about 40% of their mass in 42 hr of incubation as a result of decrease in blood cells and hepatocytes. Proteins from the cytosol and particulate elements of the tissue were found in the culture medium. About 60% of this protein was degraded to peptides during culture. The transfer of malate and lactate dehydrogenases from tissue to medium paralleled that of proteins. Glutamate dehydrogenase was lost from the mitochondria and in part leaked through the cell membrane into the medium. Net loss of activity of the three enzymes occurred, probably as a consequence of proteolytic degradation. Of 12 enzymes in liver tissue, the specific activities of eight—soluble malate dehydrogenase, glutamate dehydrogenase, succinate dehydrogenase, phosphopyruvate carboxylase, hexosediphosphatase, glucose-6-phosphatase, tyrosine, aminotransferase, and alanine aminotransferase—were unchanged or increased. Glycogen synthetase, aspartate aminotransferase, pyruvate kinase, and lactate dehydrogenase decreased. Although changes in membrane permeability may have had some influence on the results reported, the predominant effect was due to loss of protein from tissue as a result of discharge of total contents of some of the cells into the medium. The residual explanted tissue retained its structural integrity. It is concluded that fetal rat liver in organ culture provides a suitable model system for controlled studies with this organ in vitro. This investigation was supported by grants from the National Institute of Child Health and Human Development (RO 1 HD09715), National Cancer Institute (CA 14194), and United States Public Health Service General Research Support Grant RR 5589.     

Reissner’s fibre supports the survival of chick cortical neurons in primary mixed cultures

Reissner's fibre, a thread-like structure present in the central canal of the spinal cord, is a product of the condensation of specific glycoproteins that are released by specialized ependymal cells into the cerebrospinal fluid. These secretory ependymocytes constitute the subcommissural organ, a circumventricular organ that lines the roof of the third ventricle of the brain. The subcommissural organ/Reissner's fibre complex is a permanent structure in the vertebrate central nervous system. The addition of bovine Reissner's fibre itself or of soluble material released by Reissner's fibre to primary mixed cultures of chick cerebral cortical cells markedly enhances neuronal survival. The responsive cells have been identified as neurons by labelling them with antibodies to neurofilament proteins. This neuronal survival effect is dose-dependent and does not require the presence of serum in the culture medium. Affinity-purified polyclonal antibodies raised against bovine Reissner's fibre partially block the effect of Reissner's fibre on neuronal survival. These results suggest that Reissner's fibre is involved in developmental processes of the central nervous system.     

Maturation of vomeronasal receptor neurons in vitro by coculture with accessory olfactory bulb neurons

To analyze the mechanisms of perception and processing of pheromonal signals in vitro, we previously developed a new culture system for vomeronasal receptor neurons (VRNs), referred to as the vomeronasal pocket (VN pocket). However, very few VRNs were found to express the olfactory marker protein (OMP) and to have protruding microvilli in VN pockets, indicating that these VRNs are immature and that VN pockets are not appropriate for pheromonal recognition. To induce VRN maturation in VN pockets, we here attempted to coculture VN pockets with a VRN target-accessory olfactory bulb (AOB) neurons. At 3 weeks of coculture with AOB neurons, the number of OMP-immunopositive VRNs increased. By electron microscopy, the development of microvilli in VRNs was found to occur coincidentally with OMP expression in vitro. These results indicate that VRN maturation is induced by coculture with AOB neurons. The OMP expression of VRNs was induced not only by AOB neurons but also by neurons of other parts of the central nervous system (CNS). Thus, VRN maturation requires only CNS neurons. Since the maturation of VRNs was not induced in one-well separate cultures, the nonspecific induction of OMP expression by CNS neurons suggests the involvement of a direct contact effect with CNS in VRN maturation.     

