The NeurotroDhins BDNF, NT-3 and NT-4/5 Promote Survival and Morphological and Biochemical Differentiation of Striatal Neurons In Vitro

The neurotrophins, brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), neurotrophin 4/5 (NT-4/5) and nerve growth factor (NGF), were compared for their effects on the survival and differentiation of embryonic rat striatal neurons grown in low-density cultures. Treatment with BDNF for 8 days resulted in a 40% increase in overall neuronal survival, a 3- to 5-fold increase in the number of calbindin-immunoreactive neurons, and an 80% increase in GABA-positive neurons. Treatment with NT-3 or NT-4/5 produced a 2- to 3-fold increase in the number of calbindin-positive neurons and an increase in GABA-positive cell number similar to that induced by BDNF. BDNF treatment produced a striking morphological differentiation of striatal GABAergic neurons, which was characterized by a doubling of the number of neurite branch points, the total area of arborization and the perikaryal area compared to control cultures. All three of these factors increased high-affinity GABA uptake 2-fold. NGF had no effect on any of the parameters examined. Our results show that BDNF, NT-3 and NT-4/5 promote the survival and/or differentiation of calbindin-immunopositive and GABAergic striatal neurons.     

Synergistic effects of BDNF and NT-3 on postnatal spiral ganglion neurons

Neurotrophins have profound effects on the development and maintenance of neurons that compose the VIIIth cranial nerve. In the auditory division of the nerve, brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) have been localized to the sensory epithelium, and their respective high-affinity tyrosine kinase receptors (TrkB and TrkC) are expressed within the neuronal population. By using a culture methodology that allows evaluation of single neurons, we determined that BDNF and neurotrophin-4 (NT-4), which both bind to the TrkB high-affinity receptor, greatly enhanced neuron survival above control cultures. NT-3, which acts via the TrkC high-affinity receptor, also increased survival, but to a lesser extent. By testing a variety of neurotrophin concentrations and combinations, we observed that simultaneous activation of the TrkB and TrkC receptors synergistically promoted neuron survival compared to cultures that contained either neurotrophin alone at the same total concentration. Antibody labeling showed that the high-affinity Trk receptors were localized predominantly to the neurons and not to the surrounding satellite cells; furthermore, TrkB- and TrkC-specific antibodies each labeled 100% of the cultured neurons. These results suggest that synergistic interactions between BDNF and NT-3 may be crucial for spiral ganglion neuron survival during the final stages of development. J. Comp. Neurol. 386:529–539, 1997. © 1997 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.     

Highly selective effects of nerve growth factor,brain-derived neurotrophic factor,and neurotrophin-3 on intact and injured basal forebrain magnocellular neurons

Cholinergic neurons of the basal nucleus complex (BNC) respond to nerve growth factor (NCF), the first member of a polypeptide gene family that also includes brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and neurotrophin-4/5 (NT-4/5), NGF, BDNF, and NT-3 are enriched in hippocampus. In addition, NGF and, more recently, BDNF have been shown to stimulate the cholinergic differentiation and enhance the survival of BNC cells in vitro. The present investigation was designed to test, in a comparative fashion, the in vivo effects of human recombinant NGF, BDNF, and NT-3 with confirmed activities in vitro on cholinergic and γ-aminobutyric acid (GABA)-ergic BNC neurons. The specific questions asked were whether and, to what extent, biologically active recombinant neurotrophins stimulate the transmitter phenotypes of intact cholinergic and GABAergic neurons of the BNC, and whether, and to what extent, recombinant neurotrophins protect the transmitter phenotypes of axotomized cholinergic and GABAergic neurons of the BNC following complete transections of the fimbria-fornix (measured by ChAT mRNA hybridization). Our results confirm the profound stimulatory and p^75NGFR expression in both intact and axotomized cholinergic neurons and to exert minor effects on some cholinergic markers (e.g., ChAT immunoreactivity). NT-3 had no influence on GABAergic neurons. Taken together, these results indicate that, despite their significant sequence homologies and their shared abundance in target fields of BNC neurons, NGF, BDNF, and NT-3 show striking differences in their efficacies as cholinergic trophic factors. GABAergic neurons of the BNC are resistant to neurotrophins. The result of the present investigation establish that NGF excels among neurotrophins as a trophic factor for intact and injured basal forebrain cholinergic neurons. © 1994 Wiley-Liss, Inc.     

