Self-secretion of fibroblast growth factor-9 supports basal forebrain cholinergic neurons in an autocrine/paracrine manner

We examined the effect of fibroblast growth factor (FGF)-9 on primary cultures of rat basal forebrain cholinergic neurons (BFCN) obtained at embryonic day 17. FGF-9 enhanced survival of AChE-positive neurons, increased their mean soma size, and up-regulated their choline acetyltransferase (ChAT) activity. The ChAT-promoting effect of FGF-9 was approximately as potent as that of nerve growth factor (NGF) and was greater than those of basic fibroblast growth factor (bFGF), ciliary neurotrophic factor (CNTF), or glia-derived neurotrophic factor (GDNF). Simultaneous addition of FGF-9 and NGF induced extremely high ChAT levels, suggesting that FGF-9 and NGF may enhance cholinergic properties in BFCN via different pathways that can act synergistically. In immunocytochemical and in situ hybridization studies in cultured cells and also in sections of adult rat brain, BFCN showed cytoplasmic immunostaining for FGF-9 and expressed FGF-9 messenger RNA; thus, we concluded that FGF-9 acts on BFCN in an autocrine and/or paracrine manner. Although effective delivery of exogenous FGF-9 into the central nervous system remains a problem to be solved, FGF-9 may be a promising candidate for therapeutic trials in Alzheimer disease.     

Ciliary neurotrophic factor activates spinal cord astrocytes, stimulating their production and release of fibroblast growth factor-2, to increase motor neuron survival.

At focal CNS injury sites, several cytokines accumulate, including ciliary neurotrophic factor (CNTF) and interleukin-1beta (IL-1beta). Additionally, the CNTF alpha receptor is induced on astrocytes, establishing an autocrine/paracrine loop. How astrocyte function is altered as a result of CNTF stimulation remains incompletely characterized. Here, we demonstrate that direct injection of CNTF into the spinal cord increases GFAP expression and astroglial size and that primary cultures of spinal cord astrocytes treated with CNTF, IL-1beta, or leukemia inhibitory factor exhibit nuclear hypertrophy comparable to that observed in vivo. Using a coculture bioassay, we further demonstrate that CNTF treatment of astrocytes increases their ability to support ChAT(+) ventral spinal cord neurons (presumably motor neurons) more than twofold compared with untreated astrocytes. Also, the complexity of neurites was significantly increased in neurons cultured with CNTF-treated astrocytes compared with untreated astrocytes. RT-PCR analysis demonstrated that CNTF increased levels of FGF-2 and nerve growth factor (NGF) mRNA and that IL-1beta increased NGF and hepatocyte growth factor mRNA levels. Furthermore, both CNTF and IL-1beta stimulated the release of FGF-2 from cultured spinal cord astrocytes. These findings demonstrate that cytokine-activated astrocytes better support CNS neuron survival via the production of neurotrophic molecules. We also show that CNTF synergizes with FGF-2, but not epidermal growth factor, to promote DNA synthesis in spinal cord astrocyte cultures. The significance of these findings is discussed by presenting a new model depicting the sequential activation of astrocytes by cytokines and growth factors in the context of CNS injury and repair.     

Transforming growth factor-beta 3, glial cell line-derived neurotrophic factor,and fibroblast growth factor-2, act in different manners to promote motoneuron survival in vitro

Developing chick motoneurons depend on as yet unidentified factors from the periphery and the central nervous system for their survival. Using cultures of purified embryonic motoneurons, we show that basic fibroblast growth factor (FGF-2) or transforming growth factor-beta 3 (TGFβ3) each have only low survival-promoting activity when tested alone, but act synergistically to keep motoneurons alive for at least 3 days. Glial cell line-derived neurotrophic factor (GDNF), another member of the TGFβ family, was itself sufficient to maintain a population of motoneurons. However, its effect was not significantly increased by the addition of FGF-2. These results suggest that FGF-2, TGFβ3, and GDNF, which are all present in the environment of developing motoneurons, may act by different mechanisms as physiological survival factors for this population of central neurons. © 1996 Wiley-Liss, Inc.     