Cholinergic differentiation in neurogenic basal forebrain cultures

To study early events in the central nervous system (CNS) cholinergic development, cells from rat basal fore brain tissue were placed in culture at an age when neurogenesis in vivo is still active [embryonic day (E) 15]. The rapid mortality of these cells in defined medium, with 50% mortality after 5–10 h, was blocked completely by soluble proteins from the olfactory bulb (a basal forebrain target), extending earlier observations (Lambert, Megerian, Garden, and Klein, 1988). Treated cultures were capable of incorporating thymidine into DNA, and most cells incorporating ³H-thymidine (>90%) also stained positive for neurofilament, confirming neuronal proliferation in the supplemented cultures. A small percentage of ³H-thymidine labelled cells were glial fibrillary acidic protein (GFAP) positive, but growth factors that support astroglial proliferation [epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), and insulin-like growth factor (IGF-1)] were not sufficient for neuronal support. After 5 culture days with supplemented medium, almost 50% of the cells showed choline acetyltransferase (ChAT) immunofluorescence. The cholinergic neurons typically formed clusters separate from noncholinergic cells. These mature cultures did not develop if young cultures were treated with aphidicolin to block DNA synthesis. The data show that cultures of very young rat basal forebrain cells can be neurogenic, giving rise to abundant cholinergic neurons, and that early cell proliferation is essential for long-term culture survival.     

Amino acid and energy requirements for rat hepatocytes in primary culture

Summary The amino acid and energy requirements of rat hepatocytes in suspension and early culture were investigated. Among a number of potential energy substrates tested, pyruvate (20 mM) was found to be most effective in stimulating hepatocytic protein synthesis. Amino acids stimulated protein synthesis both as energy substrates and as protein precursors. An amino acid mixture was designed to provide maximal inhibition of protein degradation as well as maximal stimulation of protein synthesis. In a defined medium containing amino acids at these concentrations, and supplemented with glucocorticoid hormone and insulin, hepatocytes could be maintained—on a collagen substratum—for at least a week without any significant net loss of cells or cellular protein. The work was supported by grants from The Norwegian Cancer Society and from The Norwegian Council for Science and the Humanities. An erratum to this article is available at .     

Selection of a super-growing legume root culture that permits controlled switching between root cloning and direct embryogenesis

 Root cultures, displaying vigorous growth and high embryogenic capacity, were established in the legume forage species Lotus corniculatus (bird’s-foot trefoil). Root cloning as well as plant regeneration was achieved on hormone-free medium, in agitated culture in the dark or under stationary conditions in the light, respectively. These qualities of vigorous growth and regeneration faded with time in hormone-free culture, with slow-growing roots turning brown in color. Addition of the synthetic cytokinin-like hormone benzylaminopurine to the culture medium, however, re-established the aging tissue’s capacity for somatic embryogenesis and plant formation. During continuous initiation of new cultures, it was possible to obtain one root culture (selected from 11 960 seeds at a 65% germination rate) which did not show the typical decline of qualities after prolonged proliferation but distinguished itself by displaying even faster growth and more vigorous embryogenic plant production on hormone-free medium. There was no decline since its initiation 9 months earlier. This super-growing root culture produces plants that show no morphological differences as compared to wild-type regenerants or seedlings. Roots, dissected from plantlets derived from super-root embryogenesis, expressed all the super-root qualities again when cultured in vitro. This is the first report on somatic embryogenesis from sustained root cultures without exogenous hormone application. Such a hormone-free, continuous root culture should provide a superior experimental system for genetic or developmental studies that might be sensitive to exogenous hormones, such as somaclonal variation in transgenesis or, since introduced in a legume species, nodulation in vitro. Received: 22 September 1997 / Accepted: 21 October 1997     

Effects of triiodothyronine on the synthesis of sulfolipids by oligodendrocyte-enriched glial cultures

