Neuroinflammation and Brain Disease.

Starting from the perspective of an immune-privileged site, our knowledge of the inflammatory processes within the central nervous system has increased rapidly over the last 30 years, leading to a rather puzzling picture today. Of particular interest is the emergence of disease- and injury-specific inflammatory responses within the brain, which may form the basis for future therapeutic approaches. To advance this important topic, we invite authors to contribute research and clinical papers to the Collection "Neuroinflammation and Brain Disease".

CXCR4 and CXCR7 form a functional receptor unit for SDF-1/CXCL12 in primary rodent microglia.

AIMS: Microglial cells have been originally identified as a target for the CXC chemokine, SDF-1, by their expression of CXCR4. More recently, it has been recognized that SDF-1 additionally binds to CXCR7, which depending on the cell type acts as either a nonclassical, a classical or a scavenger chemokine receptor. Here, we asked whether primary microglial cells additionally express CXCR7 and if so how this chemokine receptor functions in this cell type. METHODS: CXCR4 and CXCR7 expression was analysed in cultured rat microglia and in the brain of animals with permanent occlusion of the middle cerebral artery (MCAO) by either Western blotting, RT-PCR, flow cytometry and/or immunocytochemistry. The function of CXCR4 and CXCR7 was assessed in the presence of selective antagonists. RESULTS: Cultured primary rat microglia expressed CXCR4 and CXCR7 to similar levels. Treatment with SDF-1 resulted in the activation of Erk1/2 and Akt signalling. Erk1/2 and Akt signalling were required for subsequent SDF-1-dependent promotion of microglial proliferation. In contrast, Erk1/2 signalling was sufficient for SDF-1-induced migration of microglial cells. Both SDF-1-dependent signalling and the resulting effects on microglial proliferation and migration were abrogated following pharmacological inactivation of either CXCR4 or CXCR7. Moreover, treatment of cultured microglia with lipopolysaccharide resulted in the co-ordinated up-regulation of CXCR4 and CXCR7 expression. Likewise, reactive microglia accumulating in the area adjacent to the lesion core in MCAO rats expressed both CXCR4 and CXCR7. CONCLUSIONS: CXCR4 and CXCR7 form a functional receptor unit in microglial cells, which is up-regulated during activation of microglia both in vitro and in vivo.

Etomidate reduces glutamate uptake in rat cultured glial cells: involvement of PKA.

BACKGROUND AND PURPOSE: Glutamate is the main excitatory neurotransmitter in the vertebrate CNS. Removal of the transmitter from the synaptic cleft by glial and neuronal glutamate transporters (GLTs) has an important function in terminating glutamatergic neurotransmission and neurological disorders. Five distinct excitatory amino-acid transporters have been characterized, among which the glial transporters excitatory amino-acid transporter 1 (EAAT1) (glutamate aspartate transporter) and EAAT2 (GLT1) are most important for the removal of extracellular glutamate. The purpose of this study was to describe the effect of the commonly used anaesthetic etomidate on glutamate uptake in cultures of glial cells. EXPERIMENTAL APPROACH: The activity of the transporters was determined electrophysiologically using the whole cell configuration of the patch-clamp recording technique. KEY RESULTS: Glutamate uptake was suppressed by etomidate (3-100 microM) in a time- and concentration-dependent manner with a half-maximum effect occurring at 2.4+/-0.6 microM. Maximum inhibition was approximately 50% with respect to the control. Etomidate led to a significant decrease of V(max) whereas the K(m) of the transporter was unaffected. In all cases, suppression of glutamate uptake was reversible within a few minutes upon washout. Furthermore, both GF 109203X, a nonselective inhibitor of PKs, and H89, a selective blocker of PKA, completely abolished the inhibitory effect of etomidate. CONCLUSION AND IMPLICATIONS: Inhibition of glutamate uptake by etomidate at clinically relevant concentrations may affect glutamatergic neurotransmission by increasing the glutamate concentration in the synaptic cleft and may compromise patients suffering from acute or chronic neurological disorders such as CNS trauma or epilepsy.

