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 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.     

Brain-derived neurotrophic factor, neurotrophin-3, and neurotrophin-4 complement and cooperate with each other sequentially during visceral neuron development

The neurotrophins nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT3), and neurotrophin-4 (NT4) are crucial target-derived factors controlling the survival of peripheral sensory neurons during the embryonic period of programmed cell death. Recently, NT3 has also been found to act in a local manner on somatic sensory precursor cells during early development in vivo. Culture studies suggest that these cells switch dependency to NGF at later stages. The neurotrophins acting on the developing placode-derived visceral nodose/petrosal (N/P) ganglion neurons are BDNF, NT3, and NT4. To assess their roles in development, we analyzed embryonic development in mice carrying a deletion in each of these genes, or combinations of them, and found that they are essential in preventing the death of N/P ganglion neurons during different periods of embryogenesis. Both NT3 and NT4 are crucial during the period of ganglion formation, whereas BDNF acts later in development. Many, but not all, of the NT3- and NT4-dependent neurons switch to BDNF at later stages. We conclude that most of the N/P ganglion neurons depend on more than one neurotrophin and that they act in a complementary as well as a collaborative manner in a developmental sequence for the establishment of a full complement of visceral neurons.     

EYA4, a novel vertebrate gene related to Drosophila eyes absent

NMDA receptors are composed of proteins from two families: NMDAR1 and NMDAR2. We used quantitative double-label in situ hybridization to examine in rat brain the expression of NMDAR1, NMDAR2A, NMDAR2B, and NMDAR2C mRNA in six neurochemically defined populations of striatal neurons: preproenkephalin (ENK) and preprotachykinin (SP) expressing projection neurons, and somatostatin (SOM), glutamic acid decarboxylase 67 (GAD67), parvalbumin (PARV), and choline acetyltransferase (ChAT) expressing interneurons. NMDAR1 was expressed by all striatal neurons: strongly in ENK, SP, PARV and ChAT neurons, and less intensely in SOM and GAD67 positive cells. NMDAR2A mRNA was present at moderate levels in all striatal neurons except those containing ChAT. Labeling for NMDAR2B was strong in projection neurons and ChAT interneurons, and only moderate in SOM, GAD67 and PARV interneurons. NMDAR2C was scarce in striatal neurons, but a low level signal was detected in GAD67 positive cells. NMDAR2C expression was also observed in small cells not labeled by any of the markers, most likely glia. These data suggest that all striatal neurons have NMDA receptors, but different populations have different subunit compositions which may affect function as well as selective vulnerability.     

Neurotrophin effects on survival and expression of cholinergic properties in cultured rat septal neurons under normal and stress conditions

These studies tested the hypothesis that survival-promoting effects of neurotrophins on basal forebrain cholinergic neurons are enhanced under stress. Septal neurons from embryonic day 14-15 rats exposed for 10-14 d to neurotrophin [nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), or neurotrophin-4 (NT-4), each at 100 ng/ml] showed a two- to threefold increase in choline acetyltransferase (ChAT) activity, with little evidence of synergistic interactions. Neurotrophins produced no significant increase in the survival of total or acetylcholinesterase (AChE)-positive neurons at moderate plating density (1200-1600 cells/mm2). However, with very low plating densities (2-28 cells/mm2) BDNF, NT-3, and NT-4 (but not NGF) increased total neuronal survival, and BDNF increased survival of AChE-positive neurons. NGF and BDNF enhanced ChAT activity and survival of cholinergic neurons after a 24 hr hypoglycemic stress, even when added 1 hr after stress onset. All four tested neurotrophins increased total neuronal survival after hypoglycemic stress. These results suggest that neurotrophins are important for preservation of central cholinergic function under stress conditions, with different neurotrophins protecting against different stresses. The stress-associated survival-promoting effects of neurotrophins were not limited to the cholinergic subpopulation.     

