Progesterone as a neuroactive neurosteroid, with special reference to the effect of progesterone on myelination

Some steroids are synthesized within the central and peripheral nervous system, mostly by glial cells. These are known as neurosteroids. In the brain, certain neurosteroids have been shown to act directly on membrane receptors for neurotransmitters. For example, progesterone inhibits the neuronal nicotinic acetylcholine receptor, whereas its 3alpha,5alpha-reduced metabolite 3alpha, 5alpha-tetrahydroprogesterone (allopregnanolone) activates the type A gamma-aminobutyric acid receptor complex. Besides these effects, neurosteroids also regulate important glial functions, such as the synthesis of myelin proteins. Thus, in cultures of glial cells prepared from neonatal rat brain, progesterone increases the number of oligodendrocytes expressing the myelin basic protein (MBP) and the 2',3'-cyclic nucleotide-3'-phosphodiesterase (CNPase). An important role for neurosteroids in myelin repair has been demonstrated in the rodent sciatic nerve, where progesterone and its direct precursor pregnenolone are synthesized by Schwann cells. After cryolesion of the male mouse sciatic nerve, blocking the local synthesis or action of progesterone impairs remyelination of the regenerating axons, whereas administration of progesterone to the lesion site promotes the formation of new myelin sheaths.     

Studies on neurosteroids XVII. Analysis of stress-induced changes in neurosteroid levels in rat brains using liquid chromatography-electron capture atmospheric pressure chemical ionization-mass spectrometry

The analysis of stress-induced changes in the brain neurosteroid levels by liquid chromatography (LC)-electron capture atmospheric pressure chemical ionization-mass spectrometry (ECAPCI-MS) is described. In the present method, neurosteroids were derivatized with a highly electron-affinitive reagent, 2-nitro-4-trifluoromethylphenylhydrazine (NFPH), to convert them to the corresponding hydrazones. The derivatized steroids showed over a 20-fold higher sensitivity in ECAPCI-MS than intact steroids measured by positive atmospheric pressure chemical ionization (APCI)-MS. Application of this method to the analysis of rat brain samples confirmed the significant increase in the levels of pregnenolone (PREG), progesterone (PROG), 5alpha-dihydroprogesterone (DHPROG), allopregnanolone (3alpha-hydroxy-5alpha-pregn-20-one; AP), and epiallopregnanolone (3beta-hydroxy-5alpha-pregn-20-one; EpiAP) in the fixated rats. The din stress, which we examined as a new short-term mental stress model, also elevated the brain neurosteroid levels. It is known that various types of stress lower the gamma-aminobutyric acid type A (GABA(A)) receptor function and induce the neuronal overexcitation. The increase in the brain level of AP, a potent positive modulator of GABA(A) receptors, may be the defensive response against acute stress. The increase in the brain concentration of its precursors, PREG, PROG, and DHPROG, may be associated with the acceleration of the AP synthesis. Thus, the present studies suggest that changes in the brain levels of neurosteroids may play an important role in the homeostatic mechanisms that counteract the inhibitory effect of stress on the GABA(A) receptor function.     

The effect of dehydroepiandrosterone sulfate and allopregnanolone sulfate on the binding of [(3)H]ifenprodil to the N-methyl-d-aspartate receptor in rat frontal cortex membrane

Neurosteroids have been shown to modulate the N-methyl-d-aspartate (NMDA) receptor function. Dehydroepiandrosterone sulfate (DHEAS) is shown to participate in memory and learning processes as well as preventing glutamate neurotoxicity in hippocampus. In this study we have focused on the modulatory effect of neurosteroids on ifenprodil binding to the NR2B subunit of the NMDA receptor. We show that DHEAS and allopregnanolone sulfate (ALLOPREGS) exert different effects on the [(3)H]ifenprodil binding at 10, 30 or 100 nM, corresponding to physiological concentrations. The effects include changes in the ifenprodil displacement curve, changing it from a one-site fit into a two-site fit leaving B(max), K(d) and K(off) unaffected. Our results indicate that DHEAS and ALLOPREGS induce an allosteric modulation of the NMDA receptor, an observation that might contribute to the understanding of the effects of these neurosteroids.     