Cyclic AMP differentially regulates the expression of fibroblast growth factor and epidermal growth factor receptors in cultured cortical astroglia.

Fibroblast growth factor (FGF)-2 and transforming growth factor alpha (TGFalpha) promote astroglial proliferation during brain development and reactive processes. The mitogenic potential of both growth factors is attenuated by increasing intracellular cAMP levels, an effect currently assumed to depend on the inhibition of the mitogen-activated protein kinase cascade. In the present study, we sought to determine whether cAMP interferes with the mitogenic potential of FGF-2 and TGFalpha on astroglia by affecting the expression of respective growth factor receptors. Treatment of highly enriched cultures of cortical astrocytes with dibutyryl cAMP accelerated the TGFalpha-induced internalization and subsequent functional inactivation of epidermal growth factor (EGF) receptor by transiently inhibiting EGF receptor mRNA synthesis. In apparent contrast, both short- and long-term activation of cAMP-dependent signaling pathways robustly promoted the expression of FGF receptors 1 and 2, whereas expression levels of FGF receptor 3 remained unaffected. Moreover, elevation of intracellular cAMP levels did not prevent translocation of FGF receptor 1 to the cell nucleus, a mechanism thought to be essential for FGF-2-induced cell proliferation. We propose that cAMP controls the mitogenic effects of TGFalpha and FGF-2 on astroglial cells by distinctly different mechanisms. Whereas cAMP seems to interfere with the mitogenic effects of TGFalpha on astroglial cells by affecting both the expression level and signaling of the EGF receptor, the modulatory effects of cAMP on FGF-2-induced astroglial proliferation seem to solely result from an inhibition of FGF receptor-activated signaling pathways.

Evidence for a ligand-specific signaling through GFRalpha-1, but not GFRalpha-2, in the absence of Ret.

Glial cell line-derived neurotrophic factor (GDNF) and neurturin (NTN) are two homologeous proteins that have been recognized as potent survival factors for distinct neuronal populations. GDNF and NTN act through a two-component receptor system consisting of the ligand-specific binding subunits GDNF family receptor (GFR)alpha-1 and GFRalpha-2 and the common transducing subunit c-Ret. In addition, it has been demonstrated that GDNF can signal through GFRalpha-1 in the absence of c-Ret. In the present study, we sought to determine whether a similar c-Ret-independent signaling applies for GFRalpha-2. In addition, we have characterized the ligand specificity of the c-Ret-independent action of GFRalphas. To establish an assay system for these studies, several neural cell lines were screened for the presence of GDNF and NTN receptor subunits by RT-PCR and immunoblot analysis. c-Ret expression was detectable only in Neuro2A cells, which did not express GFRalpha-1 or GFRalpha-2. The neuronal cell line LS expressed GFRalpha-2, and the glial cell line Mes42 expressed GFRalpha-1, whereas the neuronal cell line B104 expressed both GFRalpha-1 and GFRalpha-2. Stimulation of B104 and Mes42 cells with GDNF, but not with NTN, for 10 min resulted in CREB phosphorylation. In apparent contrast, neither NTN nor GDNF promoted CREB activation in LS and Neuro2A cells. Moreover, exposure of LS cells to NTN or GDNF also failed to activate AKT and ERK. Together these findings provide evidence that, in contrast to GFRalpha-1, GFRalpha-2 fails to signal in the absence of c-Ret. In addition, these observations reveal that c-Ret-independent signaling of GFRalpha-1 is ligand- specific and occurs only with GDNF.

Estrogen stimulates brain-derived neurotrophic factor expression in embryonic mouse midbrain neurons through a membrane-mediated and calcium-dependent mechanism.

