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Cytoplasmic calcium-binding proteins are thought to shield neurons against damage induced by excessive Ca2+ elevations. Yet, in theory, a mobile cellular Ca2+ buffer could just as well promote neuronal injury by facilitating the rapid dispersion of Ca2+ throughout the cytoplasm. In sharp contrast to controls, in mice lacking the gene for calbindin-D28k, synaptic responses of hippocampal CA1 pyramidal neurons which are normally extremely vulnerable to ischemia, recovered significantly faster and more completely after a transient oxygen-glucose deprivation in vitro, and sustained less cellular damage following a 12 min carotid artery occlusion in vivo. Other cellular and synaptic properties such as the altered adaptation of action potential firing, and altered paired-pulse and frequency potentiation at affected synapses in calbindin-D28k-deficient mice were consistent with a missing intraneuronal Ca2+ buffer. Our findings provide direct experimental evidence against a neuroprotective role for calbindin-D28k.

Ciliary neurotrophic factor (CNTF) is an abundant cytosolic molecule in myelinating Schwann cells of adult rodents. In newborn animals in which CNTF is not yet expressed, exogenous CNTF that is locally administered very effectively protects motoneurons from degeneration by axotomy. To evaluate whether endogenous CNTF, released after nerve injury from the cytosol of Schwann cells, supports motoneuron survival, we transected the facial nerve in 4-week-old pmn mice. In this mouse mutant a rapidly progressing degenerative disease of motoneurons starts by the third postnatal week at the hindlimbs and progresses to the anterior parts of the body, leading to death by the seventh to eighth week. Apoptotic death of motoneurons can be observed during this period, as revealed by TUNEL staining. In 6-week-old unlesioned pmn mice approximately 40% of facial motoneurons have degenerated. Facial nerve lesion dramatically increased the number of surviving motoneurons in pmn mice. This protective effect was absent in pmn mice lacking endogenous CNTF. Quantitative analysis of leukemia inhibitory factor (LIF) mRNA expression revealed that the dramatic upregulation seen in wild-type mice after peripheral nerve lesion did not occur in pmn mice. Therefore, endogenous LIF cannot compensate for the lack of CNTF in pmn crossbred with CNTF knock-out mice. Thus, endogenous CNTF released from lesioned Schwann cells supports the survival of axotomized motoneurons under conditions in which motoneurons are in the process of rapid degeneration.

Intracellular calcium-binding proteins are abundantly expressed in many neuronal populations. Previous evidence suggests that calcium-binding proteins can modulate various neuronal properties, presumably by their action as calcium buffers. The importance of calcium-binding proteins for nervous system function in an intact integrated system is, however, less clear. To investigate the physiological role of a major endogenous calcium-binding protein, calbindin D28k (calbindin) in vivo, we have generated calbindin null mutant mice by gene targeting. Surprisingly, calbindin deficiency does not affect general parameters of development and behavior or the structure of the nervous system at the light microscopic level. Null mutants are, however, severely impaired in tests of motor coordination, suggesting functional deficits in cerebellar pathways. Purkinje neurons, the only efferent of the cerebellar cortex, and inferior olive neurons, the source of the climbing fiber afferent, have previously been shown to express calbindin. Correlated with this unusual type of ataxia, confocal calcium imaging of Purkinje cells in cerebellar slices revealed marked changes of synaptically evoked postsynaptic calcium transients. Their fast, but not their slow, decay component had larger amplitudes in null mutant than in wild-type mice. We conclude that endogenous calbindin is of crucial importance for integrated nervous system function.

Calbindin-D28k (calbindin) is an intracellular calcium binding protein of unknown in vivo function. It is abundantly expressed in many populations of neurons, and it can, presumably by buffering calcium overload, protect cells against excitotoxic damage. In the midbrain, calbindin is preferentially expressed in those dopamine neurons which are spared from degeneration in Parkinson's disease and its animal models. Whether calbindin itself determines neuronal vulnerability is questioned in other lesion models where calbindin expression is not positively correlated with neuronal resistance. To study the possible neuroprotective role of calbindin in vivo, we generated calbindin-deficient mice by gene targeting and assessed the viability of midbrain dopamine neurons in both a chemical and a genetic lesion paradigm. Tyrosine hydroxylase-immunoreactive neurons were counted in calbindin null-mutant mice treated with the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and in a calbindin-deficient weaver strain (homozygous for weaver and the calbindin null mutation). The extent and pattern of neuron loss observed in MPTP-treated wild-type and homozygous weaver mice were as previously described. Surprisingly, no significant differences were observed between MPTP-treated calbindin null mutants and their wild-type littermates, or between calbindin-weaver double mutant mice and weaver mice. Thus, in all four groups the same subpopulation of tyrosine hydroxylase-positive midbrain neurons (i.e. those normally containing calbindin) were preferentially spared. Calretinin, a closely related calcium-binding protein, which is also expressed in some midbrain dopamine neurons, was not up-regulated in these surviving neurons. These findings indicate that the resistance of calbindin-containing neurons in the MPTP and weaver models is not causally related to the expression of calbindin, and that endogenous calbindin is not required for protection of these neurons.

Background: The survival and differentiation of motoneurons during embryonic development, and the maintenance of their function in the postnatal phase, are regulated by a great variety of neurotrophic molecules which mediate their effects through different receptor systems. The multifactorial support of motoneurons represents a system of high security, because the inactivation of individual ligands has either no detectable, or relatively small, atrophic or degenerative effect on motoneurons., Results: Leukaemia inhibitory factor (LIF) has been demonstrated to support motoneuron survival in vitro and in vivo under different experimental conditions. However, when LIF was inactivated by gene targeting, there were no apparent changes in the number and structure of motoneurons and no impairment of their function. The slowly appearing, relatively mild degenerating effects in motoneurons that resulted from ciliary neurotrophic factor (CNTF) gene targeting were substantially potentiated by simultaneous inactivation of the LIF gene, however. Thus, in mice deficient in LIF and CNTF, the degenerative changes in motoneurons were more extensive and appeared earlier. These changes were also functionally reflected by a marked reduction in grip strength., Conclusions: Degenerative disorders of the nervous system, in particular those of motoneurons, may be based on multifactorial inherited and/or acquired defects which individually do not result in degenerative disorders, but which become apparent when additional (cryptic) inherited disturbances or sub-threshold concentrations of noxious factors come into play. Accordingly, the inherited inactivation of the CNTF gene in a high proportion of the Japanese population may represent a predisposing factor for degenerative disorders of motoneurons.