Fetal alcohol exposure alters neurosteroid modulation of hippocampal N-methyl-D-aspartate receptors

The actions of ethanol on brain ligand-gated ion channels have important roles in the pathophysiology of alcohol-related neurodevelopmental disorders and fetal alcohol syndrome. Studies have shown that N-methyl-d-aspartate (NMDA) receptors are among the ligand-gated ion channels affected by prenatal ethanol exposure. We exposed pregnant dams to an ethanol-containing liquid diet that results in blood ethanol levels near the legal intoxication limit in most states (0.08%). Primary cultures of hippocampal neurons were prepared from the neonatal offspring of these dams, and NMDA receptor function was assessed by patch clamp electrophysiological techniques after 6-7 days in culture in ethanol-free media. Unexpectedly, we did not detect any changes in hippocampal NMDA receptor function at either the whole-cell or single-channel levels. However, we determined that fetal alcohol exposure alters the actions of the neurosteroids pregnenolone sulfate and pregnenolone hemisuccinate, which potentiate NMDA receptor function. Western immunoblot analyses demonstrated that this alteration is not due to a change in the expression levels of NMDA receptor subunits. Importantly, in utero ethanol exposure did not affect the actions of neurosteroids that inhibit NMDA receptor function. Moreover, the actions of pregnenolone sulfate on type A gamma-aminobutyric acid and non-NMDA receptor function were unaltered by ethanol exposure in utero, which suggests that the alteration is specific to NMDA receptors. These findings are significant because they provide, at least in part, a plausible mechanistic explanation for the alterations in the behavioral responses to neurosteroids found in neonatal rats prenatally exposed to ethanol and to other forms of maternal stress (Zimmerberg, B., and McDonald, B. C. (1996) Pharmacol. Biochem. Behav. 55, 541-547).     

Acute effects of neurosteroids in a rodent model of primary paroxysmal dystonia

The pathophysiology of various types of dyskinesias, including dystonias, is poorly understood. Clinical and epidemiological studies in humans revealed that the severity of dyskinesias and the frequency of paroxysmal forms of the disease are altered by factors such as the onset of puberty, pregnancy, cyclical changes and stress, indicating an underlying hormonal component. The dystonic phenotype in the dt(sz) hamster, a genetic animal model of paroxysmal dystonia, has been suggested to be based on a deficit of striatal gamma-aminobutyric acid (GABA)ergic interneurons and changes in the GABA(A) receptor complex. In this animal model, hormonal influences seem to be also involved in the pathophysiology, but an influence of peripheral sex hormones has already been excluded. Possibly, neurosteroids as endogenous regulators of the GABA(A) receptor may be critically involved in the pathophysiology of dystonia in this animal model. Therefore, in the present study, the effects of the neurosteroids allopregnanolone acetate and allotetrahydrodeoxycorticosterone (THDOC), representing positive modulators of the GABA(A) receptor, as well as of the negative GABA(A) receptor modulators pregnenolone sulfate and dehydroepiandrosterone (DHEA), on severity of dystonia were examined in dt(sz) hamsters after acute intraperitoneal injections. Allopregnanolone acetate and THDOC exerted a moderate reduction of dystonia, whereas pregnenolone sulfate and DHEA had no significant effects. Although the effects of allopregnanolone acetate and THDOC were moderate and short-lasting, the present results suggest that changes in neurosteroid levels might be involved in the initiation of dystonic episodes. Future studies have to include measurements of brain neurosteroid levels as well as of chronic neurosteroid administrations to clarify the pathophysiological role and therapeutic potential of neurosteroids in dystonia.     

Neuroactive steroids.