We have provided evidence that 17beta-estradiol (E) synthesized in the midbrain promotes the differentiation of midbrain dopamine neurons through nonclassical steroid action. Because these developmental effects resemble those reported for brain-derived neurotrophic factor (BDNF), we hypothesized that E influences dopaminergic cell differentiation through a BDNF-dependent mechanism. Competitive RT-PCR and ELISA techniques were employed to study first the developmental pattern of BDNF and trkB expression in the mouse midbrain. BDNF protein/mRNA levels peaked postnatally, whereas trkB did not fluctuate perinatally. To prove the hypothesis that E regulates BDNF expression in vivo, fetuses and newborns were treated with the aromatase antagonist CGS 16949A. CGS 16949A exposure reduced midbrain BDNF mRNA/protein levels. The coapplication of CGS 16949A and E abolished this effect. Midbrain cultures from mouse fetuses were used to investigate intracellular signaling mechanisms involved in transmitting E effects. Estrogen increased expression of BDNF but not of other neurotrophins. As concerns the related signaling mechanism, these effects were antagonized by interrupting intracellular Ca(2+) signaling with BAPTA and thapsigargin but not by the estrogen receptor antagonist ICI 182,780. Insofar as E effects on BDNF mRNA expression were inhibited by cycloheximide, it appears likely that other, not yet characterized intermediate proteins take part in the estrogenic regulation of BDNF expression. We conclude that E exerts its stimulatory effect on the differentiation of dopaminergic neurons by coordinating BDNF expression. This particular E effect appears to be transmitted through Ca(2+)-dependent signaling cascades upon activation of putative membrane estrogen receptors.

Cyclic AMP modulates the response of central nervous system glia to fibroblast growth factor-2 by redirecting signalling pathways.

Fibroblast growth factor-2 (FGF-2) acts as both a potent mitogen and differentiation factor for CNS glia. In the present study, we provide evidence that intracellular cAMP determines the proliferation-differentiation decision of astroglia to FGF-2 by either facilitating FGF-2 signalling to extracellular signal-related protein kinase (ERK) or cAMP response element binding protein (CREB). Pharmacologically increasing intracellular cAMP levels in cultured cortical astroglia by treatment with dbcAMP or forskolin attenuated FGF-2-induced ERK phosphorylation and glial cell proliferation. Similarly, FGF-2-induced glial proliferation was attenuated in the presence of the MEK inhibitor, PD98059, thus, confirming a direct correlation between FGF-2-induced ERK activation and glial cell proliferation. On the other hand, increases in intracellular cAMP levels in cortical astroglia prolonged FGF-2-induced CREB phosphorylation and subsequently potentiated the cAMP response element-dependent transcription of the immediate early gene, c-fos. Moreover, the effects of cAMP on the time-course of FGF-2-dependent CREB phosphorylation were mimicked by PD98059, suggesting that the cAMP-induced redirection of FGF-2-signalling is linked to the RAF-MEK-ERK signalling pathway.

Neurotrophin and GDNF expression increases in rat adrenal glands during experimental colitis.

OBJECTIVES: Neurotrophins and GDNF have been recently recognized as important local regulators of inflammatory processes of the gut. RESULTS: We now demonstrate that experimental TNBS-colitis is associated with the increased expression of neurotrophins and GDNF in the adrenal glands. In histological sections of the adrenals from untreated control animals, faint immunolabeling for BDNF, NT-3 and NGF was detectable in the adrenal cortex, with some additional labeling for NGF over the adrenal medulla, whereas GDNF immunolabeling was confined to the adrenal medulla. Induction of TNBS-colitis markedly increased NGF, BDNF, and NT-3 expression within the adrenal cortex after 8 h. NGF declined to basal levels after 7 days. In case of BDNF and NT-3 basal expression levels were reached after 14 days. GDNF expression was robustly upregulated in the adrenal medulla 8 h after induction of colitis and stayed elevated for up to 14 days. CONCLUSION: Together these observations suggest that neurotrophins and GDNF might act as local modulators of components of the HPA-axis during peripheral inflammation.

Neurotrophins require distinct extracellular signals to promote the survival of CNS neurons in vitro.