Gamma-Aminobutyric acid (GABA) switches from enhancing to repressing brain-derived neurotrophic factor (BDNF) mRNA synthesis during the maturation of hippocampal neurons in vitro. Interneurons do not produce BDNF themselves, but BDNF enhances their differentiation. Therefore, the question arose whether hippocampal interneurons regulate their phenotype by regulating BDNF expression and release from adjacent cells. The GABA(A) receptor agonist muscimol and BDNF increased the size and neuropeptide Y (NPY) immunoreactivity of hippocampal interneurons. However, GABAergic stimulation failed to increase NPY immunoreactivity in cultures from BDNF knockout embryos. At later developmental stages, when GABA represses BDNF synthesis, treatment with muscimol induced a decrease in cell size and NPY immunoreactivity of interneurons. Interneurons might thus control their phenotype through the regulation of BDNF synthesis in, and release from, their target neurons.

Brain-derived neurotrophic factor (BDNF) is widely expressed in the central nervous system, where its function is poorly understood. The aim of this study was to investigate the effects of BDNF on the differentiation of hippocampal nonpyramidal neurons using organotypic slice cultures prepared from postnatal rats. The application of BDNF induced an increase in immunostaining for the microtubule-associated protein (MAP)-2 in non-pyramidal neurons of the stratum oriens. BDNF promotes the elongation of the dendrites of these neurons, as demonstrated by analysis after biocytin labeling. Calbindin-D- and calretinin-containing subgroups of nonpyramidal cells in the stratum oriens were responsive to BDNF but not to nerve growth factor, as shown by an increase in the number of neurons immunostained for these proteins. BDNF also induced an increase in neuropeptide Y immunostaining of stratum oriens neurons. In contrast, BDNF had no effect on parvalbumin immunostaining, despite the fact that these cells express the BDNF receptor trkB. In addition, BDNF increased calretinin immunoreactivity in Cajal-Retzius cells situated around the hippocampal fissure. The Cajal-Retzius neurons persisted in slices beyond the time at which they degenerate in vivo. However, BDNF is not required for the survival of these cells, because they also persisted in slices from BDNF knock-out mice. The present results indicate that BDNF exerts an effect on the morphology of stratum oriens nonpyramidal cells and their calcium-binding protein levels. BDNF also regulates the calretinin content of Cajal-Retzius cells but is not necessary for their survival.

Although ciliary neurotrophic factor (CNTF) is a potent survival factor for many types of neurons and glial cells in vitro, there is currently no evidence that it participates in normal development. Here we show that CNTF greatly enhances the rate of oligodendrocyte generation. Proliferation of oligodendrocyte precursor cells purified from rodent optic nerves and cultured in platelet-derived growth factor-containing medium is significantly increased by CNTF. Similarly, the number of proliferating oligodendrocyte precursor cells in developing optic nerves of transgenic mice lacking CNTF is decreased by up to threefold and the number of oligodendrocytes is transiently decreased; proliferation is restored to normal by the delivery of exogenous CNTF into the developing optic nerve. Both oligodendrocyte number and myelination ultimately attain wild-type values in CNTF-deficient adult mice, indicating that CNTF is not necessary for either oligodendrocyte differentiation or myelination, although it normally accelerates oligodendrocyte development by enhancing the proliferation of oligodendrocyte precursor cells.

There is increasing evidence that neurotrophins (NTs) are involved in processes of neuronal plasticity besides their well-established actions in regulating the survival, differentiation, and maintenance of functions of specific populations of neurons. Nerve growth factor, brain-derived neurotrophic factor, NT-4/5, and corresponding antibodies dramatically modify the development of the visual cortex. Although the neuronal elements involved have not yet been identified, complementary studies of other systems have demonstrated that NT synthesis is rapidly regulated by neuronal activity and that NTs are released in an activity-dependent manner from neuronal dendrites. These data, together with the observation that NTs enhance transmitter release from neurons that express the corresponding signal-transducing Trk receptors, suggest a role for NTs as selective retrograde messengers that regulate synaptic efficacy.

Brain-derived neurotrophic factor (BDNF), a member of the nerve growth factor (NGF) gene family, has been shown to influence the survival and differentiation of specific classes of neurons in vitro and in vivo. The possibility that neurotrophins are also involved in processes of neuronal plasticity has only recently begun to receive attention. To determine whether BDNF has a function in processes such as long-term potentiation (LTP), we produced a strain of mice with a deletion in the coding sequence of the BDNF gene. We then used hippocampal slices from these mice to investigate whether LTP was affected by this mutation. Homo- and heterozygous mutant mice showed significantly reduced LTP in the CA1 region of the hippocampus. The magnitude of the potentiation, as well as the percentage of cases in which LTP could be induced successfully, was clearly affected. According to the criteria tested, important pharmacological, anatomical, and morphological parameters in the hippocampus of these animals appear to be normal. These results suggest that BDNF might have a functional role in the expression of LTP in the hippocampus.

Neurotrophins play a crucial role in the regulation of survival and maintenance of specific functions of various populations of neurons. Brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3) and neurotrophin 4/5 (NT-4) have been shown to support motoneuron survival during embryonic development and, after birth, to protect motoneurons from degeneration after nerve lesion. We have compared the levels of these neurotrophins in skeletal muscle by quantitative Northern blot analysis, both during embryonic development and postnatally. We localized the sites of expression of these neurotrophins by in situ hybridisation and analysed the expression of trkB in the spinal cord by in situ hybridisation and immunohistochemistry. NT-3 is most abundantly expressed both during embryonic development and in the postnatal phase, followed by NT-4. The levels of BDNF are very low, in particular after birth. After nerve lesion, NT-3 mRNA essentially remained unchanged, whereas NT-4 mRNA rapidly decreased. The slow increase in BDNF expression seems to be essentially due to the expression in Schwann cells rather than skeletal muscle, demonstrated by in situ hybridisation. Our data indicate that motoneurons can receive trophic support from several members of the neurotrophin gene family during the period of naturally occurring cell death. Postnatally, the predominant ligand acting via trkB on motoneurons is NT-4, whereas BDNF expression seems to play a role mainly after nerve lesion.