Neuroactive steroids are natural or synthetic steroids that rapidly alter the excitability of neurons by binding to membrane-bound receptors such as those for inhibitory and (or) excitatory neurotransmitters. The best-studied neuroactive steroids are a series of sedative-hypnotic 3 alpha-hydroxy ring A-reduced pregnane steroids that include the major metabolites of progesterone and deoxycorticosterone, 3 alpha-hydroxy-5 alpha-pregnan-20-one (allopregnanolone) and 3 alpha,21-dihydroxy-5 alpha-pregnan-20-one (allotetrahydroDOC), respectively. These 3 alpha-hydroxysteroids do not interact with classical intracellular steroid receptors but bind stereoselectively and with high affinity to receptors for the major inhibitory neurotransmitter in brain, gamma-amino-butyric acid (GABA). Biochemical and electrophysiological studies have shown that these steroids markedly augment GABA-activated chloride ion currents in a manner similar (but not identical) to that of anesthetic barbiturates. Several steroids have also been observed to have convulsant or proconvulsant properties, including the synthetic amidine 3 alpha-hydroxy-16-imino-5 beta-17-azaandrostan-11-one (RU5135) and the natural sulfate esters of pregnenolone and dehydroepiandrosterone. Several of these have been shown to be bicuculline or picrotoxin-like GABAA receptor antagonists. Examples of steroids that alter neuronal excitability rapidly by augmenting or inhibiting excitatory amino acid receptor-mediated responses have also been reported. Recently, allopregnanolone and allotetrahydroDOC have also been measured in brain and plasma where their levels have been shown to fluctuate in response to stress and during the estrous and menstrual cycles of rats and humans, respectively. Although the major fraction of allopregnanolone in tissue, including brain, is of adrenal and/or ovarian origin, appreciable levels of allopregnanolone can still be measured in the brains of adrenalectomized and/or oophorectomized animals. Receptor-active neurosteroids may represent an important class of neuromodulators that can rapidly alter central nervous system excitability via novel nongenomic mechanisms.     

Roles of ionotropic glutamate receptors in early developing neurons derived from the P19 mouse cell line

We cultured a P19 mouse teratocarcinoma cell line and induced its neuronal differentiation to study the function of ionotropic glutamate receptors (GluRs) in early neuronal development. Immunocytochemical studies showed 85% neuronal population at 5 days in vitro (DIV) with microtubule-associated protein 2-positive staining. Thirty percent and 50% of the cells expressed the alpha-amino-3-hydroxy-5-methyl-4-isopropinonate (AMPA) receptor subunit, GluR2/3, and the kainate (kainic acid; KA) receptor subunit, GluR5/6/7, respectively. In Western blot analysis, the temporal expression of GluR2/3 began to appear at 3 DIV, whereas GluR5/6/7 was already expressed in the undifferentiated cells. P19-derived neurons began to respond to glutamate, AMPA and KA, but not to the metabotropic GluR agonist trans-1-aminocyclopentane-1,3-decarboxylic acid, by 5 DIV in terms of increases in intracellular calcium and phospholipase C-mediated poly-phosphoinositide turnover. Furthermore, KA reduced cell death of P19-derived neurons in both atmospheric and hypobaric conditions in a phospholipase C-dependent manner. The common AMPA/KA receptor antagonist, 6-cyano-7-nitroquinoxaline-2,3-dione, but not the AMPA receptor antagonist, 1,2,3,4-tetrahydro-6-nitro-2,3-dioxo-benzo[f]quinoxaline-7-sulfonamide disodium, profoundly increased hypobaric insult-induced neurotoxicity. In a flow cytometry study, the nerve growth factor-mediated antiapoptotic effect was facilitated by AMPA, with an induction of TrkA, but not p75(NTR) expression. Therefore, AMPA and KA receptors might mediate neurotrophic functions to facilitate neurotrophic factor signaling to protect neurons against hypoxic insult in early neuronal development.     