Glial cultures were obtained from the brains of 1-week-old rats and were grown in a chemically defined, serum-free medium. We investigated the development of oligodendrocytes in these cultures and the synthesis of sulfolipids in the presence and absence of triiodothyronine (T3) in the medium: (1) In the presence of T3, the incorporation of [35S]sulfate into sulfolipids exhibited a developmental profile which is comparable to that found in the developing brain in vivo. A sharp peak of sulfolipid synthesis was observed at day 5 in vitro, which is equivalent to day 12 after birth. As observed in vivo, the percentage of label incorporated into sulfogalactosyldiradylglycerols decreased with time in culture. (2) Addition of T3 to the medium stimulated sulfolipid synthesis by oligodendrocytes in a dose-related manner (optimal T3 concentration, 30 nM). The hormone also enhanced the rates of cholesterogenesis and lipogenesis but to a lesser extent than sulfolipid synthesis. (3) The temporary omission of T3 from the medium resulted in lower rates of sulfolipid synthesis that could not be restored by readdition of T3. This inhibitory effect was most pronounced if the hormone was omitted from the medium on days 2 and 3 in culture. (4) Omission of T3 also resulted in the development of fewer oligodendrocytes in the cultures. Our results show that T3 is essential for the development of oligodendrocytes in our neurone-free culture system. They also indicate that the stimulation of myelination by thyroid hormones can, at least partially, be explained as a direct effect of T3 on oligodendrocytes, independent of an effect of T3 on neuronal growth.     

A new culturing strategy optimises Drosophila primary cell cultures for structural and functional analyses

Neurons in primary cell cultures provide important experimental possibilities complementing or substituting those in the nervous system. However, Drosophila primary cell cultures have unfortunate limitations: they lack either a range of naturally occurring cell types, or of mature physiological properties. Here, we demonstrate a strategy which supports both aspects integrated in one culture: Initial culturing in conventional serum-supplemented Schneider's medium (SM(20K)) guarantees acquisition of all properties known from 30 years of work on cell type-specific differentiation in this medium. Through subsequent shift to newly developed active Schneider's medium (SM(active)), neurons adopt additional mature properties like the ability to carry out plastic morphological changes, neurotransmitter expression and electrical activity. We introduce long-term FM-dye measurements as a tool for Drosophila primary cell cultures demonstrating the presence of increased, action potential-dependent synaptic activity in SM(active). This is confirmed by patch-clamp recordings, which in addition show that SM(active)-cultured neurons display different spiking patterns. Furthermore, we demonstrate that transmission can be evoked in SM(active) cultures, revealing the existence of synaptic plasticity. Thus, these culture conditions support developmental, structural and physiological properties known or expected from the nervous system, enhancing possibilities for future experiments complementing or substituting those in nervous systems of Drosophila.     

Identification,paracrine generation,and possible function of human breast carcinoma myofibroblasts in culture

Summary Myofibroblasts from human breast carcinomas were identified and experimentally generated in culture, and a possible function was examined. The frequency ofα-smooth muscle actin immunoreactive cells was evaluated as a measure of myofibroblast differentiation in primary culture. Few or noα-smooth muscle actin-positive stromal cells (6.1 ± 8.4%) were identified in primary cultures from normal breast tissue (n=9). In contrast, high frequencies (68.8 ± 15.1%) were observed in primary cultures from carcinomas (n=19). The frequencies of myofibroblasts in primary cultures were almost identical to those obtained in the corresponding cryostat sections (69.1 vs. 68.8%). A possible precursor cell to the myofibroblast was looked for among typical fibroblasts and vascular smooth muscle cells. Purified blood vessels containing both fibroblasts and vascular smooth muscle cells were embedded in collagen gel and incubated with medium conditioned by breast epithelial cells. Fibroblasts rather than smooth muscle cells were recruited from the blood vessels. In medium conditioned by carcinoma cell lines or in co-cultures of carcinoma cell lines and purified fibroblasts,α-smooth muscle actin and the typical myofibroblast phenotype were induced in otherwiseα-smooth muscle actin-negative fibroblasts. The effect of myofibroblasts on cellular movement—essential to neoplastic cells—was analyzed. Spontaneous motility of tumor cells (MCF-7) was entirely suppressed in a collagen gel assay. Under these conditions tumor cell motility was selectively mediated by direct cell-to-cell interaction between tumor cells and myofibroblasts. Under chemically defined conditions, interaction was dependent on the presence of plasminogen. Anti-plasminogen, soybean trypsin inhibitor, and anti-fibronectin partly neutralized the effect of plasminogen. It is concluded that elements of myofibroblast differentiation and function may be studied in culture.     