Althoughthe neurotrophins BDNF and NT-3 have been recognized as potent survival factors for distinct neuronal populations in the peripheral nervous system, they seem to have only minor effects on the survival of CNS neurons. In the present study, we provide evidence that BDNF and NT-3 require distinct additional extracellular signals in order to effectively promote the survival of several established populations of target neurons in the CNS. In dissociated cell cultures of the embryonic rat mesencephalon, BDNF promoted dopaminergic cell survival only after a delay of several days. Even after prolonged cultivation, survival promoting effects were completely absent with NT-3. Irrespective of the cultivation time, survival promoting effects of both BDNF and NT-3 on dopaminergic neurons were induced or potentiated upon simultaneous depolarization of cultured mesencephalic cells with NMDA or upon activation of cAMP/PKA-dependent signaling pathways with dibutyryl cAMP. Dibutyryl cAMP (dbcAMP), but not NMDA, also potentiated or induced the survival promoting effects of BDNF and NT-3 on cultured cerebellar granule cells. None of these substances, either alone or in combination, affected the survival of cultured cortical neurons. However, cortical cell survival increased upon depolarization with elevated potassium; an effect known to involve the induction of an autocrine BDNF loop. In both cerebellar and mesencephalic neurons, but not in cortical neurons, dbcAMP also potentiated neurotrophin-induced c-fos response, indicating intimate cross-coupling of signaling pathways activated by these different factors. Together these findings suggest that in the CNS, neurotrophins preferentially promote the survival of functionally active neurons. Our findings further reveal that the neuronal response to neurotrophins is modulated in a brain region-specific manner.

Pituitary adenylate cyclase-activating polypeptide (PACAP), a neuron-derived peptide regulating glial glutamate transport and metabolism.

In the brain, glutamatergic neurotransmission is terminated predominantly by the rapid uptake of synaptically released glutamate into astrocytes through the Na(+)-dependent glutamate transporters GLT-1 and GLAST and its subsequent conversion into glutamine by the enzyme glutamine synthetase (GS). To date, several factors have been identified that rapidly alter glial glutamate uptake by post-translational modification of glutamate transporters. The only condition known to affect the expression of glial glutamate transporters and GS is the coculturing of glia with neurons. We now demonstrate that neurons regulate glial glutamate turnover via pituitary adenylate cyclase-activating polypeptide (PACAP). In the cerebral cortex PACAP is synthesized by neurons and acts on the subpopulation of astroglia involved in glutamate turnover. Exposure of astroglia to PACAP increased the maximal velocity of [(3)H]glutamate uptake by promoting the expression of GLT-1, GLAST, and GS. Moreover, the stimulatory effects of neuron-conditioned medium on glial glutamate transporter expression were attenuated in the presence of PACAP-inactivating antibodies or the PACAP receptor antagonist PACAP 6-38. In contrast to PACAP, vasoactive intestinal peptide promoted glutamate transporter expression only at distinctly higher concentrations, suggesting that PACAP exerts its effects on glial glutamate turnover via PAC1 receptors. Although PAC1 receptor-dependent activation of protein kinase A (PKA) was sufficient to promote the expression of GLAST, the activation of both PKA and protein kinase C (PKC) was required to promote GLT-1 expression optimally. Given the existence of various PAC1 receptor isoforms that activate PKA and PKC to different levels, these findings point to a complex mechanism by which PACAP regulates glial glutamate transport and metabolism. Disturbances of these regulatory mechanisms could represent a major cause for glutamate-associated neurological and psychiatric disorders.

CNS glia are targets for GDNF and neurturin.

Glial cell line-derived neurotrophic factor (GDNF) and neurturin (NTN) are two closely related growth factors reported to selectively act on distinct neuronal populations in the CNS. Both GDNF and NTN signal through a receptor complex consisting of the signal transducing subunit, Ret, and a ligand-specific binding subunit, termed GDNF family receptor (GFR)alpha-1 and GFRalpha-2, respectively. By using RT-PCR, we observed that mRNAs encoding the subunits of both receptor complexes are widely expressed throughout the developing brain, suggesting the presence of targets for these growth factors other than the ones known today. We provide evidence that these targets include glial cells.

Spatial and temporal growth factor influences on developing midbrain dopaminergic neurons.