Fibroblast Growth Factors Regulate Calcitonin Gene-related Peptide mRNA Expression in Rat Motoneurons after Lesion and in Culture

In this study, we have investigated the effect of fibroblast growth factors (bFGF and FGF-5) and brain derived neurotrophic factor (BDNF) on the expression of calcitonin gene-related peptide (CGRP) in rat motoneurons in vivo and in vitro . Following sciatic nerve transection in adult rats, the levels of α-CGRP and β-CGRP mRNA were up- and down-regulated respectively in axotomized motoneurons, revealed by in situ hybridization histochemistry. Local administration of 1 μg bFGF was able to entirely abolish the up-regulation of α-CGRP mRNA, and to further down-regulate β-CGRP. These effects, albeit less pronounced, were still evident with 0.2 μg bFGF. In contrast, bFGF did not attenuate the lesion-induced decrease of choline acetyltransferase (ChAT) mRNA. Administration of BDNF did not significantly alter the expression of CGRP or ChAT mRNA in axotomized motoneurons. Both α- and β-CGRP mRNAs could be detected by PCR in enriched motoneuron cultures prepared from rat embryos at embryonic day 14–15. Comparing the amplification of α- and β-CGRP mRNAs with that of mRNA encoding glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in parallel samples, we found that cultures treated with FGF-5 had a lower ratio of α- and β-CGRP mRNA to GAPDH mRNA, than did control or BDNF-treated cultures. BDNF, on the other hand increased α-CGRP and decreased β-CGRP mRNA levels, though these effects were moderate compared with the effects of FGF-5. The results obtained in this study suggest that members of the FGF family of growth factors influence the expression of CGRP in rat motoneurons, and that the increase of this neuropeptide induced by axotomy may, at least in part, be due to deprivation of these target-derived factors.     

Properties of motoneurons underlying their regenerative capacity after axon lesions in the ventral funiculus or at the surface of the spinal cord

Spinal motoneurons represent neurons with axons located in both the central (CNS) and peripheral (PNS) nervous systems. Following a lesion to their axons in the PNS, motoneurons are able to regenerate. The regenerative capacity of these neurons is seen also after lesion in the ventral funiculus of the spinal cord, i.e. within the CNS compartment. Thus, after an axotomy within the ventral funiculus, motoneurons respond with a changing polarity towards production of axons, sometimes even from the dendritic tree. This capacity can be used in cases of ventral root avulsion (VRA) lesions, if a conduit for outgrowing axons is presented in the form of replanted ventral roots. In human cases, this procedure may accomplish return of function in denervated muscles. The strong regenerative capacity of motoneurons provides the basis for studies of the response in motoneurons with regard to their contents of substances related to survival and regeneration. Such studies have shown that, of the large number of receptors for neurotrophic substances and extracellular matrix molecules, mRNAs for receptors or receptor components for neurotrophin-3 (NT-3), ciliary neurotrophic factor (CNTF) and leukemia inhibitory factor (LIF) are strongly downregulated after VRA, while receptors for glial cell line-derived neurotrophic factor (GDNF) and laminins are profoundly upregulated. These results should be considered in the design of combined pharmacological and surgical approaches to lesions of motor axons at or close to the CNS–PNS interface.     