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.     

Neurotrophin-induced trk receptor phosphorylation and cholinergic neuron response in primary cultures of embryonic rat brain neurons.

Tyrosine phosphorylation of trk type neurotrophin receptors in primary cultures of embryonic rat brain cells was studied by immunoprecipitation and immunoblotting. In cultures containing basal forebrain cholinergic neurons, but not in cultures of cerebral cortex, nerve growth factor (NGF) treatment for 4 min induced tyrosine phosphorylation of trk family proteins. Stimulation with brain-derived neurotrophic factor (BDNF) or neurotrophin-3 (NT-3), resulted in a very robust phosphorylation signal in basal forebrain and cortical cultures, suggesting actions of these neurotrophins not only on cholinergic cells but probably on most embryonic brain neurons. Trk tyrosine phosphorylation was completely abolished by 5 microM K-252b. Inhibition was rapid, being evident by 30 s following addition of the drug. Corresponding stimulatory and inhibitory effects were seen for phospholipase-C gamma 1 (PLC gamma 1) and extracellular signal-regulated kinase 1 (Erk1), two enzymes involved in second messenger mechanisms. Our findings indicate involvement of trk receptor activation in the NGF response of basal forebrain cholinergic cells and provide evidence for widespread presence of BDNF and NT-3 responsive neurons in the embryonic brain.     

Early BDNF, NT-3, and NT-4 signaling events.

Much more is known about nerve growth factor (NGF) signaling than that initiated by brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), or NT-4. We sought to study early BDNF, NT-3, and NT-4 signaling events. Using TrkB-expressing cells, we found that BDNF and NT-4 individually induced tyrosine phosphorylation of TrkB in a dose-dependent fashion. At maximally effective concentrations, BDNF or NT-4 induced robust TrkB tyrosine phosphorylation at 5 min; this progressively declined at 15, 30, and 60 min. Using immunoprecipitation, PI3-kinase and tyrosine phosphorylated PLC-gamma1 and SHC were shown to be associated with tyrosine phosphorylated TrkB in response to both BDNF and NT-4. BDNF and NT-4 induced similar intensities of phosphorylation of TrkB and signaling intermediates at equivalent doses. NT-3 treatment of TrkC-expressing cells induced very similar patterns for induction of TrkC tyrosine phosphorylation and recruitment of signaling intermediates. BDNF, NT-3, and NT-4 caused rapid tyrosine phosphorylation of ERK and SNT. These data suggest that the earliest signaling events for BDNF, NT-3, and NT-4 are very similar to those for NGF.     

Expression of nerve growth factor, brain-derived neurotrophic factor, and neurotrophin-3 in the somatosensory cortex of the mature rat: coexpression with high-affinity neurotrophin receptors

Neurotrophins, including nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin-3 (NT-3), are critical for the maintenance and plasticity of central nervous system (CNS) neurons. We tested the hypothesis that cortical neurons participate in redundant autocrine/paracrine systems. Three sets of studies determined the distribution of NGF-, BDNF-, and NT-3-expressing neurons, the frequency of neurons coexpressing NGF and BDNF, and the frequency of neurons expressing a neurotrophin and its associated high-affinity receptor. The distribution of NGF-, BDNF, and NT-3-immunoreactive neurons was identical. Neurotrophin-positive cells were parceled throughout the cortex, although the labeling frequency was not the same in all layers. More than 30% of the neurons in layers II/III, V, and VI were labeled, whereas only 5-10% of the neurons in layer IV was immunopositive for a neurotrophin. Some glia were also neurotrophin positive, particularly BDNF-positive glia. About 70% of the neurons in layers II/III and V coexpressed NGF and BDNF or coexpressed NGF and NT-3. Ligand-receptor colabeling was also common among cortical neurons. For example, nearly 70% of the NGF-, BDNF-, and NT-3-positive neurons in layer V colabeled with their respective high-affinity receptors, i.e., trkA, trkB, and trkC, respectively. Thus, (a) neurons express multiple neurotrophins and (b) cortical neurons (e.g., layer V neurons) contain the components required for autocrine/paracrine and/or anterograde communication (e.g., neurons in layer II/III support layer V neurons). These systems mean that the cortex is capable of regulating itself autonomously.     