Identification of a neurite outgrowth-promoting domain of laminin using synthetic peptides

We have identified a synthetic peptide derived from the B2-chain of mouse laminin, Arg-Asn-Ile-Ala-Glu-Ile-Ile-Lys-Asp-Ile (p20), which stimulates the neurite outgrowth-promoting activity of the native molecule. In organotypic cultures, neurons from newborn mouse brain or embryonic peripheral nervous system responded by extensive neurite outgrowth for native laminin or the peptide p20 in the culture medium. If rat cerebellar neurons were grown on laminin, 1-5 microM (1-5 micrograms/ml) of peptide p20 in the culture medium competed with laminin and inhibited neuronal attachment and neurite outgrowth, whereas higher concentrations (greater than 50 microM; greater than 50 micrograms/ml) had a specific neurotoxic effect. When peptide p20 was used as the culture substratum, neurite outgrowth in cerebellar cultures was up to 60% of that seen on native laminin. Our results indicate that a neurite outgrowth-promoting domain of laminin is located in the alpha-helical region of the B2-chain, and is active for both central and peripheral neurons.     

Specific Insulin-Mediated Regulation of Glutamine Synthetase in Cultured Chick Astroglial Cells

The expression of glutamine synthetase (GS; L-glutamate ammonia ligase; EC 6.3.1.2) in primary cultures of chick astroglial cells and neurons grown in a chemically defined medium, with and without insulin added, was investigated. An inhibitory effect of insulin toward GS activity, and specific to chick astroglial cells, was observed. Neurons in culture were not sensitive to the hormone effect. Modulation of the activating effect of hydrocortisone on glial GS by insulin was also observed. The data suggest that insulin contributes to the regulation of the metabolism of amino acid neurotransmitters via its effect on GS.     

The development of mouse spinal cord in tissue culture

Summary Neural tubes of mouse embryos at Theiler Stages 14, 15, and 16 were grown in cultures for 21 d with 0.5 μCi/ml tritiated thymidine or cold growth medium. It was found that 50 to 60% of the neurons formed in the outgrowth zone were labeled, indicating that they formed from precursor cells that proliferated in the cultures. The unlabeled neurons must have formed from cells that were already postmitotic when the cultures were started. By comparing the total number of neurons per neuromere formed in vivo and in vitro, it seems that the postmitotic precursor cells survive better in cultures and only a small percentage of proliferative precursor cells in cultures enter the postmitotic stage and form neurons. This work was supported by Grant MT4235 from the Medical Research Council of Canada.     

Neuronal survival in intact ciliary ganglia in vivo and in vitro: Ciliary neuronotrophic factor as a target surrogate

Ciliary neuronotrophic factor (CNTF) requirements for neuronal survival in the intact ciliary ganglion (CG) have been investigated in organ culture. Exogenous CNTF was not essential for neuronal survival until embryonic Day 8. Three-day cultures from 5-day ganglia were similar with or without CNTF, showing numerous neurons and extensive neuritic development. In 3-day cultures from 8-day-old ganglia, however, no neurons survived without CNTF, and the ganglia contained only nonneuronal cells and cell debris. Similar ganglia cultured with CNTF contained many neurons, surrounded by nonneuronal cells, and abundant neuritic processes. Morphologic maturation of the neurons was less advanced in CNTF-supported ganglia than in their in vivo counterparts.