Neural adhesion molecules L1 and CHL1 are survival factors for motoneurons

Many neurotrophic factors with survival activity for motoneurons in vivo were first identified using cultures of purified embryonic motoneurons. The L1 neural cell adhesion molecule has multiple roles in brain development. We showed by in situ hybridization and RT-PCR that L1 mRNA was expressed at significant levels in motoneurons of embryonic and postnatal spinal cord. We therefore cultured purified motoneurons from E14 rat embryos in the absence of trophic factors but with L1-Fc and CHL1-Fc fusion proteins. L1-Fc prevented the death of approximately half of the motoneurons that were saved by BDNF in a dose-dependent manner (EC50 = 10 pM). CHL1-Fc saved the same number of motoneurons as did L1-Fc, whereas P0-Fc had little neurotrophic activity at the same concentrations. Survival induced by L1 and CHL1 was completely inhibited by 20 microM LY294002 and PD98059, indicating that both MEK and PI3K pathways are required for signaling by these molecules. L1 can signal in other cell types through the FGF receptor FGFR1. In cultures of motoneurons, effects of suboptimal concentrations of L1 and suboptimal concentrations of FGF-2 were additive, but the effects of optimal concentrations of FGF-2 (50 ng/ml) were not further increased in the presence of L1-Fc. Thus, in this system, too, FGF and L1 may use similar signaling pathways.     

FGF9: a motoneuron survival factor expressed by medial thoracic and sacral motoneurons

In the nervous system, fibroblast growth factor-9 (FGF9) is produced mainly by neurons. By whole-mount in situ hybridization, on embryonic rat spinal cord, we observed Fgf9 expression in a subpopulation of motoneurons located in the thoracic and sacral regions of the median motor column that innervate the axial muscles. Furthermore, FGF9 prevented death of purified rat and chicken motoneurons in culture in the same concentration range as FGF2. The targets of FGF9 are more restricted than that of the other FGFs, however, because conversely to FGF1 or FGF2, FGF9 had only weak or inexistent survival effects on chicken ciliary neurons or rat DRG. FGF9 may therefore play a role as an autocrine/paracrine survival factor for motoneurons.     

Ventral and dorsal horn acetylcholinesterase neurons are maintained in organotypic cultures of postnatal rat spinal cord explants

Transverse sections of postnatal rat spinal cord have been cultured using the organotypic roller tube method. These explant cultures retain identifiable anatomical landmarks, allow identification of individual neurons, can be maintained for up to 8 weeks, and undergo maturational changes in vitro. Putative ventral horn motoneurons were identified in these cultures by localization to ventral horn regions analogous to those of motoneurons in vivo and by staining for choline acetyltransferase (ChAT) immunoreactivity and acetylcholinesterase (AChE) activity. Morphometric studies of the photomicrographic areas of cell bodies of these ventral horn neurons in intact cultures show a range of sizes up to 1635 microns 2 with the average size being 245 +/- 7 microns 2 (n = 724) (average +/- S.E.M.). The size ranges are roughly comparable to cross-sectional areas determined previously for ventral horn motoneurons in vivo. Dorsal horn regions of these cultures also developed prominent AChE activity that was absent at explantation. Biochemical analysis of ChAT and AChE activity in pooled samples of whole cultures showed ChAT activity to be 0.48 +/- 0.08 (n = 7) mumol/min/g protein and AChE activity to be 12.2 +/- 2.0 (n = 7) mumol/min/g protein at 37 degrees C (averages +/- S.E.M.). These values are comparable to previously reported values for neonatal rat spinal cord in situ. Organotypic roller tube cultures of postnatal rat spinal cord provide an attractive system for studies of survival, morphology, growth and differentiation of mammalian ventral horn neurons in vitro.     

Neurotrophin expression by spinal motoneurons in adult and developing rats

Expression of the neurotrophins NT-4, brain-derived neurotrophic factor (BDNF), and NT-3 in adult rat lumbosacral spinal cord motoneurons is reported. A sensitive in situ hybridization procedure demonstrates localization of the mRNA for each of these neurotrophins within spinal motoneurons of the adult and in early postnatal development. A majority of adult rat spinal cord lumbar motoneurons (approximately 63%) express NT-4 mRNA as assessed by counting motoneurons in the L4 and L5 segments of two adult rat spinal cords on adjacent cresyl violet-stained and in situ hybridization sections. Similarly, a majority of lumbar motoneurons (approximately 73%) express BDNF mRNA. Further analyses of adjacent lumbar spinal cord sections revealed that many, although not all motoneurons coexpress both NT-4 and BDNF mRNAs. At birth, the mRNA encoding NT-3 is expressed in motoneurons, but BDNF mRNA is not apparent until postnatal day 5 (P5) and NT-4 mRNA first appears at P9. The potential biological significance of neurotrophin mRNA expression in spinal motoneurons is supported by immunohistochemical localization of each neurotrophin protein in adult motoneurons. We discuss the potential role of spinal cord neurotrophins as autocrine or paracrine factors involved in modulating motoneuron synaptic function.     