Despite the large number of growth factors shown to affect dopaminergic cell survival and differentiation in vitro, presently little is known about the role these growth factors play during normal in vivo development of dopaminergic neurons. To address this issue, glia and neurons of both the mesencephalic home region as well as the striatal and cortical target areas have been screened for effects on dopaminergic cell survival in serum-free dissociated cell cultures of the embryonic day (E) 15 and E17 rat mesencephalon. In E15 mesencephalic cultures, the number of surviving tyrosine hydroxylase-immunoreactive dopaminergic neurons maximally increased 2.6-fold with medium conditioned by glia of the E15-E20 mesencephalon, the E17-E20 striatum, or the E20 cortex. In marked contrast, all glial-conditioned media (CM) failed to affect dopaminergic cell survival in E17 mesencephalic cultures. Similarly, E17 dopaminergic cell survival was not affected by CM derived from striatal or mesencephalic neurons. This absence of survival-promoting effects was not due to a general lack of sensitivity of the late embryonic dopaminergic neurons to growth factors. Basal survival of cultured E17 dopaminergic neurons declined with PD98059 (20 microM), a potent inhibitor of growth factor-activated microtubule-associated protein (MAP) kinase cascade. Moreover, irrespective of the age of the cultured mesencephalic tissue, dopaminergic growth factors with potential autocrine functions such as brain-derived neurotrophic factor (BDNF; 50 ng/ml) and glial cell line-derived neurotrophic factor (GDNF; 10 ng/ml) promoted dopaminergic cell survival 1.5- to 1.9-fold. These findings suggest that dopaminergic cell survival is predominantly affected by, as yet unknown, growth factors derived from mesencephalic, cortical, and striatal glia during early embryonic development, and by autocrine-acting growth factors during late developmental stages.

Changing responsiveness of developing midbrain dopaminergic neurons for extracellular growth factors.

Numerous purified growth factors as well as yet-unidentified neurotrophic activities within mesencephalic glia support the survival of dopaminergic neurons. To further characterize the functional role of these multiple growth factor influences in dopaminergic cell development, various purified growth factors as well as mesencephalic glial-conditioned medium (CM) were screened for effects on dopaminergic cell survival and glial numbers in serum-free low density cultures of the dissociated embryonic day (E) 15 and E17 rat mesencephalon. In E15 mesencephalic cultures, dopaminergic cell survival increased with brain-derived neurotrophic factor (BDNF), glial cell line-derived neurotrophic factor (GDNF), basic fibroblast growth factor (bFGF), transforming growth factor alpha (TGFalpha), insulin-like growth factor-1 (IGF-1), platelet-derived growth factor-BB (PDGF-BB), and interleukin-6 (IL-6). bFGF, TGFalpha, PDGF, and IL-6 also stimulated glial proliferation as demonstrated by autoradiographic labeling for 3H-thymidine. Moreover, CM derived from the mesencephalic glial cell line Mes42 completely prevented the death of E15 dopaminergic neurons within the initial days of cultivation. In E17 mesencephalic cultures, survival-promoting effects on dopaminergic neurons were present with BDNF, GDNF, and bFGF. TGFalpha, IGF-1, PDGF-BB, and IL-6 stimulated glial proliferation but did not affect dopaminergic cell survival. Similarly, mesencephalic glial-CM completely failed to support the survival of E17 dopaminergic neurons. These observations demonstrate that during embryonic development, dopaminergic cell survival sequentially depends on distinct sets of growth factors. The concomitant loss of sensitivity of developing dopaminergic neurons for mesencephalic glial-CM as well as TGFalpha, IGF-1, PDGF-BB, and IL-6 further provides evidence that these growth factors indirectly affect early dopaminergic neurons through glial-mediated processes and suggests a crucial role of glia during the initial stages of neuronal development.

Effects of glial cell line-derived neurotrophic factor (GDNF) on dopaminergic neurons require concurrent activation of cAMP-dependent signaling pathways.

