ANTI-WHOLE WHITE MATTER SERUM INHIBITS INCORPORATION OF GLUCOSE AND GALACTOSE INTO THE LIPIDS OF MYELINATING SPINAL CORD CULTURES

Myelin formation was inhibited in fetal mouse spinal cord cultures in the presence of serum from rabbits with experimental allergic encephalomyelitis produced by inoculation of whole bovine spinal cord white matter in complete Freund's adjuvant. Controls were exposed to decomplemented serum. Replacement of serum in inhibited cultures on the 18th day in vitro (DIV) with control serum (disinhibited) resulted in the appearance of visible myelin within 2–3 days. From 20 to 23 DIV, d -[U-¹⁴C]glucose or d -[U-¹⁴C]galactose was present in all media. Total protein, DNA, gangliosides and galactolipids were reduced by 21% in inhibited cultures, and activity of 2′,3′-cyclic nucleotide 3′-phosphohydrolase was reduced by 50%. There was little reduction in the incorporation of glucose carbon (21–23 DIV) into several lipid classes examined. Labelling of cerebrosides by galactose carbon in inhibited cultures was only 12% of that of controls while there was no reduction in the labelling of neutral lipid–cholesterol and the glycerophosphatides. Galactolipid labelling by [¹⁴C]galactose in the disinhibited cultures was intermediate between inhibited and control cultures. Differences in the effects of inhibiting medium on the incorporation of glucose and galactose carbon indicate that ceramide synthesis is less affected than is galactose incorporation to form cerebroside.     

Characterization of oligodendrocytes in primary cultures from brain hemispheres of newborn rats

Characterization of putative oligodendrocytes obtained in primary cultures of brain hemispheres from newborn rats is reported. Most of the oligodendrocytes are scattered in the culture dish until around 20 days after seeding, the time at which they start to form aggregates made up of one to three layers of cells upon the astrocytes. At the electron microscopic level the oligodendrocytes ultrastructure appears undifferentiated but very different from that of the underlying astrocytes. These oligodendrocytes do not react to W1 Wolfgram protein and myelin basic proteins antisera until the sixth day after seeding. On Day 8, a few oligodendrocytes give a positive reaction; after 4 weeks most of them react. These results represent a further step in the identification of oligodendrocytes in culture and in the characterization of their development in vitro.     

PROTEIN PROFILES OF RAT EMBRYO CEREBRAL CELLS DURING DIFFERENTIATION IN CULTURE

The protein composition of the particulate fraction of dissociated foetal rat cerebral cells during maturation in culture was investigated. SDS polyacrylamide gel electrophoresis showed a general decrease in the histonal components and significant changes in composition of a group of polypeptides with molecular weights ranging from 42 to 60 K. Two of these polypeptides coelectrophoresed with tubulin and actin whereas a 48 K polypeptide comigrated with the major component of the Wolfgram myelin protein. Its relative quantity appeared to approach a plateau after 8 days in culture. The myelin basic and proteolipid proteins were below detection levels in cultured cells at any time point investigated. A group of polypeptides with estimated molecular weights of 47, 51 and 52 K possibly representing synaptic proteins increased with time in culture. The appearance of a prominent band (60 K) in brain cultures and in other cells of divergent origin was demonstrated. This protein may be related to the process of cell adaptation to culture conditions. The developmental changes in the protein profile are discussed in the context of an in vitro myelinogenesis and synaptogenesis and compared with whole brain particulate and subcellular fractions.     

BIOCHEMICAL CHARACTERIZATION OF A MYELIN FRACTION ISOLATED FROM RAT BRAIN AGGREGATING CELL CULTURES

Abstract— Subcellular fractions isolated from rat brain aggregating cell cultures were studied by electron microscopy and showed the presence of typical myelin membranes. The chemical composition of purified culture myelin was similar to the fraction isolated from rat brain in terms of CNP specific activity, protein and lipid composition. The ratio of small to large components of myelin basic protein was comparable in culture and in vivo. These two proteins incorporated radioactive phosphorus. The major myelin glycoprotein was present and during development in culture its apparent molecular weight decreased although it never reached the position observed in myelin isolated from adult rats. In culture, the yield of myelin did not increase substantially between 33 and 50 days and was comparable to that of 15-day-old rat brain. The ratio basic protein to proteolipid protein resembled immature myelin and the cerebroside content was very low. A 'floating fraction' was isolated from the cultures and contained some myelin but mostly single membranes. Although these results indicate that myelin maturation is delayed in vitro this culture system provides substantial amounts of purified myelin to allow a complete biochemical analysis and metabolic studies during development.     