Peripheral nerve avulsion injuries as experimental models for adult motoneuron degeneration

We have used adult rat peripheral nerve avulsion models to evaluate the effects of neuroprotective molecules on motoneuron degeneration. The right facial nerves of adult Fischer 344 male rats were avulsed and adenoviral vectors encoding glial cell line‐derived neurotrophic factor (GDNF), brain‐derived neurotrophic factor (BDNF), transforming growth factor‐β2 (TGFβ2), and growth inhibitory factor (GIF) were injected into the facial canal. The treatment with the vectors significantly prevented the loss of lesioned facial motoneurons, improved choline acetyltransferase (ChAT) immunoreactivity and suppressed the induction of nitric oxide synthase activity in these neurons. In separate experiments, animals were orally administered a solution of a neuroprotective compound T‐588 after avulsion. Both free oral administration and oral tube administration of T‐588 improved the survival of injured motoneurons and ameliorated their ChAT immunoreactivity. These results indicate that the gene transfer of GDNF, BDNF, TGFβ2, and GIF and oral administration of T‐588 may prevent the degeneration of motoneurons in adult humans with motoneuron injury and motor neuron diseases.     

CNTF rescues motoneurons from ontogenetic cell death in-vivo, but not in-vitro

We studied the effect of CNTF (ciliary neurotrophic factor, human recombinant and chick) on the survival of motoneurons in the embryonic chick lumbar spinal cord during the period of ontogenetic cell death. Daily applications of 5 micrograms CNTF to the chorionic-allantoic membrane from embryonic day 6 (E6) to E9 maintained approximately 15,500 motoneurons as opposed to 13,200 in controls. In contrast, CNTF failed to promote the survival of cells in spinal cord cultures enriched for motoneurons. These results suggest that CNTF may regulate motoneuron survival in-vivo, but its mode of action remains to be elucidated.     

Regulation of putative muscle-derived neurotrophic factors by muscle activity and innervation: in vivo and in vitro studies

The normal embryonic development of spinal cord motoneurons (MNs) involves the proliferation of precursor cells followed by the degeneration of approximately 50% of postmitotic MNs during the period when nerve-muscle connections are being established. The death of MNs in vivo can be ameliorated by activity blockade and by treatment with muscle extracts. Muscle activity and innervation have been suggested to regulate the availability of putative muscle-derived neurotrophic agent(s), and MNs are thought to compete for limited amounts of these trophic agents during normal development. Thus, activity and innervation are thought to regulate MN survival by modulating trophic factor availability. We have tested this notion by examining MN survival in vivo and ChAT development in spinal cord neurons in vitro following treatments with partially purified muscle extracts from normally active, paralyzed (genetically or pharmacologically), aneural, denervated, slow tonic, and fast-twitch muscles from embryonic and postnatal animals. Extracts from active and chronically inactive embryonic avian and mouse muscles were found to be equally effective in promoting the in vivo survival of MNs in the chick embryo. Similarly, extracts from fast-twitch and slow tonic postnatal avian muscles did not differ in their ability to promote both MN survival in vivo and ChAT activity in vitro. Although aneural and control embryonic muscle extract had similar effects on ChAT development in vitro, aneural muscle extract contained somewhat less MN survival-promoting activity when tested in vivo. By contrast, denervated postnatal muscle extract was more effective in promoting both MN survival in vivo and ChAT activity in vitro than age-matched control muscle extract.(ABSTRACT TRUNCATED AT 250 WORDS)     

Preclinical testing of neuroprotective neurotrophic factors in a model of chronic motor neuron degeneration.