Dexamethasone inhibits ischemia-induced transient reduction of neurotrophin-3 mRNA in rat hippocampal neurons

Dexamethasone (DEX) increases the expression of neurotrophin-3 (NT-3) in normal rat hippocampal neurons, whereas transient forebrain ischemia reduces the NT-3 mRNA level. The effect of DEX on the expression of NT-3 mRNA in injured brain cells after ischemia has not been investigated, however. Using in situ hybridization and ribonuclease protection assay methods, we studied NT-3 mRNA expression in rats with and without DEX administration after transient forebrain ischemia. Without DEX treatment, NT-3 mRNA was down-regulated in the hippocampal neurons at 2, 4, 12 h and returned to basal levels 24 h following ischemia. With DEX treatment, however, NT-3 mRNA showed no change at 2, 4 and 12 h and increased 24 h after ischemia. The results indicate that DEX inhibits ischemia-induced NT-3 mRNA down-regulation during the first 12 h and up-regulates NT-3 mRNA 24 h after ischemia. DEX administration might be effective in influencing some of the pathophysiological effects of ischemia in the hippocampus.     

A competitive enzyme-linked immunosorbent assay for measuring the levels of serum antibody to Haemophilus influenzae type b

1. The protein family of the neurotrophins, consisting of nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF) and Neurotrophin-3, -4/5, and -6 (NT-3; NT-4/5; NT-6) is well known to enhance the survival and to stabilize the phenotype of different populations of neurons in the central and the peripheral nervous system. These effects are mediated via binding to specific tyrosine kinase receptors (Trks) and to the low-affinity p75 neurotrophin receptor. 2. The neurotrophins NGF, BDNF, and NT-3 and the BDNF and NT-3 selective receptors (TrkB, TrkC) are expressed at high levels in neurons of the basal forebrain, the hippocampus, and the neocortex of the mammalian brain. The expression and secretion of NGF and BDNF in these brain areas is regulated by (physiological levels of) neuronal activity. 3. Exogenous application of the neurotrophins to hippocampal and neocortical neurons can enhance excitatory glutamatergic synaptic transmission via activation of Trk receptors. In addition, long-term potentiation (a potential cellular correlate for learning and memory formation in mammals) in the rodent hippocampus depends on endogenous supply of neurons with BDNF. 4. Judged by the analysis of electrophysiological data, the BDNF- and NT-3-induced enhancement of glutamatergic synapses is mediated by increasing the efficacy of glutamate release from the presynaptic neuron. However, neurotrophin-dependent postsynaptic enhancement of NMDA (but not AMPA) receptor responsiveness has also been shown. 5. On the molecular level, neither the pre- nor the postsynaptic modulation of glutamatergic synapses by neurotrophins is well understood. However, neurotrophins were shown to acutely affect intraneuronal Ca2+ levels and to influence molecular components of the transmitter release machinery, which could underly the presynaptic modifications, whereas BDNF-induced phosphorylation of NMDA-type glutamate receptors could account for the postsynaptic effects. 6. Taken together, these results suggest that the activity-dependent release of neurotrophins at frequently used synapses could modulate the synaptic efficacy at these junctions. Thus, neurotrophins might operate as locally released feedback modulators of synaptic transmission, and this could be a cellular correlate for certain aspects of information processing in the mammalian brain.     

Production and characterization of recombinant rat brain-derived neurotrophic factor and neurotrophin-3 from insect cells

Rat brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) were engineered for expression in a baculovirus-infected Spodoptera frugiperda insect cell system. The BDNF and NT-3 from the culture supernatants were purified by ion-exchange and reverse-phase chromatography to apparent homogeneity. The purification procedure yielded approximately 2 mg of pure rat BDNF or NT-3 per liter of culture supernatant. A single N-terminus only was found for either secreted molecule and was analogous to that predicted from the corresponding cDNA sequence. The recombinant neurotrophins obtained were also homogeneous with regard to molecular weight and amino acid sequence. In their native conformation, the insect cell-produced rat BDNF and NT-3 molecules were homodimers consisting of 119 amino acid polypeptide chains. Thus, although the genes transfected into the S. frugiperda cells coded for proBDNF or proNT-3, the BDNF and NT-3 recovered after purification were > 95% fully processed, mature protein. Mature recombinant rat BDNF and NT-3 were found not to be significantly glycosylated. Pure, recombinant rat BDNF and NT-3 promoted the survival of embryonic dorsal root ganglion neurons in the low picomolar range. Because recombinant rat BDNF and NT-3 can be obtained in large quantities, purified to near homogeneity, and are identical in amino acid sequence to the corresponding human proteins, they are suitable for evaluation in animal models.     