Dehydroepiandrosterone with other neurosteroids preserve neuronal mitochondria from calcium overload

This current study was designed to test whether the dehydroepiandrosterone (DHEA) and other neurosteroids could improve mitochondrial resistance to ischemic damage and cytoplasmic Ca(2+) overload. To imitate these mechanisms at mitochondrial level we treated the saponin permeabilized neurons either with the respiratory chain inhibitor, 1-methyl-4-phenylpyridinium or raised free extra-mitochondrial [Ca(2+)]. Loss of mitochondrial membrane potential (as an indicator of loss of function) was detected by JC-1. The results demonstrate that DHEA partly prevented Ca(2+) overload induced loss of mitochondrial membrane potential but not the loss of potential induced by the inhibitor of the respiratory chain. A similar effect was observed in the presence of other neurosteroids, pregnenolone, pregnanolone and allopregnanolone. DHEA inhibited also the Ca(2+) accumulation to the mitochondria in the presence of Ca(2+) efflux inhibitors. Thus, in the present work we provide evidence that DHEA with several other neurosteroids protect the mitochondria against intracellular Ca(2+) overload by inhibiting Ca(2+) influx into the mitochondrial matrix.     

神经活性甾体别孕烯醇酮对脑皮质神经元损伤的保护作用

目的：利用N-甲基-D-天门冬氨酸（NMDA）诱发新生小鼠脑皮质神经元损伤模型,探讨神经活性甾体别孕烯醇酮对脑皮质神经元的保护作用及其机制。方法：应用RT-PCR和Western blot法检测别孕烯醇酮对β2-γ-氨基丁酸受体（β2-GABA-R）表达和对蛋白激酶B（PKB,又称为Akt）磷酸化的影响。应用Western blot和DNA-Ladder方法检测NMDA诱发的神经元凋亡及别孕烯醇酮对NMDA诱发凋亡的影响。结果：Western blot和RT-PCR分析表明0.5×10-6mol/L-5×10-6mol/L别孕烯醇酮使Akt磷酸化增加并促进β2-GABA-R mRNA的表达。1×10-6mol/L别孕烯醇酮预处理小鼠脑皮质神经元有抗凋亡作用,但5×10-6mol/L别孕烯醇酮预处理小鼠脑皮质神经元使NMDA诱发的DNA-Ladder减弱明显,并能有效抵抗NMDA诱发的活化型PRAP、Caspase-3、Caspase-9的增加。结论：别孕烯醇酮可通过促进β2-GABA-R表达和增加Akt磷酸化抵抗NMDA诱发的脑皮质神经元凋亡。     

Quantification of neurosteroids in rat plasma and brain following swim stress and allopregnanolone administration using negative chemical ionization gas chromatography/mass spectrometry

A simplified method for the quantitative analysis of neurosteroids in rat plasma and brain is described. The method uses negative chemical ionization gas chromatography/mass spectrometry and involves the synthesis of pentafluorobenzyloxime/trimethylsilyl ether derivatives with excellent chromatographic and electron-capturing properties. Deuterium-labeled analogs of the steroids of interest were synthesized and used as internal standards. The steroids (allopregnanolone, epiallopregnanolone, pregnenolone, testosterone, and dehydroepiandrosterone) were isolated from the plasma or brain matrix by a rapid and straightforward solid-phase extraction procedure. The mass spectrometer was operated in a selective ion monitoring mode, allowing for picograms of neurosteroids to be quantified from biological extracts. The method was linear (typical R(2) = 0.999) over the concentration range (100 to 8000 pg from 0.3 ml plasma and 250 to 8000 pg from 100 mg brain tissue) with good precision and accuracy. In experimental protocols, the procedure was suitable for measuring concentrations of endogenous neurosteroids in rat plasma and brain. Significant elevations (P < 0.001) were observed in the frontal cortex for allopregnanolone and pregnenolone following a swim stress and for allopregnanolone and epiallopregnanolone following allopregnanolone injection (8 mg/kg, sc). The present method allows accurate determination of neurosteroids and will be helpful in elucidating the role of neurosteroids in health and disease.     