Maternal deficiency of protein or protein and calories during lactation: effect upon CNS myelin subfraction formation in rat offspring.

The present study has examined the effects of maternal protein and protein-calorie deficiency during lactation on the development of CNS myelin subfractions in rat offspring. The offspring of both the protein and protein-calorie deficient rats had decreased brain and body weights, as well as delayed CNS myelination. Delayed active CNS myelination was demonstrated by the fact that 53-day-old nutritionally stressed pups incorporated significantly more [³H]leucine and [¹⁴C]glucose into all myelin subfractions than age-matched controls. Delayed myelination was also supported by the tremendous accretion of myelin proteins in the nutritionally deprived pups between 25 and 53 days of age. Despite the delayed active synthesis of myelin, the myelin deficit persisted in the offspring of protein deficient rats. These offspring had a deficiency of light + medium myelin throughout the study. At 17 days, both groups of nutritionally stressed rats had an excess of the high molecular weight proteins in heavy myelin. Heavy myelin from 17 day offspring of protein-calorie deficient rats had a deficiency of basic proteins, while that from the offspring of protein deficient rats had a deficiency of proteolipid protein. The protein composition of all myelin subfractions was normal at 53 days.     

Nuclear and mitochondrial DNA synthesis and energy metabolism in primary rat glial cell cultures

DNA synthesis in nuclei and mitochondria purified from serum-supplemented rat glial cell cultures at different days after plating was studied. Furthermore in mitochondria, some enzymatic activities related to energy transduction (citrate synthase, malate dehydrogenase, total NADH-cytochromec reductase, cytochrome oxidase and glutamate dehydrogenase) were measured. For DNA labeling [methyl-³H]thymidine was added to the culture medium at different days after plating. During the culture times studied the specific activity of total, nuclear, and mitochondrial DNA decreased from 8 days in vitro (DIV) to 21 DIV and increased at 30 DIV. The specific activity of nuclear DNA was always higher than that of mitochondrial DNA. The specific activity of the above mentioned mitochondrial enzymes increased from 8 DIV up to 21 DIV and decreased at 30 DIV, suggesting a relationship between the energy metabolism and the differentiation of glial cells in culture.The AA. would like to dedicate this paper to the memory of Dr. Ida Serra, Associate Professor of Biochemistry at the Medical Faculty, University of Catania, who prematurely died, after this paper was submitted for publication.     

CERAMIDE GLYCOSYLTRANSFERASES IN CULTURED RAT CEREBELLUM: CHANGES WITH AGE, WITH DEMYELINATION, AND WITH INHIBITION OF MYELINATION BY 5-BROMO-2''-DEOXYURIDINE OR EXPERIMENTAL ALLERGIC ENCEPHALOMYELITIS SERUM

—UDP-galactose:ceramide galactosyltransferase (CGalT) (E.C. 2.4.1.62) and UDP-glucose:ceramide glucosyltransferase (CGlcT) activities were measured in myelinating cultures of newborn rat cerebellum. Specific activities were measured at various days in vitro and the pattern of activities compared with that reported for in vivo tissue. Cultures demyelinated by incubation with media containing 22% serum from rabbits in which experimental allergic encephalomyelitis (EAE) was induced by injection with whole guinea-pig spinal cord, had 28% of CGalT specific activity and 86% of CGlcT specific activity measured in control cultures. Cultures in which myelination was inhibited by maintenance on media containing 0.15 mm -5-bromo-2′-deoxyuridine (BUdR) had 10% of CGalT specific activity and 118% of CGlcT specific activity of control cultures. Cultures in which myelination was inhibited by maintenance on media containing 2% EAE serum had 12% of the CGalT specific activity of control cultures. The data suggest that in vitro CGalT is predominantly a glial enzyme while CGlcT occurs primarily in neurons, and that the reduced CGalT activity may be involved in the mechanism of myelination inhibition by BUdR and by EAE serum.     