Glial cell line-derived neurotrophic factor (GDNF) is a highly selective neurotrophic factor for midbrain dopaminergic neurons and might thus be of potential use in the therapy of Parkinson's disease. In this study, we present evidence that the survival-promoting action of GDNF on dopaminergic neurons requires the concurrent activation of cAMP-dependent signaling pathways. In serum-free low density cultures of the dissociated embryonic day 15 mesencephalon, dopaminergic neurons undergo constant cell death as evidenced by a 90% reduction in tyrosine hydroxylase-immunoreactive (TH-IR) cell numbers between days 1 and 9 of cultivation. This decline was not affected by GDNF (5 ng/ml) within the initial 3 days of cultivation, but was in part attenuated with prolonged treatment. In contrast, stimulation of 3-day-old mesencephalic cultures with GDNF induced c-fos expression in 73% of all TH-IR neurons, indicative for the early presence of efficient signal-transduction coupling in these neurons. Combined treatment of mesencephalic cultures with dibutyryl cyclic AMP (dbcAMP; 100 microM) and GDNF accelerated the onset of the survival effects of GDNF on dopaminergic neurons, resulting in a 1.5-fold increase in the number of surviving TH-IR neurons at 3 days in vitro. In addition, activation of cAMP-dependent signal pathways significantly potentiated the survival-promoting effects of GDNF on dopaminergic neurons in older cultures. dbcAMP alone had no effect on dopaminergic cell survival. Taken together, our findings suggest that the action of GDNF on midbrain dopaminergic neurons is modulated by other extracellular signals.

Growth factor-induced c-fos expression defines distinct subsets of midbrain dopaminergic neurons.

Growth factors are considered pivotal for the development, maintenance, and function of mesencephalic dopaminergic neurons. Recent studies have identified a plethora of growth factors which support the survival and differentiation of embryonic dopaminergic neurons. However, the exact cellular targets of these growth factors, and, thus, their precise mechanisms of action, remain largely unknown. To identify these cellular targets, we analysed, at the single cell level, growth factor-induced c-fos expression in dissociated mesencephalic cell cultures derived from a fos-lac Z transgenic mouse line. Pharmacological interference with cell-cell communication was utilized to control for direct growth factor effects. beta-Galactosidase-expressing cells were phenotypically characterized by immunocytochemistry to specific neural cell markers. Glia cell line-derived neurotrophic factor, basic fibroblast growth factor, brain-derived neurotrophic factor, and neurotrophin-3 directly induced Fos expression in differently sized, yet overlapping, populations of tyrosine hydroxylase-immunoreactive dopaminergic neurons. In an additional subpopulation of dopaminergic neurons, neurotrophin-3 induced fos-lac Z expression indirectly through a glutamate-mediated activation of N-methyl-D-aspartate receptors. Consistent with their proposed glial-mediated mode of action, transforming growth factor alpha and platelet-derived growth factor induced Fos expression predominantly in glia but only in a very small number of dopaminergic neurons. These findings demonstrate that individual dopaminergic neurons represent the direct targets of different sets of extracellular growth factors. Our findings further establish that growth factors affect dopaminergic neurons by indirect mechanisms which require specific cell-cell communication. These data also suggest a potential role for growth factors in the establishment of the morphological and functional diversity of midbrain dopaminergic neurons.

Evidence for a novel neurotrophic factor for dopaminergic neurons secreted from mesencephalic glial cell lines.