Brain microglia/macrophages express neurotrophins that selectively regulate microglial proliferation and function

Although microglia-mediated cytotoxicity has been extensively investigated, little is known about the potential microglial role in neuronal and glial support. Characterization of trophin elaboration by microglia and identification of responsive populations may define novel functions. We now report that microglia/brain macrophages express neurotrophins of the nerve growth factor (NGF) gene family in vitro and in vivo, suggesting that these cells promote development and normal function of neurons and glia. Moreover, neurotrophins promote microglial proliferation and phagocytic activity in vitro. We found that microglia express neurotrophins in a region-specific manner and that within any region only subpopulations elaborate trophins. Using an antiserum specific for neurotrophin-3 (NT-3) with the microglial/macrophage marker OX-42 on postnatal day 10 in vivo, double-labeled cells were identified in the cerebral cortex, globus pallidus, and medulla; NT-3 was undetectable in OX-42-positive cells in the ependyma, the external capsule, choroid plexus, and meninges. In contrast, ramified microglia in the adult brain did not exhibit NT-3 immunoreactivity, suggesting developmental regulation of microglial NT-3 expression. In situ hybridization studies on purified microglial cultures confirmed that only subpopulations express the NGF and NT-3 genes, substantiating the existence of microglial heterogeneity. We tentatively conclude that microglial subtypes serve trophic roles in the normal brain, in addition to exerting well documented deleterious actions in illness and injury. Microglia were also responsive to neurotrophins: brain-derived neurotrophic factor (BDNF) and NT-3 increased [3H]thymidine incorporation in vitro, and NT-3 promoted proliferation. Moreover, NT-3 induced phagocytic activity, suggesting that the factor plays a role in processes associated with cellular activation.     

Expression of trkB and trkC mRNAs by adult midbrain dopamine neurons: a double-label in situ hybridization study

The documented trophic actions of the neurotrophins brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and neurotrophin-4/5 (NT-4/5) upon ventral mesencephalic dopamine neurons in vitro and in vivo are presumed to be mediated through interactions with their high-affinity receptors TrkB (for BDNF and NT-4/5) and TrkC (for NT-3). Although both neurotrophin receptor mRNAs have been detected within the rat ventral midbrain, their specific association with mesencephalic dopaminergic cell bodies remains to be elucidated. The present study was performed to determine the precise organization of trkB and trkC mRNAs within rat ventral midbrain and to discern whether the neurotrophin receptor mRNAs are expressed specifically by dopaminergic neurons. In situ hybridization with isotopically labeled cRNA probes showed that trkB and trkC mRNAs were expressed in all mesencephalic dopamine cell groups, including all subdivisions of the substantia nigra and ventral tegmental area, and in the retrorubral field, rostral and caudal linear raphe nuclei, interfascicular nucleus, and supramammillary region. Combined isotopic/nonisotopic double-labeling in situ hybridization demonstrated that virtually all of the tyrosine hydroxylase (the catecholamine biosynthetic enzyme) mRNA-containing neurons in the ventral midbrain also expressed trkB or trkC mRNAs. Additional perikarya within these regions expressed the neurotrophin receptor mRNAs but were not dopaminergic. The present results demonstrate that essentially all mesencephalic dopaminergic neurons synthesize the neurotrophin receptors TrkB and TrkC and thus exhibit the capacity to respond directly to BDNF and NT-3 in the adult midbrain in vivo. Moreover, because BDNF and NT-3 are produced locally by subpopulations of the dopaminergic cells, the present data support the notion that the neurotrophins can influence the dopaminergic neurons through autocrine or paracrine mechanisms.     

Crystal structure of neurotrophin-3 homodimer shows distinct regions are used to bind its receptors