METHYLASES OF MYELIN BASIC PROTEIN AND HISTONE IN RAT BRAIN

—The two enzymes methylating myelin basic protein and histone were purified 170- and 250-fold respectively from the cell sap fraction of rat brain. These enzymes methylated only arginine residues of the two proteins. The enzyme activities were present in all organs tested. Testis has the highest, brain a moderate and liver the lowest activity. Most of the activities were present in the cell sap fraction in brain, liver and testis. Methylation of myelin basic protein and histone was examined in both the cell sap and solubilized nuclear fraction of rat brain during life span after birth. The myelin basic protein methylating activity in the cell sap fraction increased during myelination. Histone methylase from the nuclear fraction was highest at birth and dropped rapidly thereafter. The other activities remained unchanged. The natural occurrence of N^G-mono- and N^G,N^G-dimethylarginine residues in histones prepared from rabbit liver was demonstrated.     

Electric membrane properties of adult mouse DRG neurons and the effect of culture duration

The electrical membrane properties (EMP) of adult mouse dorsal root ganglion (DRG) neurons were characterized by an extensive electrophysiological investigation of 450 cells. The neurons were divided into two types: an M-type having an action potential with monophasic falling phase and a B-type with a more complex biphasic or triphasic falling phase. Compared to M-type, B-type were “slow” neurons with a higher specific membrane resistance (R_m), and a longer time constant (τ), duration of action potential (Δt), and absolute refractory period (ARP). B-type also had a larger amplitude action potential, afterhyperpolarization and positive overshoot. The action potential of the M-type neuron had only a Na⁺ component while that of the B-type had both a Na⁺ and a Ca²⁺ component. After two days in culture, M-type neurons exhibited phase bright cytoplasmic granules, which were seldom observed for B-type neurons. Although neuron survival remained constant during the first six days in culture (DIV), the relative frequency of occurrence of the M-type decreased from 82 to 50%. Thereafter, it decreased more gradually to a final value of approximately 20% after 40 DIV. It was concluded that at least during the first 6 DIV and possibly through to 40 DIV, M-type neurons transformed into B-type. Both M- and B-type neurons showed significant and similar changes in their EMP with increasing DIV (up to 40 DIV). For M- and B-types combined, R_m increased approximately 142%, τ by 204%, and no significant change in specific membrane capacitance was observed. Rheobasic threshold depolarization decreased 58%, while the resting membrane potential decreased by only 19%. These changes in the EMP of adult neurons are strikingly similar to changes in EMP observed in adult denervated muscle and in cultures of either embryonic nerve or muscle. This similarity suggested that the adult DRG neurons in cell culture undergo progressive dedifferentiation because of isolation from their usual trophic interactions. Determination of neuronal membrane electrical characteristics provides a new method for evaluating the effects of various possible trophic agents, e.g., hormones and tissue extracts, on the state of differentiation of neurons in cell culture.     

A small peptide mimetic of brain‐derived neurotrophic factor promotes peripheral myelination

The expression of the neurotrophins and their receptors is essential for peripheral nervous system development and myelination. We have previously demonstrated that brain‐derived neurotrophic factor (BDNF) exerts contrasting influences upon Schwann cell myelination in vitro – promoting myelination via neuronally expressed p75NTR, but inhibiting myelination via neuronally expressed TrkB. We have generated a small peptide called cyclo‐d PAKKR that structurally mimics the region of BDNF that binds p75NTR. Here, we have investigated whether utilizing cyclo‐d PAKKR to selectively target p75NTR is an approach that could exert a unified promyelinating response. Like BDNF, cyclo‐d PAKKR promoted myelination of nerve growth factor‐dependent neurons in vitro, an effect dependent on the neuronal expression of p75NTR. Importantly, cyclo‐d PAKKR also significantly promoted the myelination of tropomyosin‐related kinase receptor B‐expressing neurons in vitro, whereas BDNF exerted a significant inhibitory effect. This indicated that while BDNF exerted a contrasting influence upon the myelination of distinct subsets of dorsal root ganglion (DRG) neurons in vitro, cyclo‐d PAKKR uniformly promoted their myelination. Local injection of cyclo‐d PAKKR adjacent to the developing sciatic nerve in vivo significantly enhanced myelin protein expression and significantly increased the number of myelinated axons. These results demonstrate that cyclo‐d PAKKR promotes peripheral myelination in vitro and in vivo, suggesting it is a strategy worthy of further investigation for the treatment of peripheral demyelinating diseases.     