Modulation of GABA(A) receptor function by neuroactive steroids: evidence for heterogeneity of steroid sensitivity of recombinant GABA(A) receptor isoforms

Neuroactive steroids are potent, selective allosteric modulators of gamma-aminobutyric acid type A (GABA(A)) receptor function in the central nervous system, and may serve as endogenous anxiolytic and analgesic agents. In order to study the influence of subunit subtypes of the GABA(A) receptor on modulation of receptor function by neuroactive steroids, we expressed human recombinant GABA(A) receptors in Xenopus oocytes. GABA-activated membrane current, and the modulatory effects of the endogenous neurosteroid 5alpha-pregnan-3alpha-ol-20-one (allopregnanolone) and the synthetic steroid anesthetic 5alpha-pregnan-3alpha-ol-11,20-dione (alphaxalone) were measured using two-electrode voltage-clamp recording techniques. Allopregnanolone had similar effects to potentiate GABA-activated membrane current in the alpha1beta1gamma2L and alpha1beta2gamma2L receptor isoforms. In contrast, alphaxalone was much more effective as a positive allosteric modulator on the alpha1beta1gamma2L receptor isoform. In the absence of the gamma2L subunit subtype, allopregnanolone had much greater efficacy, but its potency was decreased. Allopregnanolone was much more effective on the alpha1beta1 receptor isoform compared with the alpha1beta2 receptor isoform. The potency for alphaxalone to potentiate the GABA response was not altered in the absence of the gamma2L subunit subtype, although its efficacy was greatly enhanced. Both allopregnanolone and alphaxalone produced nonparallel leftward shifts in the GABA concentration-response relationship in the absence of the gamma2L subunit, decreasing the EC50 concentration of GABA and increasing the maximal response. Only alphaxalone increased the maximal GABA response when the gamma2L subunit subtype was present. The 3beta-pregnane isomers epipregnanolone and isopregnanolone both inhibited the ability of allopregnanolone and alphaxalone to potentiate GABA(A) receptor function. However, the degree of block produced by the 3beta-pregnane steroid isomers was dependent on the type of receptor isoform studied and the neuroactive steroid tested. Isopregnanolone, the 3beta-isomer of allopregnanolone, was significantly more effective as a blocker of potentiation caused by allopregnanolone compared with alphaxalone in all receptor isoforms tested. Epipregnanolone had a greater efficacy as a blocker at the alpha1beta2gamma2L receptor isoform compared with the alpha1beta1gamma2L receptor isoform, and also produced a greater degree of block of potentiation caused by allopregnanolone compared with alphaxalone. Our results support the hypothesis that the heteromeric assembly of different GABA(A) receptor isoforms containing different subunit subtypes results in multiple steroid recognition sites on GABA(A) receptors, which in turn produces distinctly different modulatory interactions between neuroactive steroids acting at the GABA(A) receptor. The alpha and gamma subunit subtypes may have the greatest influence on allopregnanolone modulation of GABA(A) receptor function, whereas the beta and gamma subunit subtypes appear to be most important for the modulatory effects of alphaxalone.     

Platelet activating factor-induced neuronal apoptosis is initiated independently of its G-protein coupled PAF receptor and is inhibited by the benzoate orsellinic acid

The bioactive lipid mediator platelet activating factor (PAF) is recognized as a key effecter of neuronal apoptosis, yet it is not clear whether its G-protein coupled receptor (PAFR) initiates or prevents PAF neurotoxicity. Using PAFR-/- and congenic wild-type mice, we show that PAF triggers caspase-3/7 activity and neuronal death in PAFR-/- but not PAFR+/+ cerebellar granule neurons. Restoring receptor expression by recombinant adenoviral infection protected cells from PAF challenge. Neuronal death was not mediated by nitric oxide or N-methyl-d-aspartate receptor signaling given that N-nitro-l-arginine methyl ester and MK-801 did not inhibit PAF-induced neuronal loss in PAFR-/- neurons. To intervene in PAFR-independent neurotoxicity, the anti-apoptotic actions of three structurally distinct PAF antagonists were compared to a panel of plant and fungal benzoic acid derivatives. We found that the PAF antagonist BN 52021 but not FR 49175 or CV 3988 inhibited PAFR-independent neurotoxicity. Orsellinic acid, a fungal-derived benzoic acid, blocked PAF-mediated neuronal apoptosis without affecting PAFR-mediated neuroprotection. These findings demonstrate that PAF can transduce apoptotic death in primary neurons independently of its G-protein coupled receptor, that PAFR activation is neuroprotective, and that orsellinic acid effectively attenuates PAFR-independent neuronal apoptosis.     