Neurotrophin-3 (NT-3) is a cystine knot growth factor that promotes the survival, proliferation, and differentiation of developing neurons and is a potential therapeutic for neurodegenerative diseases. To clarify the structural basis of receptor specificity and the role of neurotrophin dimerization in receptor activation, the structure of the NT-3 homodimer was determined using X-ray crystallography. The orthorhombic crystals diffract to 2.4 A, with dimer symmetry occurring about a crystallographic 2-fold axis. The overall structure of NT-3 resembles that of the other neurotrophins, NGF and BDNF; each protomer forms a twisted four-stranded beta sheet, with three intertwined disulfide bonds. There are notable differences, however, between NT-3 and NGF in the surface loops and in three functionally important regions, shown in previous mutagenesis studies to be critical for binding. One such difference implies that NT-3's binding affinity and specificity depend on a novel hydrogen bond between Gln 83, a residue important for binding specificity with TrkC, and Arg 103, a residue crucial for binding affinity with TrkC. NT-3's extensive dimer interface buries much of the otherwise solvent-accessible hydrophobic surface area and suggests that the dimeric state is stabilized through the formation of this hydrophobic core. A comparison of the dimer interface between the NT-3 homodimer and the BDNF/NT-3 heterodimer reveals similar patterns of hydrogen bonds and nonpolar contacts, which reinforces the notion that the evolutionarily conserved neurotrophin interface resulted from the need for receptor dimerization in signal initiation.     

Glucocorticoids differentially increase nerve growth factor and basic fibroblast growth factor expression in the rat brain

Adrenocorticotropin hormone (ACTH) and adrenal steroids may influence trophic processes operative in neuronal plasticity. Because nerve growth factor (NGF) and basic fibroblast growth factor (bFGF) participate in neuronal trophism, we have investigated whether adrenal steroids induce the expression of these two trophic factors in the rat brain. The systemic administration of dexamethasone (DEX) elicited a rapid (within 3 hr) and sustained accumulation of bFGF and NGF mRNA in the cerebral cortex and hippocampus. Regional studies showed that DEX increases bFGF but not NGF mRNA in the cerebellum, striatum, and hypothalamus. In situ hybridization studies revealed that DEX increases NGF mRNA in superficial layers of the cerebral cortex and in the dentate gyrus of the hippocampus, and bFGF mRNA throughout the brain, suggesting that DEX induces NGF mRNA in neurons and bFGF in glial cells. ACTH administered systemically elicited a temporal and regional induction in NGF and bFGF mRNA similar to that obtained with DEX. Increases in NGF and bFGF mRNAs were also observed after administration of corticosterone and, albeit to a lesser extent, aldosterone, suggesting that the pituitary-adrenocortical axis plays an important role in the regulation of NGF and bFGF expression in the brain. Our data suggest that NGF and bFGF represent a link by which the adrenal cortical system can exert trophic action on the CNS.     

A new role for neurotrophins: involvement of brain-derived neurotrophic factor and neurotrophin-4 in hair cycle control

Neurotrophins exert many biological effects not directly targeted at neurons, including modulation of keratinocyte proliferation and apoptosis in vitro. Here we exploit the cyclic growth and regression activity of the murine hair follicle to explore potential nonneuronal functions of neurotrophins in the skin, and analyze the follicular expression and hair growth-modulatory function of BDNF, NT-4, and their high-affinity receptor, TrkB. The cutaneous expression of BDNF and NT-4 mRNA was strikingly hair cycle dependent and peaked during the spontaneous, apoptosis-driven hair follicle regression (catagen). During catagen, BDNF mRNA and immunoreactivity, as well as NT-4-immunoreactivity, were expressed in the regressing hair follicle compartments, whereas TrkB mRNA and immunoreactivity were seen in dermal papilla fibroblasts, epithelial strand, and hair germ. BDNF or NT-4 knockout mice showed significant catagen retardation, whereas BDNF-overexpressing mice displayed acceleration of catagen and significant shortening of hair length. Finally, BDNF and NT-4 accelerated catagen development in murine skin organ culture. Together, our data suggest that BDNF and NT-4 play a previously unrecognized role in skin physiology as agents of hair growth control. Thus, TrkB agonists and antagonists deserve exploration as novel hair growth-modulatory drugs for the management of common hair growth disorders.     

Anandamide and other N-acylethanolamines in mouse peritoneal macrophages

Exposure to stressful events and elevated level of stress hormones are associated with impaired spatial memory and neuronal damage in the hippocampus. These neurons are considered to be maintained by neurotrophins such as nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin-3 (NT-3) and trk family of neurotrophin receptors. Male Wistar rats (6 weeks old) were exposed to immobilization stress for 8 h and their brains were processed for in situ hybridization histochemistry. Exposure to long-lasting immobilization stress reduced mRNA levels for neurotrophins and their high affinity receptors in the brain, especially in the hippocampus. Our results provide, some new information that may be relevant to the pathogenesis of stress-induced disturbances of memory and learning.     