Many neurotrophic factors have been shown to enhance survival of embryonic motor neurons or affect their response to injury. Few studies have investigated the potential effects of neurotrophic factors on more mature motor neurons that might be relevant for neurodegenerative diseases. Using organotypic spinal cord cultures from postnatal rats, we have demonstrated that insulin-like growth factor-I (IGF-I) and glial-derived neurotrophic factor (GDNF) significantly increase choline acetyltransferase (ChAT) activity, but brain-derived neurotrophic factor (BDNF), neurotrophin-4 (NT-4/5), and neurotrophin-3 (NT-3) do not. Surprisingly, ciliary neurotrophic factor (CNTF) actually reduces ChAT activity compared to age-matched control cultures. Neurotrophic factors have also been shown to alter the sensitivity of some neurons to glutamate neurotoxicity, a postulated mechanism of injury in the neurodegenerative disease, amyotrophic lateral sclerosis (ALS). Incubation of organotypic spinal cord cultures in the presence of the glutamate transport inhibitor threo-hydroxyaspartate (THA) reproducibly causes death of motor neurons which is glutamate-mediated. In this model of motor neuron degeneration, IGF-I, GDNF, and NT-4/5 are potently neuroprotective, but BDNF, CNTF, and NT-3 are not. The organotypic glutamate toxicity model appears to be the best preclinical predictor to date of success in human clinical trials in ALS.     

Differential regulation of trophic factor receptor mRNAs in spinal motoneurons after sciatic nerve transection and ventral root avulsion in the rat

After sciatic nerve lesion in the adult rat, motoneurons survive and regenerate, whereas the same lesion in the neonatal animal or an avulsion of ventral roots from the spinal cord in adults induces extensive cell death among lesioned motoneurons with limited or no axon regeneration. A number of substances with neurotrophic effects have been shown to increase survival of motoneurons in vivo and in vitro. Here we have used semiquantitative in situ hybridization histochemistry to detect the regulation in motoneurons of mRNAs for receptors to ciliary neurotrophic factor (CNTF), leukemia inhibitory factor (LIF), glial cell line-derived neurotrophic factor (GDNF), brain-derived neurotrophic factor (BDNF), and neurotrophin-3 (NT-3) 1-42 days after the described three types of axon injury. After all types of injury, the mRNAs for GDNF receptors (GFRalpha-1 and c-RET) and the LIF receptor LIFR were distinctly (up to 300%) up-regulated in motoneurons. The CNTF receptor CNTFRalpha mRNA displayed only small changes, whereas the mRNA for membrane glycoprotein 130 (gp130), which is a critical receptor component for LIF and CNTF transduction, was profoundly down-regulated in motoneurons after ventral root avulsion. The BDNF full-length receptor trkB mRNA was up-regulated acutely after adult sciatic nerve lesion, whereas after ventral root avulsion trkB was down-regulated. The NT-3 receptor trkC mRNA was strongly down-regulated after ventral root avulsion. The results demonstrate that removal of peripheral nerve tissue from proximally lesioned motor axons induces profound down-regulations of mRNAs for critical components of receptors for CNTF, LIF, and NT-3 in affected motoneurons, but GDNF receptor mRNAs are up-regulated in the same situation. These results should be considered in relation to the extensive cell death among motoneurons after ventral root avulsion and should also be important for the design of therapeutical approaches in cases of motoneuron death.     

Effects of ciliary neuronotrophic factor on rat spinal cord neurons in vitro: survival and expression of choline acetyltransferase and low-affinity nerve growth factor receptors.