Development of gabaergic connections in vitro: Increasing efficacy of synaptic transmission is not accompanied by changes in miniature currents

Development of inhibitory synaptic transmission was studied using a dissociated cell culture from the superior colliculus of neonatal rat. Patch-clamp recordings in the whole-cell configuration were performed to measure evoked (single-cell-activated) inhibitory postsynaptic currents (IPSCs), miniature IPSCs and current responses to maximal concentrations of exogenous γ-aminobutyric acid (GABA). Over a period of 3 weeks in vitro (DIV3-24), the fraction of synaptically coupled neurons raised from 0% to 76%. Evoked IPSCs were first observed at DIV5. They had an average amplitude of 33.9 pA during the first week (n = 13) and 129.7 pA during the fourth week (n = 48). This increase by a factor of 3.8 represents a significant rise in the efficacy of GABAergic transmission during in vitro development. However, no developmental change has been observed in the average amplitudes of miniature somatic IPSCs. The latter remained at an average level of about 9 pA (symmetrical chloride concentration and a driving force of 68 mV). No increase was found also in whole-cell current densities induced by saturating concentrations of exogenous GABA. Our results suggest that under the given conditions, synapse maturation was primarily the result of presynaptic sprouting. This conclusion is further supported by bouton counts in immunostained collicular cultures, where the number of axosomatic and axodendritic GABAergic contacts per neuron increased from 0.54 and 0.37, respectively, at DIV3, to 13.84 and >23.1, at DIV24. The overall density of GABAergic neurons decreased during this period from about 41,000/cm² to 15,600 cm², indicating that a growing number of contacts is formed by a declining number of presynaptic neurons. © 1992 John Wiley & Sons, Inc.     

Human schwann cells in vitro. II. Myelination of sensory axons following extensive purification and heregulin-induced expansion

Co-culture conditions are well established in which Schwann cells (SCs) derived from immature or adult rats proliferate and form myelin in response to contact with sensory axons. In a companion article, we report that populations of adult-derived human Schwann cells (HASCs) fail to function under these co-culture conditions. Furthermore, we report progressive atrophy of neurons in co-cultures containing populations of either human fibroblasts. Two factors that might account for the insufficiency of the co-culture system to support HASC differentiation are the failure of many HASCs to proliferate and the influence of contaminating fibroblasts. To minimize fibroblast contamination of neuron-HASC co-cultures, we used fluorescence-activated cell sorting to highly purify HASC populations (to more than 99.8%). To stimulate expansion of the HASC population, a mitogenic mixture of heregulin (HRGβ1 amino acid residues 177-244; 10 nM), cholera toxin (100 ng/mL), and forskolin (1 μM) was used. When these purified and expanded HASCs were co-cultured with embryo-derived rat sensory neurons, neuronal shrinkage did not occur and after 4 to 6 weeks some myelin segments were seen in living co-cultures. This myelin was positively identified as human by immunostaining with a monoclonal antibody specific to the human peripheral myelin protein P₀ (antibody 592). Although this is the first reported observation of myelination by HASCs in tissue culture, it should be noted that myelination occurred more slowly and in much less abundance than in comparable cultures containing adult rat-derived SCs. We anticipate that further refinements of the HASC co-culture system that enhance myelin formation will provide insights into important aspects of human SC biology and provide new opportunities for studies of human peripheral neuropathies. © 1995 John Wiley & Sons, Inc.     

Brain reaggregate cultures: Biochemical evidence for myelin membrane synthesis

Myelin membrane synthesis was studied using mechanically dissociated fetal rodent CNS which formed spherical reaggregates while being maintained in rotating culture flasks. These reaggregate cultures exhibited myelinogenesis in vitro after precisely the same period of time needed for myelin synthesis to commence in vivo. The myelin membrane related enzymes, 2′,3′ cyclic nucleotide phosphohydrolase (CNP) and cerebroside sulfotransferase (CST), appear similar in their specific activities and follow the same developmental patterns that these enzymes exhibit in vivo. In addition, phosphorylation of myelin basic protein occurs by the third week in vitro which agrees with previously published in vivo studies. These experiments indicate that this nerve-cell culture system may be an appropriate model for studying the biological regulation of myelinogenesis as well as a variety of other nervous-system functions.     