Neurotrophins and their receptors in the tench retina during optic nerve regeneration

To understand the role of neurotrophins in the visual system, we investigated the distribution of both neurotrophins and their receptors within the retina of a fish that has the capacity to spontaneously regenerate its optic nerve axons after lesion. Intact retinas and retinas from tench, whose optic nerve had been crushed, were analyzed by immunohistochemistry and in situ hybridization. Trk receptors were mainly immunolocalized in cells of the inner nuclear and ganglion cell layers, a distribution coincident with that of their mRNAs. Nerve growth factor (NGF) immunoreactivity was detected exclusively in Müller cell processes, and brain-derived neurotrophic factor (BDNF) was found in both neuronal bodies and Müller cell processes. Neurotrophin-3 (NT-3) was detected in most of the cell nuclei, and neurotrophin-4/5 (NT-4/5) was localized in fibers and in a few cells in the inner retina. An increase in both TrkA protein and mRNA was detected during axonal regeneration within the retinal ganglion cell layer, reaching a maximum 30 days postcrush and returning to normal levels by day 90, when optic nerve regeneration is almost completed in this fish. None of the other neurotrophins and receptors showed appreciable changes. The heterogeneous distribution patterns of neurotrophins and their receptors in fish retina, their differences from the distribution observed in other species, and the TrkA changes after optic nerve crush suggest an important role for these molecules in the normal physiology of the fish retina and during the regeneration process.     

Brain-derived neurotrophic factor and neurotrophin-3 enhance somatostatin gene expression through a likely direct effect on hypothalamic somatostatin neurons

Although neurotrophins (NTs) have been extensively studied as neuronal survival factors in some areas of the central nervous system, little is known about their function or cellular targets in the hypothalamus. To understand their functional significance and sites of action on hypothalamic neurons, we examined the effects of their cognate ligands on neuropeptide content and messenger RNA (mRNA) expression in somatostatin neurons present in fetal rat hypothalamic cultures. Treatments were performed in defined insulin-free medium between days 6 and 8 of culture, since the maximal effects of NTs on somatostatin content and mRNA expression were observed after 48-h incubations. Brain-derived neurotrophic factor and NT-3, but not nerve growth factor, induced a dose-dependent increase in somatostatin content, which was influenced by plating density. The same treatment increased somatostatin mRNA and immunostaining intensity of somatostatin neurons, but had no effect on the number of these labeled neurons. The increased levels of somatostatin (peptide and mRNA) induced by NTs were not blocked by tetrodotoxin or by glutamate receptor antagonists, suggesting that endogenous neurotransmitters (e.g. glutamate) were not involved in these effects. In contrast, the stimulatory effects were completely blocked by K-252a, an inhibitor of tyrosine kinase (Trk) receptors, whereas the less active analog K-252b was ineffective. Double-labeling studies demonstrated that both TrkB or TrkC receptors were located on somatostatin neurons. Our results show that, in rat hypothalamic cultures, brain-derived neurotrophic factor, and NT-3 have a potent stimulatory effect on peptide synthesis in somatostatinergic neurons, likely through direct activation of TrkB and TrkC receptors.     

NT-4/5 and LIF, but not NT-3 and BDNF, promote NPY mRNA expression in cortical neurons in the absence of spontaneous bioelectrical activity