gamma-Aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the adult mammalian central nervous system. However, GABA depolarizes immature rat hippocampal neurons and increases intracellular Ca2+ ([Ca2+]i). Here we show, that GABA and the GABAA receptor agonist muscimol induce c-Fos immunoreactivity and increase BDNF mRNA expression in embryonic hippocampal neurons cultured for 5 days. In contrast, after 3 weeks in culture, GABA and muscimol failed to induce c-fos and BDNF expression. Fura-2 fluorescence microscopy revealed that muscimol produces a dihydropyridine-sensitive transient increase in [Ca2+]i, comparable to the effect of the non-NMDA receptor agonist kainic acid in neurons cultured for 5 days, but not in 3-week-old cultures. The increase in c-Fos immunoreactivity and BDNF mRNA levels by GABA were dependent upon the activation of voltage-gated Ca2+ channels, as shown using the L-type specific Ca2+ channel blocker nifedipine. The differential regulation of c-fos and BDNF expression by GABA and muscimol in developing and mature hippocampal neurons is due to a switch in the ability of GABAA receptors to activate voltage-gated Ca2+ channels. These observations support the hypothesis that GABA might have neurotrophic effects on embryonic or perinatal hippocampal neurons, which are mediated by BDNF.

We have detected scattered brain-derived neurotrophic factor mRNA-producing neurons in the medial septal nucleus, which contains cholinergic neurons that are responsive to brain-derived neurotrophic factor and nerve growth factor. In the brainstem, many adrenergic neurons showed a positive signal for brain-derived neurotrophic factor messenger RNA. Several hypothalamic nuclei contain brain-derived neurotrophic factor messenger RNA-positive neurons, among them paraventricular, median preoptic, vetromedial and dorsomedial nuclei. Osmotic stimulus, which activates vasopressin-producing neurons increased brain-derived neurotrophic factor messenger RNA levels in the paraventricular nucleus demonstrating that this factor is regulated by neuronal activity not only in the hippocampus and cortex but also in the hypothalamus.

Hippocampal levels of mRNA encoding nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) are rapidly induced by enhanced neuronal activity following seizures and glutamate or muscarinic receptor activation. However, the levels of neurotrophin-3 (NT-3) mRNA acutely decrease after limbic seizures suggesting that a different mode of regulation may exist for these neurotrophins. Here we show that BDNF and neutrotrophin-4 (NT-4), but not NT-3 itself, up-regulate NT-3 mRNA in cultured hippocampal neurons. In the rat hippocampus, the muscarinic receptor agonist, pilocarpine increased BDNF mRNA levels rapidly and those of NT-3 with a delay of several hours. Injection of BDNF into neonatal rats elevated NT-3 mRNA in the hippocampus which demonstrates that BDNF is able to enhance NT-3 expression in vivo. The regulation of NT-3 by BDNF and NT-4 enlargens the neurotrophic spectrum of these neurotrophins to include neuron populations responsive primarily to NT-3.

Ciliary neurotrophic factor (CNTF) was first identified and partially purified from embryonic chick eye tissues. Subsequently, it was shown that CNTF is also present in large amounts in sciatic nerves of adult rats and rabbits, which led to its final purification and cloning. CNTF is not secreted by the classical secretory pathway involving the endoplasmatic reticulum and Golgi complex, but can be detected in high quantities within the cytoplasm of myelinating Schwann cells and astrocytes using immunohistochemistry. CNTF supports survival and/or differentiation of a variety of neuronal cell types including sensory, sympathetic, and motoneurons. Also, nonneuronal cells, such as oligodendrocytes, microglial cells, liver cells, and skeletal muscle cells, respond to exogenously administered CNTF, both in vitro and in vivo. During development, expression of CNTF is very low, if indeed it is expressed at all, and the phenotype of mice lacking endogenous CNTF after inactivation of the CNTF gene by homologous recombination suggests that CNTF does not play a crucial role for responsive cells during embryonic development. However, motoneurons are lost postnatally in mice lacking endogenous CNTF, suggesting that CNTF acts physiologically on the maintenance of these cells. The ability of exogenous CNTF to protect against motoneuron loss following lesion or in other animal models indicates that CNTF might be useful in the treatment of human motoneuron disorders, provided appropriate means of administration can be found.

Spinal motoneurons innervating skeletal muscle were amongst the first neurons shown to require the presence of their target cells to develop appropriately. Isolated embryonic chick and rat motoneurons have been used to identify neurotrophic factors and cytokines capable of supporting the survival of developing motoneurons. Such factors include ciliary neurotrophic factor (CNTF), which is present physiologically in high amounts in myelinating Schwann cells of peripheral nerves, and brain-derived neurotrophic factor (BDNF) which is synthesized in skeletal muscle and, after peripheral nerve lesion, in Schwann cells. These factors have been further analyzed for their physiological significance in maintaining motoneuron function in vivo, and for their potential therapeutic usefulness in degenerative motoneuron disease. Both CNTF and BDNF are capable of rescuing injured facial motoneurons in newborn rats. Furthermore, CNTF prolongs survival and improves motor function of pmn mice, an animal model for degenerative motoneuron disease, by preventing degeneration of motoneuron axons and somata. Thus treatment of human motoneuron disease with neurotrophic factors should be possible, provided that rational means for application of these factors can be established considering also the appearance of potential side effects.

The aim of our study was to evaluate whether ciliary neurotrophic factor (CNTF) can substitute for leukaemia inhibitory factor (LIF) in maintaining pluripotential embryonic stem (ES) cells in culture. Two subclones of D3 ES cells were used to assess cell proliferation and differentiation in the presence of CNTF, LIF or Buffalo rat liver (BRL) cell-conditioned medium, or in the absence of exogenous differentiation inhibiting factors. ES cells maintained in medium supplemented with CNTF for up to four weeks were injected into blastocysts to investigate their in vivo pluripotency in terms of chimaera formation. CNTF inhibited ES cell differentiation in a dose-dependent manner. The most effective concentration was 10 ng CNTF per ml of medium. The effects of CNTF on ES cell differentiation and proliferation were comparable to those of LIF at the same concentration. BRL cell-conditioned medium was less effective at preventing ES cell differentiation but induced their proliferation very markedly. Both ES cell clones efficiently formed chimaeras after long-term culture with CNTF as the only differentiation inhibiting agent. The ability of these ES cells to colonize the germ-line is the ultimate proof that CNTF can preserve the pluripotency of ES cells.