Our previous studies have shown that primary mesencephalic glia secrete factors that promote dopaminergic cell survival and differentiation in vitro. To obtain enough starting material to identify the neurotrophic activity, embryonic day (E)14.5 rat mesencephalic glia were stimulated with acidic fibroblast growth factor to increase number of cells. These cells were replated in the absence of neurons and immortalized by transfection with the SV 40 large T-antigen. Clonal cell lines were established and characterized for immunoreactivity (IR) to various glial and non-glial markers. Media conditioned by these cell lines were tested for survival-promoting effects on dopaminergic neurons in serum-free cultures of the dissociated E14.5 rat mesencephalon. All cell lines expressed IR for the astrocytic marker, GFAP, the oligodendroglial marker, CNP, and for A2B5, a marker for O-2A progenitor cells, but were negative for the neuronal marker, microtubule associated protein-2, and the fibroblast marker, fibronectin. Moreover, treatment of serum-free cultures of the dissociated E14.5 mesencephalon with glial cell line-CM conditioned medium (CM) delayed dopaminergic cell death in a dose-dependent manner, resulting in a maximal twofold to sixfold increase in the number of surviving tyrosine hydroxylase-IR neurons at various days in vitro. This increase in dopaminergic cell survival was not mimicked by GDNF, BDNF or NT-3 within the initial 3 days of cultivation. Moreover, initial biochemical characterization demonstrated that the neurotrophic activity is restricted to the high MW fraction of >50 kD of glial cell line-CM. Since the apparent MW of this factor exceeds the size of most known growth factors, it may represent a novel dopaminergic neurotrophic factor.

Regulation of glial-derived dopaminergic growth factors by glucocorticoids and protein kinase C.

Mesencephalic glia secrete factors that support the survival and differentiation of cultured dopaminergic neurons. Crucial to the understanding of the role of glial-derived growth factors in normal and pathophysiological conditions is knowledge about the physiological regulation of their synthesis and secretion. To address this issue, several substances have been tested for effects on the secretion of dopaminergic growth factors from the mesencephalic glial cell line, Mes42. Regulatory influences were assessed by comparing the effects of conditioned medium (CM) obtained from pretreated and untreated Mes42 cells on the survival of tyrosine hydroxylase-immunoreactive (TH-IR) neurons in serum-free low density cultures of the dissociated Embryonic Day 15 rat mesencephalon. This screening demonstrated that corticosterone and dexamethasone decreased the neurotrophic activity of Mes42-CM on TH-IR neurons by 40-60% in a dose-dependent manner. In contrast, the neurotrophic activity of Mes42-CM on TH-IR neurons was enhanced with tetradecanoylphorbol acetate (TPA). Moreover, regulatory effects of glucocorticoids and TPA on secretion of dopaminergic growth factors were not restricted to mesencephalic glial cell lines but also were present in primary mesencephalic glia. Pretreatment of Mes42 cells with 17 beta-estradiol, testosterone, progesterone, basic fibroblast growth factor, transforming growth factor alpha, insulin-like growth factor-I, or activation of cAMP-dependent protein kinases was without effect on the survival promoting activity of Mes42-CM on dopaminergic neurons. These findings suggest that the secretion of dopaminergic growth factors from mesencephalic glia is regulated by glucocorticoids and protein kinase C-dependent second messenger systems.

Development of phenylethanolamine N-methyltransferase (PNMT) in cultures of dissociated embryonic rat medulla oblongata.

The adrenergic phenotypic marker, phenylethanolamine N-methyltransferase (PNMT) is expressed in a subgroup of catecholaminergic neurons in the brain, as well as in the chromaffin cells of the adrenal medulla. Although PNMT in the rat adrenal is regulated by glucocorticoids, PNMT in the rat brainstem appears not to be regulated by glucocorticoids. Furthermore, little is known about factors required for the differentiation of this specific class of central neuron. The identification of such factors has been hampered not only by the heterogeneity of cell types in the brainstem, of which only a smaller number express PNMT, but also by the lack of a well characterized in vitro system in which the development of these neurons can be studied under defined conditions. The present study addresses this issue by establishing and characterizing a culture system for the study of adrenergic neurons. Dissociated cultures were prepared from embryonic rat medulla oblongata and the expression and development of PNMT was studied using immunocytochemistry and radioisotopic assay of PNMT activity. The survival of PNMT-immunoreactive (IR) neurons in vitro was found to be critically dependent on embryonic age. Numerous PNMT-IR neurons were observed in cultures prepared only from embryos of 46-51 somites (embryonic day E13-13.5). In contrast, cultures containing numerous neurons immunoreactive for tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine biosynthesis, could be successfully established from medulla oblongata of any age between E13 and E16.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of acidic and basic fibroblast growth factors (aFGF, bFGF) on glial precursor cell proliferation: age dependency and brain region specificity.