We have studied the effects of ciliary neuronotrophic factor (CNTF) and nerve growth factor (NGF) on cultures of E14 rat spinal cord cells maintained for 7 days. The trophic factors were supplied at the day of seeding and every other day thereafter. Treatments with CNTF (human recombinant or purified from rat sciatic nerve, 100 TU/ml) resulted after 7 days in an increase, relative to control cultures, of: (i) the total number of neurons (identified by neurofilament protein and neuron-specific enolase immunostaining) that were not stained with choline, acetyltransferase (ChAT) and low affinity nerve growth factor receptor (LNGFR) antibodies; (ii) the number of motoneurons (0.5% of the neuronal population) as identified by size (greater than 25 microns), morphology and immunostaining for ChAT and LNGFR; and (iii) a population of small- to medium-sized (less than 25 microns), ChAT- and LNGFR-positive neurons, representing 5-10% of the total neuronal population. NGF treatments (mouse submaxillary beta NGF; 10-3000 TU/ml) were without effect on all 3 neuronal populations. Experiments in which CNTF administration was delayed revealed that the population of ChAT- and LNGFR-negative neurons and the population of motoneurons, were both dependent on CNTF for their survival. The third population, small ChAT and LNGFR-positive neurons, was not dependent on CNTF for survival but was induced by CNTF to express its two markers. These observations indicate that CNTF is a neuronotrophic factor for motoneurons, but that the effect of CNTF is not restricted to that cell population. In addition to its survival promoting effect, CNTF has also a regulatory role on the expression of ChAT and LNGFR for some spinal cord neurons.     

Dedifferentiation of intrinsic response properties of motoneurons in organotypic cultures of the spinal cord of the adult turtle

Explant cultures from the spinal cord of adult turtles were established and used to study the sensitivity of the intrinsic response properties of motoneurons to the changes in connectivity and milieu imposed by isolation in culture. Transverse sections 700 microm thick were explanted on cover slips and maintained in roller-tube cultures in medium containing serum and the growth factors brain-derived neurotrophin factor (BDNF), neurotrophin-3 (NT3), glial cell line-derived neurotrophic factor (GDNF) and ciliary neurotrophic factor (CNTF). The gross morphology of acute sections was maintained after 4 weeks in culture. Cell bodies of motoneurons remained stainable in fixed cultures with an antibody against choline acetyltransferase (ChAT) throughout the culture period. During culture, motoneurons maintained stable resting membrane potentials and were contacted by functional synapses. The ability to generate action potentials was also preserved as was delayed inward rectification and generation of calcium spikes in the presence of tetra-ethyl ammonium (TEA). In response to depolarization, however, motoneurons presented strong outward rectification, and only 41% of the cells recorded from maintained the ability to fire repetitively. By the second week in culture, a fraction of motoneurons displayed fast and slow transient outward rectification and low-threshold calcium spikes, features not seen in turtle motoneurons in acute slices. On the other hand, properties mediated by L-type Ca2+ channels disappeared during the first few days in culture. Our observations show that the phenotypical intrinsic response properties of mature spinal motoneurons are modified in explant cultures. The properties acquired resemble the properties in juvenile motoneurons in several species of terrestrial vertebrates.     

Fibroblast Growth Factor Treatment Produces Differential Effects on Survival and Neurite Outgrowth from Identified Bulbospinal Neurons in Vitro