COMPOSITION OF CEREBRAL LIPIDS IN MURINE LEUCODYSTROPHY: THE QUAKING MUTANT

The composition of sphingolipids and phospholipids of mouse brain during myelination was determined in the Quaking mutant, which manifests a genetic disorder of myelin formation, and in littermate controls. The biochemical changes during myelination in the brains of the controls corresponded quantitatively with previous findings in a different strain of mice. The Quaking mutant exhibited concentrations of sphingolipids and phospholipids in brain which were comparable to those of controls in the early stage of myelination but the tissue content failed to increase with maturation. The greatest differences occurred in the cerebrosides which at 65 days of postnatal age were only 10 per cent of control levels. During development the pattern of cerebral levels of sphingomyelin, plasmalogen and total phospholipid in the mutants tended to resemble that of the cerebrosides. The defect in the Quaking mutant is compatible with a failure in maturation of myelin. These findings have been compared with those in the Jimpy mutant, a different genetic disorder of myelin in the mouse previously studied in a similar fashion. The Jimpy mutant is characterized by a quantitatively more pronounced deficiency of myelin lipids and a decline in cerebrosides during brain development.     

THE BIOSYNTHESIS AND CONCENTRATION OF GALACTOSYL DIGLYCERIDE IN GLIAL AND NEURONAL ENRICHED FRACTIONS OF ACTIVELY MYELINATING RAT BRAIN

—In continuation of our studies on the association of the galactosyl diglycerides of brain with myelination, we have measured the biosynthesis and concentration of these glyceride glycolipids, in oligodendroglial, astroglial, neuronal, and myelin enriched fractions from brains of rats of postnatal age 16, 19 and 29 days. The relative purity of cell fractions and myelin derived from 50 to 60 brains of each age-group was checked by phase contrast microscopy and 2′,3′-cyclic nucleotide-3′-phosphohydrolase activity. The relative purity was comparable to that reported by other investigators for cell fractions from bovine brain. Of the three cell types, the oligodendroglia had the highest and the neurons had the lowest capacity to enzymatically synthesize and to accumulate monogalactosyl diglyceride. The amount of monogalactosyl diglyceride found in myelin compared to that found in oligodendroglial fraction greatly increased during development between 16 and 29 days of age. The biosynthesis of galactosyl ceramide but not glucosyl ceramide was highest in oligodendroglial enriched cell fraction. However, ceramide glucosyl-transferase activity, which was greatly affected by the method used for cellular separation, was highest in a microsomal fraction derived from grey matter. Our results support the contention that the oligodendroglial cells are the site of synthesis of myelin constituents of the central nervous system, and that there is a temporal relationship between this site of synthesis and the site of deposition (myelin).     

Death and survival of heterozygous Lurcher Purkinje cells In vitro

The differentiation and survival of heterozygous Lurcher (+/Lc) Purkinje cells in vitro was examined as a model system for studying how chronic ionic stress affects neuronal differentiation and survival. The Lurcher mutation in the δ2 glutamate receptor (GluRδ2) converts an orphan receptor into a membrane channel that constitutively passes an inward cation current. In the GluRδ2^+/Lc mutant, Purkinje cell dendritic differentiation is disrupted and the cells degenerate following the first week of postnatal development. To determine if the GluRδ2^+/Lc Purkinje cell phenotype is recapitulated in vitro, +/+, and +/Lc Purkinje cells from postnatal Day 0 pups were grown in either isolated cell or cerebellar slice cultures. GluRδ2^+/+ and GluRδ2^+/Lc Purkinje cells appeared to develop normally through the first 7 days in vitro (DIV), but by 11 DIV GluRδ2^+/Lc Purkinje cells exhibited a significantly higher cation leak current. By 14 DIV, GluRδ2^+/Lc Purkinje cell dendrites were stunted and the number of surviving GluRδ2^+/Lc Purkinje cells was reduced by 75% compared to controls. However, treatment of +/Lc cerebellar cultures with 1‐naphthyl acetyl spermine increased +/Lc Purkinje cell survival to wild type levels. These results support the conclusion that the Lurcher mutation in GluRδ2 induces cell autonomous defects in differentiation and survival. The establishment of a tissue culture system for studying cell injury and death mechanisms in a relatively simple system like GluRδ2^+/Lc Purkinje cells will provide a valuable model for studying how the induction of a chronic inward cation current in a single cell type affects neuronal differentiation and survival. © 2009 Wiley Periodicals, Inc. Develop Neurobiol, 2009     

Long‐term culture of mouse cortical neurons as a model for neuronal development,aging, and death