Epigenetic factors are known to influence the differentiation of neocortical neurons. The present study analyses the role of spontaneous bioelectrical activity (SBA) and neurotrophic factors on the expression of neuropeptide Y (NPY) in rat visual cortical neurons using organotypic monocultures prepared from newborn animals and in situ hybridization to detect the NPY messenger ribonucleic acid (mRNA). Spontaneously active cortex cultures display NPY mRNA expression in about 7% of all cortical neurons from 10 days in vitro (DIV) on. Blocking the SBA by chronic application of 10 mM Mg2+ for 3-30 DIV reduces the percentage of NPY neurons to about 2%. Allowing an initial phase of SBA (1-20 DIV) followed by an SBA blockade (for 21-50 DIV) results in 2% labelled neurons, indicating a dramatic reduction of NPY mRNA expression in the absence of SBA. Surprisingly, the reverse experiment (a period of SBA blockade for 1-20 DIV followed by a period of SBA recovery for 21-40 DIV) does not cause an upregulation of NPY mRNA expression. However, allowing cultures to differentiate as spontaneously active cultures, then applying a transient period of SBA blockade which is followed by a second period of SBA, does rescue the NPY mRNA expression in 7% of the cortical neurons. We conclude that SBA is a main trigger for NPY mRNA expression and it is particularly important during an early postnatal period of differentiation. We then analysed whether neurotrophic factors known to modulate cortical neuropeptide expression are able to do so in the absence of SBA. Supplementing chronically blocked cultures with the neurotrophins, brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), neurotrophin-4/5 (NT-4/5) and the cytokine, leukaemia inhibitory factor (LIF), reveals that BDNF and NT-3 are unable to increase the percentage of NPY neurons. In contrast, LIF and NT-4/5 increase the percentage of NPY neurons to 4 and 6-7%, respectively. Moreover, neurons treated with NT-4/5 display a very high level of NPY mRNA expression in somata and in the dendritic trees. The data suggest a complex interplay and a hierarchy of epigenetic factors in regulating the neurochemical architecture of the developing neocortex.     

Neurotrophins rescue cerebellar granule neurons from oxidative stress-mediated apoptotic death: selective involvement of phosphatidylinositol 3-kinase and the mitogen-activated protein kinase pathway

Cerebellar granule neurons maintained in medium containing serum and 25 mM K+ reliably undergo an apoptotic death when switched to serum-free medium with 5 mM K+. New mRNA and protein synthesis and formation of reactive oxygen intermediates are required steps in K+ deprivation-induced apoptosis of these neurons. Here we show that neurotrophins, members of the nerve growth factor gene family, protect from K+/serum deprivation-induced apoptotic death of cerebellar granule neurons in a temporally distinct manner. Switching granule neurons, on day in vitro (DIV) 4, 10, 20, 30, or 40, from high-K+ to low-K+/serum-free medium decreased viability by >50% when measured after 30 h. Treatment of low-K+ granule neurons at DIV 4 with nerve growth factor, brain-derived neurotrophic factor (BDNF), neurotrophin-3, or neurotrophin-4/5 (NT-4/5) demonstrated concentration-dependent (1-100 ng/ml) protective effects only for BDNF and NT-4/5. Between DIV 10 and 20, K+-deprived granule neurons showed decreasing sensitivity to BDNF and no response to NT-4/5. Cerebellar granule neuron death induced by K+ withdrawal at DIV 30 and 40 was blocked only by neurotrophin-3. BDNF and NT-4/5 also circumvented glutamate-induced oxidative death in DIV 1-2 granule neurons. Granule neuron death caused by K+ withdrawal or glutamate-triggered oxidative stress was, moreover, limited by free radical scavengers like melatonin. Neurotrophin-protective effects, but not those of antioxidants, were blocked by selective inhibitors of phosphatidylinositol 3-kinase or the mitogen-activated protein kinase pathway, depending on the nature of the oxidant stress. These observations indicate that the survival-promoting effects of neurotrophins for central neurons, whose cellular antioxidant defenses are challenged, require activation of distinct signal transduction pathways.     

Opposing roles for endogenous BDNF and NT-3 in regulating cortical dendritic growth

Neurons within each layer of cerebral cortex express multiple members of the neurotrophin family and their corresponding receptors. This multiplicity could provide functional redundancy; alternatively, different neurotrophins may direct distinct aspects of cortical neuronal growth and differentiation. By neutralizing endogenous neurotrophins in organotypic slices of developing cortex with Trk receptor bodies (Trk-IgGs), we found that BDNF and NT-3 oppose one another in regulating the dendritic growth of pyramidal neurons. In layer 4, both endogenous and exogenous NT-3 inhibited the dendritic growth stimulated by BDNF. In contrast, in layer 6 both endogenous and exogenous BDNF inhibited dendritic growth stimulated by NT-3. These antagonistic actions of endogenous BDNF and NT-3 provide a mechanism by which dendritic growth and retraction can be dynamically regulated during cortical development, and suggest that the multiple neurotrophins expressed in developing cortex represent distinct components of an extracellular signaling system for regulating dendritic growth.