In the rat hippocampus, nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) are synthesized by neurons in an activity-dependent manner. Glutamate receptor activation increases whereas GABAergic stimulation decreases NGF and BDNF mRNA levels. Here we demonstrate that NGF and BDNF mRNA and NGF protein are up-regulated in the rat hippocampus by the activation of muscarinic receptors. Conversely, NGF and BDNF enhance the release of acetylcholine (ACh) from rat hippocampal synaptosomes containing the nerve endings of the septal cholinergic neurons. NGF also rapidly increases the high-affinity choline transport into synaptosomes. The reciprocal regulation of ACh, NGF and BDNF in the hippocampus suggests a novel molecular framework by which the neurotrophins might influence synaptic plasticity.

Ciliary neurotrophic factor (CNTF) supports the survival of motoneurons in vitro and in vivo. Recombinant CNTF is an investigational drug for the treatment of amyotrophic lateral sclerosis. We determined the pharmacokinetics of radioiodinated CNTF after intravenous injection into rats. CNTF shows a biphasic clearance with an initial plasma half-life of 2.9 minutes and is removed from the circulation by the liver. No accumulation of radioactivity was detectable in nerve tissue or skeletal muscle after intravenous injection of 0.1 microgram and 0.5 microgram of CNTF. Radioactive degradation products accumulate in the skin. Liver cells express specific binding proteins for CNTF, and the incorporation and degradation of intravenously injected CNTF by the liver may occur after association of CNTF with the soluble CNTF receptor alpha in the circulation. Probably as a consequence of its binding to hepatocytes, CNTF induces acute-phase responses in liver. The short half-life and the inflammatory side effect may limit the clinical usefulness of systematically administered CNTF in the treatment of human motoneuron disorders.

Neurotrophin-3 (NT-3) is a member of the neurotrophin gene family and is highly expressed in the developing rat cerebellum. Here we show that brain-derived neurotrophic factor (BDNF) increased by approximately 10-fold the NT-3 mRNA levels in cultured cerebellar granule neurons isolated from postnatal rats, whereas nerve growth factor (NGF) and NT-3 itself had no effect. The effect of BDNF was additive to that of triiodothyronine (T3), which also increased NT-3 mRNA in these neurons. The drug K252a inhibited the BDNF-mediated stimulation of NT-3 expression, suggesting an involvement of trkB receptors. Nuclear run-on experiments showed that BDNF enhanced NT-3 transcription, whereas the stability of NT-3 mRNA remained unchanged. The data presented are the first demonstration that one neurotrophin regulates the expression of another and provide evidence that NT-3 production in granule neurons is regulated by both BDNF and T3.

We have studied the effects of different neurotrophins on the survival and proliferation of rat cerebellar granule cells in culture. These neurons express trkB and trkC, the putative neuronal receptors for brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) respectively. Binding studies using iodinated BDNF and NT-3 demonstrated that both BDNF and NT-3 bind to the cerebellar granule neurons with a similar affinity of approximately 2 x 10(-9) M. The number of receptors per granule cell was surprisingly high, approximately 30 x 10(-4) and 2 x 10(5) for BDNF and NT-3, respectively. Both NT-3 and BDNF elevated c-fos mRNA in the granule neurons, but only BDNF up-regulated the mRNA encoding the low-affinity neurotrophin receptor (p75). In contrast to NT-3, BDNF acted as a survival factor for the granule neurons. BDNF also induced sprouting of the granule neurons and significantly protected them against neurotoxicity induced by high (1 mM) glutamate concentrations. Cultured granule neurons also expressed low levels of BDNF mRNA which were increased by kainic acid, a glutamate receptor agonist. Thus, BDNF, but not NT-3, is a survival factor for cultured cerebellar granule neurons and activation of glutamate receptor(s) up-regulates BDNF expression in these cells.

The structure of the rat ciliary neurotrophic factor (CNTF) gene and the regulation of CNTF mRNA levels in cultured glial cells were investigated. The rat mRNA is encoded by a simple two-exon transcription unit. Sequence analysis of the region upstream of the transcription start-site did not reveal a typical TATA-box consensus sequence. Low levels of CNTF mRNA were detected in cultured Schwann cells, and CNTF mRNA was not increased by a variety of treatments. Three-week-old astrocyte-enriched cell cultures from new-born rat brain contained easily detectable CNTF mRNA. In astrocyte-enriched cultures, upregulation of CNTF mRNA levels was observed after treatment with IFN-gamma. CNTF mRNA levels were down-regulated in these cells by treatments that elevate intracellular cyclic AMP and by members of the fibroblast growth factor (FGF) family. The implications of these results for potential in vivo functions of CNTF are discussed.

CNTF is a cytosolic molecule expressed postnatally in myelinating Schwann cells and in a subpopulation of astrocytes. Although CNTF administration prevents lesion-mediated and genetically determined motor neuron degeneration, its physiological function remained elusive. Here it is reported that abolition of CNTF gene expression by homologous recombination results in a progressive atrophy and loss of motor neurons in adult mice, which is functionally reflected by a small but significant reduction in muscle strength.