Neurotrophins regulate proliferation and survival of two microglial cell lines in vitro

Microglia are thought to play a key role in the development and regeneration of the central nervous system although the mechanisms regulating their presence and activity are not fully understood. Substantial evidence suggests that members of the neurotrophin family such as nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin-3 and -4 (NT-3/4) have a dramatic effect on both neurons and perineuronal cells. This study employed two murine microglial lines, BV-2 and N9, to examine the action of these neurotrophins on the mitotic activity and survival of microglia in vitro. Neurotrophins were incorporated into the media at the time of plating and cell number and levels of mitochondrial dehydrogenase activity (MTT) were determined at various time points in vitro. NGF increased cell number and MTT levels of both cell lines in a dose-dependent manner. BV-2 was more sensitive to NGF than N9. Similar responses were elicited by BDNF, although the sensitivity of each cell line was different than that found for NGF. NT-3 and NT-4 had no effect on cell proliferation. However, NT-4 had an effect on the survival of BV-2 and N9 cells. The response of these cells to neurotrophins was blocked by K252a, a tyrosine kinase inhibitor, suggesting that actions of neurotrophins were mediated by high-affinity tyrosine kinase receptors (Trk). Immunolocalization studies revealed positive Trk (pan) reactivity in the above cell lines and in primary microglia, but an absence of the low-affinity p75 neurotrophin receptor. Western blot analysis supported the above observations. These studies suggest that in addition to their neurotrophic actions, NGF and BDNF may also regulate microglial dynamics, thereby influencing the surrounding milieu during neuronal regeneration.     

Function and evolution in the NGF family and its receptors.

The gene family of neurotrophins includes nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and neurotrophin-4 (NT-4). Recently, neurotrophin-5 (NT-5), a possible mammalian homologue to NT-4 described in the frog Xenopus, has been cloned in man and rat. The neurotrophins stimulate survival and differentiation of a range of target neurons by binding to cell surface receptors. The structure of NGF has recently been clarified from crystallographic data. The similarities between the different neurotrophins are substantial with the variable regions, giving specificity to each of the family members, being localized to some exposed loop regions. Low-affinity binding (Kd of 10(-9) M) of all tested neurotrophins is mediated via a 75 K glycoprotein (LNGFR) that has been cloned and characterized. A 140 K tyrosine protein kinase encoded by the proto-oncogene trk has been found to bind NGF with high affinity (Kd of 10(-11) M) and to evoke the cellular neurotrophic responses. In addition, a protein encoded by the trk-related gene trkB has been shown to bind BDNF. Recently, a third member of the trk family, trkC, has been cloned and demonstrated to function as a high-affinity receptor for NT-3. The expression of trk and LNGFR mRNA are co-localized in the rat brain to the medial septal nucleus and the nucleus of Broca's diagonal band containing the NGF-responsive magnocellular cholinergic neurons projecting to hippocampus and cerebral cortex. In sharp contrast, the pattern of expression of trkB is widely spread in many areas of the cortex as well as lateral septum. The trkB protein might serve general functions in large areas of the cortex. Site-directed mutagenesis and expression of recombinant chimaeric neurotrophin proteins have made it possible to localize a likely region for the interaction between NGF and the LNGFR. This region could be altered, resulting in the total loss of LNGFR binding by the mutant NGF protein without affecting the binding to the trk receptor which was sufficient for the full biological activity. Cladistic analysis of likely phylogenies within the neurotrophins shows BDNF and NT-4 to be most closely related whereas NGF may be the sister group to NT-3, BDNF, and NT-4. Neurotrophins offer obvious clinical possibilities for treatment of neurodegenerative diseases.     