In vivo evidence for the production of sulfated steroids in the frog brain

It is well established that sulfated neurosteroids are potent regulators of neuronal activity but the biosynthesis of sulfate esters of steroids in the central nervous system (CNS) has received little attention. In particular, the localization of hydroxysteroid sulfotransferase (HST), the enzyme which is responsible for the formation of sulfated steroids, has never been determined in the brain. We took advantage of the availability of an antiserum raised against rat liver HST to investigate the distribution of this enzyme in the CNS of the frog Rana ridibunda. Two populations of HST-positive neurons were localized in the anterior preoptic area and the magnocellular nucleus of the hypothalamus. Numerous HST-immunoreactive fibers were visualized throughout the telencephalon and the diencephalon. Reversed-phase high performance liquid chromatography (HPLC) analysis of frog telencephalon and hypothalamus extracts combined with radioimmunoasssay (RIA) detection showed the presence of substantial amounts of DHEAS-immunoreactive material which coeluted with synthetic DHEAS. The concentrations of DHEAS detected in the telencephalon and hypothalamus were respectively eight and five times higher than in the serum. The present study demonstrates the occurrence of HST-immunoreactive material in neurons of the frog telencephalon and diencephalon. This report also provides evidence for the presence of HST bioactivity, in vivo, in the frog brain.     

Neurosteroids: biosynthesis, metabolism and function of pregnenolone and dehydroepiandrosterone in the brain

Pregnenolone (P) and dehydroepiandrosterone (D) accumulate in the brain as unconjugated steroids and their sulfate (S) and fatty acid (L) esters. The microsomal acyl-transferase activity is highest in immature (1-3 weeks old) male rats. The immunocytochemical and biochemical evidence for P biosynthesis by differentiated oligodendrocytes is reviewed. The importance of P synthesis for its brain accumulation is assessed by the intracysternal injection of the inhibitor aminoglutethimide. Primary glial cell cultures convert P to 20-OH-P, PL, progesterone, 5 alpha-pregnane-3,20-dione and 3 alpha-hydroxy-5 alpha-pregnane-20-one (Polone). Astroglial cell cultures also produce these metabolites, whereas neurons from 17-day mouse embryos only form 20-OH-P. P and D are converted to the corresponding 7 alpha-hydroxylated metabolites by a very active P-450 enzyme from rat brain microsomes. Several functions of neurosteroids are documented. P decreases in olfactory bulb of intact male rats exposed to the scent of estrous females. D inhibits the aggressive behavior of castrated male mice towards lactating female intruders. The D analog 3 beta-methyl-androst-5-en-17-one, which cannot be metabolized into sex steroids and is not demonstrably androgenic or estrogenic is at least as efficient as D. Both compounds elicit a marked decrease of PS in rat brain. The Cl- conductance of gamma-aminobutyric (GABAA) receptor is stimulated by GABA agonists, an effect which is enhanced by Polone and antagonized by PS. Thus, P metabolites in brain as well as steroids of extraencephalic sources may be involved physiologically in GABAA receptor function. The neurosteroids accumulated in brain may be precursors of sex steroid hormones and progesterone receptors have been localized in glial cells. P and D do not bind to any known intracellular receptor. A heat stable P binding protein has been found in brain cytosol with distinct ligand specificity. A binding component specific for steroids sulfates, including Polone S, DS and PS, in the order of decreasing affinity is localized in adult rat brain synaptosomal membranes. Its relationship to the GABAA receptor is under current investigation.     