In previous experiments it has been demonstrated that the synthesis of BDNF (brain-derived neurotrophic factor) and NGF in neurons of the hippocampus is regulated by neuronal activity. The glutamate system is predominantly responsible for upregulation and the GABAergic system for downregulation both in vitro and in vivo (Zafra et al., 1990, 1991). The aim of the present study is to examine the extent to which the cholinergic system is also involved in the regulation of NGF and BDNF mRNA and whether the regulatory contribution of the cholinergic system changes during development. Partial transection of the fimbria fornix bundle in the second postnatal week resulted in a reduction of BDNF and NGF mRNA levels in the hippocampus, suggesting that septal cholinergic input is involved in the regulation of hippocampal BDNF and NGF mRNA levels. Because the fimbria fornix bundle also contains fibers other than cholinergic ones, we further evaluated the importance of the cholinergic influence by injecting pilocarpine, a muscarinic agonist. Pilocarpine markedly increased hippocampal BDNF and NGF mRNA levels in both early postnatal and adult rats. In situ hybridization experiments demonstrated that pilocarpine led to an increase in BDNF expression in the CA1-CA4 regions of the hippocampus and in the dentate gyrus. However, pilocarpine increased NGF mRNA only in those neurons of the dentate gyrus and CA1-CA4 regions that also expressed NGF mRNA in the controls.(ABSTRACT TRUNCATED AT 250 WORDS)

Motoneurons played an essential role in establishing the concept of target-mediated support of innervating neurons. However, it took several decades until molecules were identified which trophically support motoneurons in vitro and in vivo. The most potent molecule identified so far is ciliary neurotrophic factor (CNTF). It is expressed as a cytosolic molecule in myelinating Schwann cells rather than in skeletal muscle in the postnatal period and therefore does not qualify as a target-derived neurotrophic factor regulating motoneuron survival during embryonic development. However, the inactivation of CNTF by gene targeting experiments results in progressive atrophy and degeneration of motoneurons, demonstrating that CNTF plays an essential role as a maintenance factor for motoneurons postnatally. Secretory molecules which are expressed in skeletal muscle during embryonic development and which support motoneurons in culture and partially also in vivo include members of the NGF gene family (BDNF, NT-3, NT-4/5), FGF-5, IGF-I, and LIF. The evaluation of the physiological importance of these molecules is under investigation.

We have studied the effects of MK-801, a noncompetitive antagonist of N-methyl-D-aspartate-type glutamate receptors, on brain-derived neurotrophic factor (BDNF) mRNA levels in the rat brain. MK-801 decreased BDNF mRNA in the hippocampus and in the superficial layers of the cerebral cortex. However, in single cells of the middle layer of the cerebral cortex and the midline thalamic nuclei BDNF mRNA levels were markedly increased by MK-801. The highest density of these cells was found in the limbic cortex, especially in the retrosplenial and medial entorhinal cortex. Pentobarbital (an enhancer of gabaergic functions) and scopolamine (a muscarinic receptor antagonist) blocked the effects of MK-801 on BDNF mRNA levels in the retrosplenial cortex, but the nicotinic and dopaminergic receptor antagonists mecamylamine and haloperidol, respectively, were ineffective. Pilocarpine, a muscarinic cholinergic agonist increased BDNF mRNA in some, but not all, cortical areas, where MK-801 had elicited an increase in BDNF mRNA. Thus, the observations made with MK-801 demonstrate that depending on the neuronal connections and the transmitter systems involved, a given compound can elicit either a decrease or an increase in BDNF mRNA levels. This may open up pharmacological possibilities to a regionally more refined regulation of the neurotrophin synthesis.

We have investigated the expression of the mRNAs for brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), neurotrophin-3 (NT-3) and neurotrophin-4 (NT-4) in the hippocampus before and after induction of long term potentiation (LTP) of synaptic transmission in the dentate gyrus through stimulation of the perforant path (PP). A unilateral PP stimulation produced a bilateral increase in the mRNA for both BDNF and NGF in granular neurones of the dentate gyrus but not in other neurones in the hippocampus. The mRNA for neurotrophin-3 (NT-3) was bilaterally decreased by LTP but that of NT-4 remained at the basal level. These results suggest that individual neurotrophic factors may play different roles in neuronal plasticity.

Motoneurons innervating the skeletal musculature were among the first neurons shown to require the presence of their target cells to develop appropriately. But the characterization of molecules allowing motoneuron survival has been difficult. Ciliary neurotrophic factor prevents the death of motoneurons, but its gene is not expressed during development. Although the presence of a neurotrophin receptor on developing motoneurons has suggested a role for neurotrophins, none could be shown to promote motoneuron survival in vitro. We report here that brain-derived neurotrophic factor can prevent the death of axotomized motoneurons in newborn rats, suggesting a role for this neurotrophin for motoneuron survival in vivo.

Brain-derived neurotrophic factor (BDNF) and NGF are both expressed by neurons in the hippocampus. In previous studies, it has been demonstrated that both BDNF and NGF mRNA levels are regulated by neuronal activity. Upregulation is predominantly regulated by the glutamate (NMDA and non-NMDA receptors); downregulation, predominantly by the GABA system (Zafra et al., 1990, 1991). In neuronal cultures of the rat hippocampus, potassium depolarization and kainic acid-mediated increases in BDNF and NGF mRNA were eliminated in a dose-dependent manner by the calcium channel blocker nifedipine. Conversely, calcium ionophores (Bay-K8644 and ionomycin) augmented BDNF and NGF mRNA levels by a calmodulin-mediated mechanism. In view of the fact that many potential modulators (conventional transmitters and neuropeptides) of neuronal and astrocytic BDNF and NGF mRNA synthesis may act via the adenylate cyclase system, we studied the effect of forskolin, an activator of adenylate cyclase. Indeed, forskolin enhanced the effects of calcium ionophores and kainic acid on BDNF and NGF mRNA levels. Cytokines, such as interleukin-1 and transforming growth factor-beta 1, which have previously been shown to increase NGF mRNA markedly in astrocytes, were without effect on neuronal BDNF and NGF mRNA levels. In contrast to neuronal cultures, where the regulation of BDNF and NGF mRNA was generally very similar, the regulation in astrocytes was distinctly different. All the cytokines that produce a marked increase in NGF mRNA were without effect on astrocyte BDNF mRNA levels, which under basic conditions were below the detection limit. However, norepinephrine produced a marked elevation of BDNF mRNA in astrocytes, an effect that was further enhanced by glutamate receptor agonists.(ABSTRACT TRUNCATED AT 250 WORDS)

Specific sensory input has profound transient and long-lasting effects on the function of corresponding sensory cortical areas both during development and in adulthood. To study whether neurotrophic factors might play a role in such processes, we investigated the effects of light on the nerve growth factor and brain-derived neurotrophic factor (BDNF) mRNA levels in rat visual cortex. Keeping adult rats in the dark or preventing normal activity of retinal ganglion cells by intraocular injection of tetrodotoxin significantly decreased the levels of BDNF mRNA in the visual cortex but not in other cortical areas. Exposure to light after a period in darkness rapidly restored the mRNA to control levels. These alterations in visual input had no effect on nerve growth factor mRNA. The mRNA of trkB, the putative signal-transducing receptor unit for BDNF, was also decreased in darkness, although less than BDNF mRNA. BDNF mRNA levels increased in the visual cortex of newborn rats after eye-opening. This increase is retarded, although not completely abolished, by rearing the pups in darkness. Thus, the levels of BDNF mRNA are rapidly regulated by sensory input during development and in adulthood. BDNF may therefore play an important role in formation and in activity-dependent modulation of specific connections in the visual cortex.

Nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) are molecules which regulate the development and maintenance of specific functions in different populations of peripheral and central neurons, amongst them sensory neurons of neural crest and placode origin. Under physiological conditions NGF is synthesized by peripheral target tissues, whereas BDNF synthesis is highest in the CNS. This situation changes dramatically after lesion of peripheral nerves. As previously shown, there is a marked rapid increase in NGF mRNA in the nonneuronal cells of the damaged nerve. The prolonged elevation of NGF mRNA levels is related to the immigration of activated macrophages, interleukin-1 being the most essential mediator of this effect. Here we show that transsection of the rat sciatic nerve also leads to a very marked increase in BDNF mRNA, the final levels being even ten times higher than those of NGF mRNA. However, the time-course and spatial pattern of BDNF mRNA expression are distinctly different. There is a continuous slow increase of BDNF mRNA starting after day 3 post-lesion and reaching maximal levels 3-4 wk later. These distinct differences suggest different mechanisms of regulation of NGF and BDNF synthesis in non-neuronal cells of the nerve. This was substantiated by the demonstration of differential regulation of these mRNAs in organ culture of rat sciatic nerve and Schwann cell culture. Furthermore, using bioassays and specific antibodies we showed that cultured Schwann cells are a rich source of BDNF- and ciliary neurotrophic factor (CNTF)-like neurotrophic activity in addition to NGF. Antisera raised against a BDNF-peptide demonstrated BDNF-immunoreactivity in pure cultured Schwann cells, but not in fibroblasts derived from sciatic nerve.

Ciliary neurotrophic factor (CNTF) supports the survival of embryonic motor neurons in vitro and in vivo, and prevents lesion-mediated degeneration of rat motor neurons during early post-natal stages. Here we report that CNTF greatly reduces all the functional and morphological changes in pmn/pmn mice, an autosomal recessive mutant leading to progressive caudo-cranial motor neuron degeneration. The first manifestations of progressive motor neuronopathy in homozygous pmn/pmn mice become apparent in the hind limbs at the end of the third post-natal week, and all the mice die up to 6 or 7 weeks after birth from respiratory paralysis. Treatment with CNTF prolongs survival and greatly improves motor function of these mice. Moreover, morphological manifestations, such as loss of motor axons in the phrenic nerve and degeneration of facial motor neurons, were greatly reduced by CNTF, although the treatment did not start until the first symptoms of the disease had already become apparent and substantial degenerative changes were already present. The protective and restorative effects of CNTF in this mouse mutant give new perspectives for the treatment of human degenerative motor neuron diseases with CNTF.

Ciliary neurotrophic factor (CNTF) is expressed in high quantities in Schwann cells of peripheral nerves during postnatal development of the rat. The absence of a hydrophobic leader sequence and the immunohistochemical localization of CNTF within the cytoplasm of these cells indicate that the factor might not be available to responsive neurons under physiological conditions. However, CNTF supports the survival of a variety of embryonic neurons, including spinal motoneurons in culture. Moreover we have recently demonstrated that the exogenous application of CNTF protein to the lesioned facial nerve of the newborn rat rescued these motoneurons from cell death. These results indicate that CNTF might indeed play a major role in assisting the survival of lesioned neurons in the adult peripheral nervous system. Here we demonstrate that the CNTF mRNA and protein levels and the manner in which they are regulated are compatible with such a function in lesioned peripheral neurons. In particular, immunohistochemical analysis showed significant quantities of CNTF at extracellular sites after sciatic nerve lesion. Western blots and determination of CNTF biological activity of the same nerve segments indicate that extracellular CNTF seems to be biologically active. After nerve lesion CNTF mRNA levels were reduced to less than 5% in distal regions of the sciatic nerve whereas CNTF bioactivity decreased to only one third of the original before-lesion levels. A gradual reincrease in Schwann cells occurred concomitant with regeneration.

In the central nervous system brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF) are predominantly located in neurons. Here we demonstrate that the balance between the activity of the glutamatergic and gamma-aminobutyric acid (GABA)ergic systems controls the physiological levels of BDNF and NGF mRNAs in hippocampal neurons in vitro and in vivo. The blockade of the glutamate receptors and/or stimulation of the GABAergic system reduces BDNF and NGF mRNAs in hippocampus and NGF protein in hippocampus and septum. The reduction of NGF in the septum reflects the diminished availability of NGF in the projection field of NGF-dependent septal cholinergic neurons. These neurons do not synthesize NGF themselves but accumulate it by retrograde axonal transport. The refined and rapid regulation of BDNF and NGF synthesis by the glutamate and GABA transmitter systems suggests that BDNF and NGF might be involved in activity-dependent synaptic plasticity.

Ciliary neurotrophic factor (CNTF) is a potent survival molecule for a variety of embryonic neurons in culture. The developmental expression of CNTF occurs clearly after the time period of the physiological cell death of CNTF-responsive neurons. This, together with the sites of expression, excludes CNTF as a target-derived neuronal survival factor, at least in rodents. However, CNTF also participates in the induction of type 2 astrocyte differentiation in vitro. Here we demonstrate that the time course of the expression of CNTF-mRNA and protein in the rat optic nerve (as evaluated by quantitative Northern blot analysis and biological activity, respectively) is compatible with such a glial differentiation function of CNTF in vivo. We also show that the type 2 astrocyte-inducing activity previously demonstrated in optic nerve extract can be precipitated by an antiserum against CNTF. Immunohistochemical analysis of astrocytes in vitro and in vivo demonstrates that the expression of CNTF is confined to a subpopulation of type 1 astrocytes. The olfactory bulb of adult rats has comparably high levels of CNTF to the optic nerve, and here again, CNTF-immunoreactivity is localized in a subpopulation of astrocytes. However, the postnatal expression of CNTF in the olfactory bulb occurs later than in the optic nerve. In other brain regions both CNTF-mRNA and protein levels are much lower.