A long‐term cell culture system was used to study maturation, aging, and death of cortical neurons. Mouse cortical neurons were maintained in culture in serum‐free medium (Neurobasal supplemented with B27) for 60 days in vitro (DIV). The levels of several proteins were evaluated by immunoblotting to demonstrate that these neurons matured by developing dendrites and synapses and remained continuously healthy for 60 DIV. During their maturation, cortical neurons showed increased or stable protein expression of glycolytic enzyme, synaptophysin, synapsin IIa, α and β synucleins, and glutamate receptors. Synaptogenesis was prominent during the first 15 days and then synaptic markers remained stable through DIV60. Very early during dendritic development at DIV3, β‐synuclein (but not α‐synuclein) was localized at the base of dendritic growth cones identified by MAP2 and α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazole (AMPA) receptor GluR1. In mature neurons, α and β synucleins colocalized in presynaptic axon terminals. Expression of N‐methyl‐D ‐aspartate (NMDA) and AMPA receptors preceded the formation of synapses. Glutamate receptors continued to be expressed strongly through DIV60. Cortical neurons aging in vitro displayed a complex profile of protein damage as identified by protein nitration. During cortical neuron aging, some proteins showed increased nitration, while other proteins showed decreased nitration. After exposure to DNA damaging agent, young (DIV5) and old (DIV60) cortical neurons activated apoptosis mechanisms, including caspase‐3 cleavage and poly(ADP)‐ribose polymerase inactivation. We show that cultured mouse cortical neurons can be maintained for long term. Cortical neurons display compartmental changes in the localization of synucleins during maturation in vitro. These neurons sustain protein nitration during aging and exhibit age‐related variations in the biochemistry of neuronal apoptosis. © 2002 Wiley Periodicals, Inc. J Neurobiol 51: 9–23, 2002     

Myelination in developing cultured newborn rat cerebellum inhibited by 5-bromodeoxyuridine

Incorporation of 5-bromodeoxyuridine (BUdR) into developing newborn rat cerebellum organ cultures inhibited formation of myelin without apparent effect on morphological maturation. Inhibition of the appearance of myelin, which usually begins on 10–11 days in vitro (DIV), occurred only when BUdR was present during 6 and 7 DIV, thus suggesting a ‘critical’ cell division on those days for the differentiating oligodendrocytes that will produce myelin.     

ASTROGLIAL UPTAKE IS MODULATED BY EXTRACELLULAR K+

Abstract— Developmental changes of myelin proteins in chick sciatic nerve were studied at the stage of myelination by sodium dodecylsulfate (SDS)-polyacrylamide gel electrophoresis. The myelin of adult hen peripheral nervous system (PNS) contained two glycoproteins (BR-P0 and PASII), both of which are unique to PNS myelin, in addition to the basic encephalitogenic protein, BP, which is common to CNS and PNS myelin. The other basic protein (BF-P2) found in the PNS of other species was not definitely detectable in hen PNS. At the early stages of myelination (from 14 to 18 embryonic days) the amounts of myelin proteins increased rapidly in parallel with the increase in number of layers of the myelin sheath of the PNS. At 14 embryonic days high molecular weight proteins were dominant, while myelin specific proteins were barely detectable in the PNS myelin fraction. At 18 embryonic days, however, BR-PO, BP and PASII proteins became the main protein components of the PNS myelin, whereas high molecular weight proteins decreased in quantitative importance during development. At the early stage of myelination other glycoproteins were also detectable in the PNS myelin. Radioactive fucose was actively incorporated into the two glycoproteins, BR-P0 and PASII, at the early stage of myelination in vivo. These results suggested that myelin proteins especially glycoproteins, may play an important role in PNS myelin formation.     

Action of steroid hormones on growth and differentiation of CNS and spinal cord organotypic cultures

Summary During the prenatal period, gonadal steroid environment induces dramatic sexually dimorphic changes in the nervous system.We have usedin vitro methods to study the mechanism and timing of hormonal influences on neuronal sprouting and myelination during the prenatal period.Organotypic cultures of hypothalamus and lumbar spinal cord (SC) slices from rat fetuses were grown on plasma clot or in hyaluronic acid and exposed to estogen (17 estradiol) and testosterone (T) during cultivation. Both steroid hormones were active: 17 estradiol enhanced sprouting of hypothalamic neuronal fibers and increased the amount of synapses. In SC cultures T induced regeneration of thick nerve processes and an early onset of myelination, mainly of peripheral myelin.