Acidic fibroblast growth factor (aFGF) and basic fibroblast growth factor (bFGF) are present in high levels in most areas of the embryonic rodent brain. To begin to understand the role of these growth factors in brain development, the effects of aFGF and bFGF on dissociated cell cultures prepared from embryonic and neonatal rat brain were studied. Addition of aFGF and heparin or bFGF alone to serum-free cultures of the dissociated Embryonic Day (E) 14.5 mesencephalon stimulates cell proliferation, as judged by [3H]thymidine autoradiography, leading to a maximal 75-fold increase in the total number of cells. This effect is dose-dependent with half-maximal increases at concentrations of about 5-6 ng/ml of aFGF or bFGF and is inhibited by the FGF antagonist HBGF-1U. The effect of aFGF on cell proliferation in cultures prepared from E14.5 mesencephalon is similar to that in cultures prepared from E14.5 cortex. However, in cultures prepared from E14.5 rhombencephalon or diencephalon, the proliferative effect of aFGF is much reduced. In all brain areas studied, the proliferative effect of aFGF declines with increasing age. Immunocytochemical analysis of E14.5 mesencephalic cultures demonstrated that the aFGF-induced increase in cell number is due to the proliferation of A2B5-immunoreactive (IR) glial precursor cells, but not of neuronal precursors, fibroblasts, or microglial cells. Moreover, differentiated glial fibrillary acidic protein-IR astrocytes and 2',3'-cyclic nucleotide 3'-phosphohydrolase-IR oligodendrocytes were not observed in cultures continuously treated with aFGF or bFGF, but were observed in high numbers after removal of the growth factors. These results suggest (1) that aFGF and bFGF are potent mitogens for glial precursor cells in all embryonic brain regions, (2) that the magnitude of the effects of aFGF depends on embryonic age and brain region, and (3) that both growth factors inhibit the differentiation of astrocyte or oligodendrocyte precursors. These observations made in vitro strongly support the hypothesis that FGF plays a critical role in gliogenesis and the timing of glial differentiation in the brain.

The neurotrophic effects of fibroblast growth factors on dopaminergic neurons in vitro are mediated by mesencephalic glia.

Neurotrophic support is generally believed to result from a direct action of growth factors on developing neurons. However, there is increasing evidence that growth factors can indirectly affect neuronal development by glial-mediated processes. To investigate a possible role of glia in mediating neurotrophic effects on dopaminergic neurons, four purified growth factors were screened for dual effects on the survival and differentiation of dopaminergic neurons and on the proliferation of mesencephalic glial cells in vitro. Dissociated embryonic day 14.5 rat mesencephalon was grown at low cell density without serum, conditions under which both glial growth and neuronal survival are not optimal. Treatment of these cultures with acidic fibroblast growth factor (aFGF) or basic fibroblast growth factor (bFGF) increased the number of surviving tyrosine hydroxylase-immunoreactive (TH-IR) neurons by 90-110% [corrected] at 8 d in vitro in a dose-dependent manner. The effects of these factors were not additive. High-affinity dopamine uptake was increased by bFGF, but not by aFGF. Length of TH-IR neurites was not affected by either aFGF or bFGF. Both growth factors induced proliferation of mesencephalic astrocytes as demonstrated by autoradiographic labeling with 3H-thymidine combined with immunocytochemistry for glial fibrillary acidic protein (GFAP). In contrast, platelet-derived growth factor (PDGF) and interleukin-1 had no effect on the survival or differentiation of dopaminergic neurons or the proliferation of mesencephalic astrocytes. Inhibition of glial proliferation abolished the neurotrophic effects exerted by aFGF or bFGF on dopaminergic neurons. Moreover, conditioned medium derived from mesencephalic glial cultures replated in the virtual absence of neurons also contained neurotrophic activity.(ABSTRACT TRUNCATED AT 250 WORDS)