Both nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) exert neurotrophic actions on the cholinergic neurons of the basal forebrain nuclei. These neurotrophic factors are synthesized by hippocampal and cortical neurons that are located in the projection field of the basal forebrain cholinergic neurons. Both in vivo and in vitro the levels of NGF- and BDNF-mRNAs are increased up to 20-fold by kainic acid via non-NMDA glutamate receptors. Enhancement of the effectiveness of the GABAergic system by benzodiazepam or direct GABA agonists blocks the effect of kainic acid and reduces the basic levels of NGF- and BDNF-mRNAs. Whereas the increases in both NGF- and BDNF-mRNAs above normal levels are mediated by non-NMDA receptors, maintenance of the normal levels of NGF- and BDNF-mRNAs seems to be mediated predominantly by NMDA receptors. The regulation of NGF and BDNF synthesis via specific transmitter systems is discussed in the context of the refined tuning of synaptic functions, the potential implications for memory functions, and the possible therapeutic consequences for the treatment of Alzheimer's disease.

We have demonstrated that the extensive degeneration of motoneurons in the rat facial nucleus after transection of the facial nerve in newborn rats can be prevented by local ciliary neurotrophic factor (CNTF) administration. CNTF differs distinctly from known neurotrophic molecules such as NGF, BDNF and NT-3 in both its molecular characteristics (CNTF is a cytosolic rather than a secretory molecule) and its broad spectrum of biological activities. CNTF is expressed selectively by Schwann cells and astrocytes of the peripheral and central nervous system, respectively, but not by target tissues of the great variety of CNTF-responsive neurons. CNTF mRNA is not detectable by Northern blot or PCR analysis during embryonic development and immediately after birth. However, during the second post-natal week, a more than 30-fold increase in CNTF mRNA and protein occurs in the sciatic nerve. Since the period of low CNTF levels in peripheral nerves coincides with that of high vulnerability of motoneurons (i.e. axonal lesion results in degeneration of motoneuron cell bodies), insufficient availability of CNTF may be the reason for the rate of lesion-induced cell death of early post-natal motoneurons. Highly enriched embryonic chick motoneurons in culture are supported at survival rates higher than 60% by CNTF, even in single cell cultures, indicating that CNTF acts directly on motoneurons. In contrast to CNTF, the members of the neurotrophin gene family (NGF, BDNF and NT-3) do not support the survival of motoneurons in culture. However, aFGF and bFGF show distinct survival activities which are additive to those of CNTF, resulting in the survival of virtually all motoneurons cultured in the presence of CNTF and bFGF.

The mRNAs of nerve growth factor (NGF) and brain derived neurotrophic factor (BDNF) exhibit a similar, though not identical, regional and cellular distribution in the rodent brain. In situ hybridization experiments have shown that BDNF, like NGF, is predominantly expressed by neurons. The neuronal localization of the mRNAs of these two neurotrophic molecules raised the question as to whether neuronal activity might be involved in the regulation of their synthesis. After we had demonstrated that depolarization with high potassium (50 mM) resulted in an increase in the levels of both BDNF and NGF mRNAs in cultures of hippocampal neurons, we investigated the effect of a large number of transmitter substances. Kainic acid, a glutamate receptor agonist, was by far the most effective in increasing BDNF and NGF mRNA levels in the neurons, but neither N-methyl-D-aspartic acid (NMDA) nor inhibitors of the NMDA glutamate receptors had any effect. However, the kainic acid mediated increase was blocked by antagonists of non-NMDA receptors. Kainic acid also elevated levels of BDNF and NGF mRNAs in rat hippocampus and cortex in vivo. These results suggest that the synthesis of these two neurotrophic factors in the brain is regulated by neuronal activity via non-NMDA glutamate receptors.

In previous studies, it has been demonstrated that ciliary neurotrophic factor (CNTF) has a potent survival effect on various populations of neurons in culture, in particular, neurons isolated from chick ciliary, dorsal root sensory, and sympathetic ganglia (Barbin et al., 1984). After recent investigations demonstrated that CNTF prevents the degeneration of motoneurons in newborn rats after axonal lesion (Sendtner et al., 1990), the question arose as to whether CNTF also has a survival effect on embryonic chick motoneurons at the developmental stage where physiological cell death occurs. To study this, it was essential to develop an isolation and culture procedure for the survival of chick E6 spinal motoneurons in which non-neuronal cells were eliminated and the motoneurons were highly enriched. In these cultures, virtually all of the initially plated motoneurons survived for at least 3 d in the presence of muscle extract, which was chosen as a positive control. Retrograde labeling of the motoneurons prior to their isolation showed that there is more than an 80% enrichment for motoneurons by the method used. The retrogradely labeled neurons also fulfilled the morphological criteria (diameter of neurons, appearance of processes) to identify motoneurons independent of retrograde labeling. Under these conditions, CNTF at a concentration of 1.5 ng/ml (EC50, 0.023 ng/ml) supported maximally 64% of the initially plated spinal motoneurons after 3 d and 53% after 6 d (the longest time period investigated). Other neurotrophic factors, such as NGF, brain-derived neurotrophic factor (BDNF), and neurotrophin-3, had no survival effect at all, even at concentrations up to 10 micrograms/ml for NGF and BDNF.(ABSTRACT TRUNCATED AT 250 WORDS)

The period of natural cell death in the development of rodent motor neurons is followed by a period of sensitivity to axonal injury. In the rat this early postnatal period of vulnerability coincides with that of very low ciliary neurotrophic factor (CNTF) levels in the sciatic nerve before CNTF increases to the high, adult levels. The developmental time course of CNTF expression, its regional tissue distribution and its cytosolic localization (as suggested by its primary structure) favour a role for CNTF as a lesion factor rather than a target-derived neurotrophic molecule like nerve growth factor. Nevertheless CNTF exhibits neurotrophic activity in vitro on different populations of embryonic neurons. To determine whether the vulnerability of motor neurons to axotomy in the early postnatal phase is due to insufficient availability of CNTF, we transected the axons of newborn rat motor neurons and demonstrated that local application of CNTF prevents the degeneration of the corresponding cell bodies.