Retinoic acid-induced neural differentiation of embryonal carcinoma cells.

We have previously shown that the P19 line of embryonal carcinoma cells develops into neurons, astroglia, and fibroblasts after aggregation and exposure to retinoic acid. The neurons were initially identified by their morphology and by the presence of neurofilaments within their cytoplasm. We have more fully documented the neuronal nature of these cells by showing that their cell surfaces display tetanus toxin receptors, a neuronal cell marker, and that choline acetyl-transferase and acetyl cholinesterase activities appear coordinately in neuron-containing cultures. Several days before the appearance of neurons, there is a marked decrease in the amount of an embryonal carcinoma surface antigen, and at the same time there is a substantial decrease in the volumes of individual cells. Various retinoids were able to induce the development of neurons in cultures of aggregated P19 cells, but it did not appear that polyamine metabolism was involved in the effect. We have isolated a mutant clone which does not differentiate in the presence of any of the drugs which are normally effective in inducing differentiation of P19 cells. This mutant and others may help to elucidate the chain of events triggered by retinoic acid and other differentiation-inducing drugs.     

Retinoic acid induces embryonal carcinoma cells to differentiate into neurons and glial cells

Murine embryonal carcinoma cells can differentiate into a varied spectrum of cell types. We observed the abundant and precocious development of neuronlike cells when embryonal carcinoma cells of various pluripotent lines were aggregated and cultured in the presence of nontoxic concentrations of retinoic acid. Neuronlike cells were also formed in retinoic acid-treated cultures of the embryonal carcinoma line, P19, which does not differentiate into neurons in the absence of the drug. The neuronal nature of these cells was confirmed by their staining with antiserum directed against neurofilament protein in indirect immunofluorescence experiments. Retinoic acid-treated cultures also contained elevated acetylcholinesterase activity. Glial cells, identified by immunofluorescence analysis of their intermediate filaments, and a population of fibroblastlike cells were also present in retinoic acid-treated cultures of P19 cells. We did not observe embryonal carcinoma, muscle, or epithelial cells in these cultures. Neurons and glial cells appeared in cultures exposed to retinoic acid for as little as 48 h. We found no evidence for retinoic acid toxicity, suggesting that the effect of the drug was to induce the development of neurons and glia rather than to select against cells differentiating along other developmental pathways.     

Characterization of neurotransmitter phenotype during neuronal differentiation of embryonal carcinoma cells

Embryonal carcinoma cells are useful in the study of embryogenesis and development, and their differentiation into neurons serves as a model of neuronal development. Retinoic acid was used to differentiate P19S18O1A1 embryonal carcinoma cells into neuronal, glial, and fibroblast-like cells and the phenotype of the neuronal population was examined. Neuron-specific enolase was present in the neuronal cells, suggesting that these neurons had reached some degree of maturity. A population (approximately 70%) of the neurons showed positive immunocytochemistry for tyrosine hydroxylase, dopamine beta-hydroxylase and phenylethanolamine N-methyltransferase, three enzymes in the pathway of catecholamine synthesis. Therefore a population of the neurons appeared to be adrenergic. These neurons also showed a low level of histofluorescence for endogenous catecholamines and exhibited an exogenous catecholamine reuptake system. In order to determine the phenotype of other neuron-like cells found to be negative for the adrenergic properties examined, immunocytochemistry for neuropeptides and neurotransmitters known to coexist within central neurons was performed. Serotonin, vasoactive intestinal peptide, glutamic acid decarboxylase, and choline acetyltransferase were all absent from retinoic acid-treated P19S18O1A1 neuronal cultures. These studies, along with those that compare the effects of retinoic acid and other growth modulators on neuronal differentiation of embryonal carcinoma cells, should aid in the understanding of neuronal induction and development in vivo.