Thyroid hormone regulation of NGF, NT-3 and BDNF RNA in the adult rat brain.

The effects of peripherally administered thyroid hormone (TH; 500 micrograms/kg; i.p.; q.d.) on the relative abundances of nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin-3 (NT-3) RNA were determined by rtPCR in the cortex and hippocampus of young adult rats. Corresponding changes in choline acetyltransferase (ChAT) activity were measured since NGF and BDNF have been shown to enhance the expression of this marker enzyme of central cholinergic pathways. Abundance levels of NGF and NT-3, relative to cyclophilin (cycl), were increased significantly (+50%, P < 0.05) in the hippocampus following TH treatment. Despite enhanced abundance of NGF in the hippocampus, ChAT activity was unchanged, whereas ChAT activity was modestly increased by 28% in the cortex without corresponding changes in NGF, NT-3 or BDNF. These results demonstrate that TH administration is capable of inducing the accumulation of NT-3, in addition to NGF but that the induction levels of RNA cannot be directly correlated with responsivity of the cholinergic system as measured by ChAT activity.     

Autocrine—paracrine Regulation of Hippocampal Neuron Survival by IGF-1 and the Neurotrophins BDNF, NT-3 and NT-4

In contrast to sympathetic and sensory neurons in the peripheral nervous system, the neurotrophic requirements for neurons in the central nervous system (CNS) have not been clearly identified. The inactivation of specific neurotrophic factors and their receptors by gene targeting has shown that there are no major changes in neuron numbers in the CNS. This suggests an overlap between the action of different neurotrophic factors in the brain during development. Here we have studied the survival of hippocampal neurons prepared from embryonic rats, using different culture conditions. Whereas the hippocampal neurons survive well in culture when plated at high density, they die at lower cell densities in the absence of appropriate neurotrophic factors. Under the latter conditions, both insulin-like growth factor-1 (IGF-1) and the neurotrophins—brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3) and neurotrophin-4 (NT-4)—rescued a large proportion of cultured neurons. In addition, hippocampal neurons from BDNF knockout mice exhibited enhanced cell death compared with cells from wild-type animals. BDNF and IGF-1 both increased the survival of the hippocampal neurons lacking BDNF, showing complementary action for these factors in supporting survival. Blocking antibodies against NT-3 and IGF-1 decreased hippocampal neuron survival at low cell densities, showing autocrine or paracrine action of the factors. At higher cell densities, however, the antibodies had no effect, demonstrating that there is a sufficient amount of endogenous factors supporting survival under these conditions. The present results show that hippocampal neurons depend for survival on local neurotrophic factors such as IGF-1, BDNF and NT-3, which act in an autocrine/paracrine manner. The multifactorial support of hippocampal neurons ensures a maximal degree of neuron survival even in the absence of an individual factor.     

Distinct usages of phospholipase C gamma and Shc in intracellular signaling stimulated by neurotrophins

Nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3), members of the neurotrophin family, bind to and activate TrkA, TrkB and TrkC, respectively, members of the Trk receptor tyrosine kinase family, to exert various effects including promotion of differentiation and survival, and regulation of synaptic plasticity in neuronal cells. Many reports have suggested that different neurotrophins show distinct biological functions, although molecular mechanisms by which neurotrophins exert their different functions remain unclear. In the present study, we found distinct usages of phospholipase Cgamma (PLCgamma) and Shc in intracellular signaling stimulated by neurotrophins. BDNF stimulated much stronger interactions of PLCgamma with Trk than NGF and NT-3 in PC12 cells stably expressing TrkB and cultured cerebral cortical neurons, respectively, although BDNF, NGF and NT-3 induced similar levels of tyrosine phosphorylation of Trk. Furthermore, the cultured cortical neurons showed large PLCgamma-dependent increases in intracellular Ca(2+) levels in response to BDNF compared with NT-3. In Shc signaling, NGF, but not BDNF, displayed interactions between Trk and Shc in a phenylarsine oxide (PAO; an inhibitor of tyrosine phosphatase)-dependent manner in TrkB-expressing PC12 cells. These results indicated that neurotrophins stimulate distinct kinds of interactions between Trk and PLCgamma and between Trk and Shc. These differences may lead to the distinct biological functions of neurotrophins.     

Cultured basal forebrain cholinergic neurons from postnatal rats show both overlapping and non-overlapping responses to the neurotrophins

Basal forebrain cholinergic neurons respond in vitro and in vivo to nerve growth factor (NGF) and to brain-derived neurotrophic factor (BDNF). It is not clear to what extent the neurons that respond to these two factors, or to neurotrophin-3 or−45 (NT-3;NT-45) are identical or only partially overlapping populations. We have addressed this issue in cultures of basal forebrain neurons derived from 2-week-old postnatal rats, using choline acetyltransferase (ChAT) and acetylcholinesterase (AChE) as cholinergic markers. Cholinergic neuron survival was enhanced in the presence of NGF, BDNF andNT-45.NT-45 was as effective as BDNF. NT-3 was without effect at this age, although in cultures derived from embryonic forebrain, cholinergic differentiation was induced by NT-3. Cotreatment with NGF and BDNF resulted in small, but consistent, increases in the number of ChAT-positive neurons, compared with either factor alone.NT-45 was also found to be additive with NGF, whereas cotreatment with BDNF andNT-45 showed no addivity. NT-3 had no additive effects with any other neurotrophin on any cholinergic parameters in postnatal cultures. Taken together, the results indicate the existence in postnatal rat brain of a large overlapping population of cholinergic neurons that are responsive to ligands for the neurotrophin receptors TrkA (NGF) and TrkB (BDNF andNT-45), but not TrkC (NT-3), and small distinct populations that show specificity for NGF or BDNF but not both. We hypothesize that cholinergic neurons projecting into different regions of the hippocampus may derive trophic support from distinct neurotrophins.     

BDN F Down-regulates Neurotrophin Responsiveness, TrkB Protein and TrkB mRNA Levels in Cultured Rat Hippocampal Neurons

Regulation of Trk receptors by their ligands, the neurotrophins, was investigated in dissociated cultures of embryonic day 18 rat hippocampal neurons. Cultures were exposed to brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3) or NT-4/5 for 24 h upon plating followed by factor washout. As determined by immunohistochemical staining and phosphotyrosine blotting, the functional responses to acute stimulation with BDNF, NT-3 and NT-4/5, including c-Fos induction and phosphorylation of Trk and extracellular signal-regulated kinase (ERK) proteins, were significantly decreased after 6 days in culture by prior exposure to BDNF. As determined by Western and Northern blot analysis respectively, there was a parallel down-regulation of TrkB protein as well as of trkB and trkC mRNA levels in BDNF-pretreated cultures. Exposure to NT-3 or NT-4/5 at the same concentrations as BDNF did not down-regulate any of the measured cellular responses or TrkB protein and/or trkB and trkC mRNA levels. Regulation of hippocampal neuronal TrkB protein does not appear to be just a developmental phenomenon, as infusion of BDNF into the hippocampus of adult rats for 6 days produced an 80% decrease in levels of full-length TrkB protein. We thus show that exposure of hippocampal neurons to BDNF, both in culture and in the adult brain, results in down-regulation of TrkB. At least in vitro , this leads to long-term functional desensitization to BDNF. NT-3 and NT-4/5. as well as down-regulation of trkB and trkC mRNA.     