The in vivo application of appropriate trophic factors may enhance regeneration of bulbospinal projections after spinal cord injury. Currently, little is known about the sensitivities of specific bulbospinal neuron populations to the many identified trophic factors. We devised novel in vitro assays to study trophic effects on the survival and neurite outgrowth of identified bulbospinal neurons. Carbocyanine dye crystals implanted into the cervical spinal cord of embryonic day (E)5 chick embryos retrogradely labeled developing bulbospinal neurons. On E8, dissociated cultures containing labeled bulbospinal neurons were prepared. Fibroblast growth factor (FGF)-2 (but not FGF-1) promoted the survival of bulbospinal neurons. FGF receptor expression was widespread in the E8 brainstem, but not detected in young bulbospinal neurons, suggesting that nonneuronal cells mediated the FGF-stimulated survival response. Astrocytes synthesize a variety of trophic factors, and astrocyte-conditioned medium (ACM) also promoted the survival of bulbospinal neurons. As might be expected, FGF-2 function blocking antibodies did not suppress ACM-promoted survival, nor did an ELISA detect FGF-2 in ACM. This suggests that nonneuronal cells synthesize other factors in response to exogenous FGF-2 which promote the survival of bulbospinal neurons. Focusing on vestibulospinal neurons, dissociated (survival assay) or explant (neurite outgrowth assay) cultures were prepared. FGF-2 promoted both survival and neurite outgrowth of identified vestibulospinal neurons. Interestingly, FGF-1 promoted neurite outgrowth but not survival; the converse was true of FGF-9. Thus, differential effects of specific growth factors on survival or neurite outgrowth of bulbospinal neurons were distinguished.     

Members of several gene families influence survival of rat motoneurons in vitro and in vivo

The survival and functional maintenance of spinal motoneurons, both during the period of developmental cell death and in adulthood, have been shown to be dependent on trophic factors. In vitro experiments have previously been used to identify several survival factors for motoneurons, including CNTF, LIF, and members of the neurotrophin, FGF, and IGF gene families. Some of these factors have also been shown to be active in vivo, either on chick motoneurons during embryonic development or on lesioned facial and spinal motoneurons of the newborn rat. Here we demonstrate that lesioned newborn rat facial motoneurons can be rescued by NT-4/5, IGF-I and LIF. Furthermore, in contrast to chick motoneurons, the survival of isolated embryonic rat motoneurons can be maintained by the neurotrophins BDNF, NT-3, and NT-4/5. IGF-I and FGF-5 were also active in this system, each supporting more than 50% of the originally plated neurons. The responsiveness of motoneurons to multiple factors in vitro and in vivo suggests that motoneuron survival and function are regulated by the coordinated actions of members of different gene families. © 1993 Wiley-Liss, Inc.     

Synergistic effects of brain-derived neurotrophic factor and ciliary neurotrophic factor on cultured basal forebrain cholinergic neurons from postnatal 2-week-old rats.

Brain-derived neurotrophic factor (BDNF), a member of the neurotrophin family, and ciliary neurotrophic factor (CNTF), a member of the neurocytokine family, are known to have synergistic effects on motoneurons, but such synergistic effect has not been studied in detail especially in the brain. In the present study, we examined the synergistic effects of BDNF and CNTF on the survival of basal forebrain cholinergic neurons cultured from postnatal 2-week-old (P2w) rats. Although BDNF is well-known to promote the survival of basal forebrain cholinergic neurons in P2w culture, CNTF had little effect on the survival of choline acetyltransferase (ChAT)-positive neurons and did not increase ChAT activity in the culture. However, CNTF enhanced BDNF-mediated promotion of cell survival of cholinergic neurons when added concomitantly. BDNF alone induced only a three-fold increase in ChAT activity in control cultures, but the concomitant addition of CNTF resulted in an eight-fold increase. CNTF did not enhance BDNF-mediated cell survival of total neurons from the basal forebrain, hippocampus or cerebellum, suggesting that the synergistic effects of CNTF on the BDNF-mediated increase of viability might be strong in basal forebrain cholinergic neurons. CNTF also enhanced the neurotrophin-4/5-mediated increase of ChAT activity, but not the nerve growth factor (NGF)-mediated one. Furthermore, the BDNF-mediated increase was also enhanced by leukemia inhibitory factor but not by interleukin-6. Similar synergistic pattern between neurotrophins and cytokines were also observed in the induction of ChAT activity in embryonic basal forebrain culture. These results suggest that TrkB, a functional high-affinity receptor of BDNF and NT-4/5, and LIFR beta, a receptor component contained in CNTF and LIF receptor complex, might be involved in the observed synergistic effects.