Brain-derived neurotrophic factor (BDNF) is a small, basic protein purified from the mammalian brain that has been shown previously to support the survival of cultured spinal sensory neurons (Barde et al., 1982). In current studies, BDNF was tested for its ability to support the survival of cultured CNS cells isolated from the perinatal rat retina. Both immunofluorescent labeling of Thy-1 and prior retrograde labeling with HRP were used as retinal ganglion cell markers in vitro. With embryonic day (E) 17 retinas, it was found that BDNF allowed the survival of a small subpopulation of neurons (about 7% of the cells plated at this age) identified by the immunofluorescent labeling of Thy-1. No detectable effects were seen when either the total number of cells or the number of tetanus toxin-positive neurons was measured. BDNF also had an effect on cultured neurons retrogradely labeled after HRP injections in the superior colliculi of neonatal rats. The BDNF-responsive population was therefore detected only in retinal cultures with specific markers and identified as consisting of retinal ganglion cells. These cells could be enriched about 80-fold by density gradient centrifugation, and purified ganglion cell cultures were shown to be responsive to BDNF. Whereas with E17 retinas, the number of surviving Thy-1 positive neurons could be kept constant for at least 4 d, the survival of postnatal neurons was only transiently increased by BDNF. We conclude that in the retina, BDNF affects only the survival of ganglion cells in vitro by a direct action on these cells. The results are discussed in terms of target-derived neurotrophic support during development.

To determine the spectrum of activity of brain-derived neurotrophic factor (BDNF) among first-order sensory neurons, explants of the nine distinct populations of sensory neurons from embryonic chicks of 3-14 d incubation (E3-E14) were grown in collagen gels with and without BDNF in the culture medium. The explants responded to BDNF with profuse neurite outgrowth and comprised those in which neurons are derived from neural crest (the dorsomedial part of the trigeminal ganglion, rostromedial part of the trigeminal mesencephalic nucleus, jugular ganglion, and lumbosacral dorsal root ganglia) and from epibranchial placodes (the ventrolateral part of the trigeminal ganglion and the geniculate, petrosal, and nodose ganglia). This response was first clearly observed in the ventrolateral trigeminal and nodose ganglia as early as E4, but did not appear until later in other explants. The dorsomedial trigeminal, jugular, and geniculate ganglia were the latest to develop a response, which was not apparent until E8. In all explants the response was maximal between E10 and E12, and there was a decline in the magnitude of the response from E12 to E14. Although explants of the vestibular ganglion failed to respond to BDNF, the survival and growth of vestibular neurons in dissociated neuron-enriched cultures were promoted by BDNF. To investigate whether all first-order sensory neurons respond to BDNF or whether BDNF-responsive neurons comprise a distinct subset, we studied the influence of BDNF and NGF on the survival and growth of the placode-derived and the neural crest-derived neurons of the trigeminal ganglion in dissociated neuron-enriched culture.(ABSTRACT TRUNCATED AT 250 WORDS)

Explants of adult or 10-day-old rat sciatic and optic nerves were implanted as "bridges" through a silicon grease seal in a three-compartment chamber culture system, leading from a narrow center chamber to two adjacent side chambers. Dissociated newborn rat sympathetic or sensory neurons were plated into the center chamber and grown in the presence of optimal concentrations of nerve growth factor (NGF). By light microscopy, nerve fibers were seen to grow out of the sciatic nerve explants in the side chambers after 2 to 3 weeks. Electron microscopy showed large numbers of axons present inside the sciatic nerves, irrespective of the presence and number of living Schwann cells. Besides their tendency to fasciculate, axons grew with high preference on Schwann cell membranes and the Schwann cell side of the basal lamina, a situation identical to in vivo regeneration. In contrast to the sciatic nerves, no axons could be found under any condition in the optic nerves. This result points to the existence of extremely poor, non-permissive substrate conditions in the differentiated optic nerves which cannot be overcome by the strong fiber outgrowth-promoting effects of NGF.

Ciliary neurotrophic factor (CNTF) was originally characterized as a survival factor for chick ciliary neurons in vitro. More recently, it was shown to promote the survival of a variety of other neuronal cell types and to affect the differentiation of E7 chick sympathetic neurons by inhibiting their proliferation and by inducing the expression of vasoactive intestinal peptide immunoreactivity (VIP-IR). In cultures of dissociated sympathetic neurons from newborn rats, CNTF induces cholinergic differentiation as shown by increased levels of choline acetyltransferase (ChAT). This increase is paralleled by a reduction of tyrosine hydroxylase (TH) activity. Moreover, CNTF promotes the differentiation of bipotential 02A progenitor cells to type-2-astrocytes in vitro. To help establish which, if any, of these functions CNTF exerts in vivo, it is necessary to determine its primary structure, cellular expression, developmental regulation and localization. The complementary DNA-deduced amino-acid sequence and subsequent expression of cDNA clones covering the entire coding region in HeLa-cells indicate that CNTF is a cytosolic protein. This, together with its regional distribution and its developmental expression, show that CNTF is not a target-derived neurotrophic factor. CNTF thus seems to exhibit neurotrophic and differentiation properties only after becoming available either by cellular lesion or by an unknown release mechanism.

The well-documented physiological role of nerve growth factor (NGF) in peripheral sympathetic and neural-crest-derived sensory neurons in vivo has its exact counterpart in vitro. This provided the conceptual basis for developing in vitro analytical procedures for the purification of new neurotrophic molecules. The experimental approaches used are discussed in the context of the purification of new neurotrophic factors, brain-derived neurotrophic factor (BDNF) and ciliary neurotrophic factor (CNTF). The importance of the modulatory role played by extracellular matrix molecules, in particular laminin, on both NGF-mediated and BDNF-mediated survival effects is also delineated. BDNF is a very basic (pI approximately 10) molecule of about 12 kDa, having physico-chemical characteristics close to those of the monomer of NGF. However, the spectrum of its biological actions is distinctly different from that of NGF. In particular, BDNF supports the survival of retinal ganglion cells and placode-derived peripheral sensory neurons which are not supported by NGF. The trophic supply of primary sensory neurons projecting to both the central nervous system and the periphery is discussed. It is hypothesized that sensory neurons receive limited quantities of neurotrophic molecules from both peripheral and central axons, a mechanism ensuring the survival of neurons adequately connected with both peripheral and central targets.