Lipopolysaccharide differentially regulates microglial trk receptor and neurotrophin expression

Activated brain microglia play a pivotal role in inflammatory and degenerative disorders, mediating immune function and producing toxic and trophic agents. We previously reported that microglia express neurotrophins and that neurotrophin-3 (NT-3) increases microglial proliferation and phagocytosis, processes associated with cellular activation. However, mechanisms regulating responsiveness to NT-3 and expression of NT-3 in activated microglia remain undefined. To investigate mechanisms governing microglial responsiveness to neurotrophins, we determined whether microglia express trk C, the high-affinity receptor for NT-3, and whether the inflammatory agent lipopolysaccharide (LPS) regulates receptor expression. Trk C mRNA was expressed by unstimulated microglia, and both trk C mRNA and protein were dramatically increased by LPS. In contrast, expression of trk A, the high-affinity receptor for nerve growth factor (NGF), was down-regulated by LPS. Consequently, the same stimulus differentially influences responsiveness of microglia to distinct trophins. In addition, LPS induced microglial NT-3 expression, suggesting that increases in both the ligand and receptor modulate NT-3 effects on microglia. Regulation was specific, since brain-derived neurotrophic factor (BDNF) and NT-4/5 expression were unaltered by LPS. In sum, our findings raise the possibility that microglial NT-3 regulates their response to inflammation through autocrine mechanisms: LPS modulates both trk C and NT-3 which, in turn, regulate microglial function. J. Neurosci. Res. 54:117–122, 1998. © 1998 Wiley-Liss, Inc.     

Neuronal signals regulate neurotrophin expression in oligodendrocytes of the basal forebrain

Previous studies suggest that oligodendrocytes express trophic molecules, including neurotrophins. These molecules have been shown to influence nearby neurons. To determine whether neuronal signals may, in turn, affect oligodendrocyte-derived trophins, we examined regulation of nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin-3 (NT-3) mRNA expression in cultured oligodendrocytes of the basal forebrain. Neuronal signals had distinct effects on individual neurotrophins. KCl elicited increases in BDNF mRNA, but did not affect expression of NGF or NT-3. The cholinergic agonist, carbachol, increased expression of NGF, but did not affect expression of BDNF or NT-3. Glutamate elicited a decrease in BDNF, but did not affect expression of NGF or NT-3. This glutamate effect is not due to toxicity, since the number of total cells was unchanged, while the number of mature myelin basic protein positive (MBP+) cells increased. Our observations suggest that individual neuronal signals distinctly influence the trophic function of oligodendrocytes.     

Brain-derived neurotrophic factor and neurotrophin-4 increase neurotrophin-3 expression in the rat hippocampus

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 neurotrophin-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.     

Exogenous BDNF, NT-3 and NT-4 differentially regulate neurite outgrowth in cultured hippocampal neurons

Multiple growth factors contribute to the differentiation of dendritic and axonal processes by a neuron. Cultured hippocampal cells elaborate dendritic and axonal processes following well-defined steps. We used this culture system to determine the specific effects of brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3) and neurotrophin-4 (NT-4) on dendritic and axonal differentiation in hippocampal pyramidal neurons. We demonstrated that each of these neurotrophins exert distinct effects on neurite outgrowth. Both BDNF and NT-3 had positive effects on the outgrowth of undifferentiated neurites, called minor neurites, and on the axonal process of hippocampal pyramidal neurons. However, the effect of NT-3 was more important than that of BDNF. On the other hand, NT-4 did not enhance axonal outgrowth but had only an effect on the outgrowth of minor neurites. Since cytoskeletal proteins play crucial roles in promoting neurite outgrowth, we examined the protein levels of some of these proteins that are associated with neurite outgrowth: beta-actin, gamma-actin, alpha-tubulin, MAP2 and tau. Surprisingly, we did not detect any change in their protein levels. Taken together, our results show that BDNF, NT-3 and NT-4 exert distinct effects on the neuritic compartments of hippocampal neurons.