Neurotrophin NT-3 Research Articles

Programmed Cell Death in Zebrafish Rohon Beard Neurons Is Influenced by TrkC1/NT-3 Signaling

Rohon Beard (RB) cells are embryonic primary sensory neurons that are removed by programmed cell death during larval development in zebrafish. RB somatosensory functions are taken over by neurons of the dorsal root ganglia (DRG), suggesting that RB cell death may be triggered by the differentiation of these ganglia, as has been proposed to be the case in Xenopus. However, here we show that the timing of RB cell death correlates with reduced expression of trkC1, the receptor for neurotrophin NT-3, but not with the appearance of DRG, which differentiate only after most RB cells die. trkC1 is expressed in subpopulations of RB neurons during development, and cell death is initiated only in trkC1-negative neurons, suggesting a role for TrkC1 and its ligand, NT-3, in RB cell survival. In support of this, antibodies that deplete NT-3 induce RB cell death while exogenous application of NT-3 reduces death. In addition, we show that RB cell death can be prevented using a caspase inhibitor, zVADfmk, showing that during normal development, RB cells die by a caspase-dependent programmed cell death pathway possibly triggered by reduced signaling via TrkC1.

Neurotrophin 3 potentiates glutamatergic responses of IHC afferents in the cochlea in vivo.

Neurotrophins have traditionally been regarded as slow-acting signals essential for neuronal survival and differentiation. Recent studies with neuronal slices, cultures and nerve ending preparations have shown that neurotrophins generate acute changes in nerve activity. Among the secondary sensory cells are the inner hair cells (IHC) and taste buds, cells which express the neurotrophic factors necessary for the survival of their innervating neurons. If in these cells neurotrophins acutely affect the nerve activity of their afferent neurons, as in the central nervous system (CNS), this may have important functional implications for the corresponding sensory transduction processes. The neurotrophin NT-3 has been reported to be expressed in IHCs. We chose an in vivo application system for the microiontophoretic supply of NT-3 in the subsynaptic region of the IHC. The effect of NT-3 on spontaneous and evoked afferent cochlear nerve activities in adult guinea pig inner ear was studied. We observed that NT-3 rapidly increases the spontaneous and glutamate-evoked firing rate of IHC afferents. Moreover, firing induced by both N-methyl-D-aspartate (NMDA) and alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) were specifically enhanced during the presence of NT-3, a process which was selectively blocked by the tyrosine kinase receptor inhibitor K252a. Because we localized NT-3 mRNA not only in IHCs but also in the spiral ganglion, we propose that similar to other sensory systems, afferent and autocrine neurotrophin activities may be responsible for survival of cochlear neurons. In addition, NT-3 in IHCs may operate as a signal-dependent, intrinsic neuromodulator and/or neuroprotector.

MEK inhibitors block BDNF-dependent and -independent expression of GABA A receptor subunit mRNAs in cultured mouse cerebellar granule neurons

Brain-derived neurotrophic factor (BDNF) can regulate the maturation of developing cerebellar granule neurons. Within 1–2 days of culture, BDNF induces the expression of granule neuron terminal differentiation markers, particularly GABA A receptor α6 subunit (GABA Aα6) mRNA. Other trophic factors including insulin-like growth factor, the neurotrophin NT-3, pituitary adenylate cyclase-activating polypeptide (PACAP), and fetal bovine serum failed to induce this early expression. The expression of other GABA A receptor subunits, including α1 and γ2, was also enhanced by exposure of developing granule neurons to BDNF. This BDNF-dependent expression of GABA A receptor subunit mRNAs could be effectively blocked by treatment with the mitogen-activated protein kinase kinase (MEK) inhibitors, PD98059 or U0126. In the absence of BDNF, GABA Aα6 expression occurs but not until 3–4 days of culture. This BDNF-independent expression of GABA Aα6 was also inhibited by PD98059. Further studies showed that the BDNF-dependent expression GABA Aα6 could also be reduced by LY294002, an inhibitor of the phosphatidylinositol 3-kinase, or depolarizing concentrations of KCl. These results thus suggest that both BDNF-dependent and -independent expressions of GABA A receptor subunits require the activation of MEK and the mitogen-activated protein kinase (MAPK) pathway. However, it is also likely that other signaling pathways modulate this maturation process.

Activity-Dependent Expression of NT-3 in Muscle Cells in Culture: Implications in the Development of Neuromuscular Junctions

Although activity-dependent expression of neurotrophins has been studied extensively in the CNS, its physiological role during synapse development is not well established. At the developing neuromuscular junction in culture, exogenous application of the neurotrophin BDNF or NT-3 has been shown to acutely potentiate synaptic transmission and chronically promote synapse maturation. Using the same cell culture model, we have investigated activity-dependent neurotrophin expression in muscle cells and its role in developing neuromuscular synapses. Membrane depolarization, elicited by either depolarizing agents or repetitive electric stimulation, rapidly and specifically increased the levels of NT-3 mRNA in developing Xenopus laevis muscle cells in culture. NT-3 gene expression also was enhanced by acetylcholine (ACh), the neurotransmitter that causes muscle membrane depolarization. The effects of depolarization were mediated by increasing intracellular calcium concentration. Moreover, factor(s) induced by membrane depolarization appeared to enhance synaptic transmission at the developing neuromuscular junction. The frequency of spontaneous synaptic currents (SSCs) recorded from neuromuscular synapses was increased significantly after treatment with conditioned medium from depolarized muscle cultures. The amplitude, rise time, and decay time of SSCs were not affected, indicating a presynaptic action of the conditioned medium. The effects of the conditioned medium were blocked, partially, by the NT-3 scavenger TrkC-IgG, suggesting that the potentiation of synaptic efficacy was attributable, at least in part, to elevated NT-3 as a consequence of muscle depolarization. Thus, activity-dependent expression of muscle NT-3 may contribute to the development of the neuromuscular synapse.

Neurotrophin-3 promotes the survival of oligodendrocyte precursors in embryonic hippocampal cultures under chemically defined conditions

Embryonic rat hippocampal cells were cultured in basal medium with or without addition of the neurotrophin NT-3. After culturing in these extreme conditions, the effects of NT-3 on the neuronal and on the glial components were assessed. Neurons survived even in the absence of NT-3 but failed to reach terminal differentiation. On the other hand, NT-3 promoted the survival but not the proliferation and/or the differentiation of oligodendrocytes precursors present in the same culture, an effect that was reversed by the addition of neutralizing antibodies against NT-3. Type I or II astrocytes were not affected by NT-3. These results reinforce the role for NT-3 in oligodendrocyte lineage development and allow to dissect the roles of this neurotrophin in survival and in proliferation/differentiation of oligodendrocytes.

Functions of basic fibroblast growth factor and neurotrophins in the differentiation of hippocampal neurons

Restrictions in neuronal fate occur during the transition from a multipotential to a postmitotic cell. This and later steps in neuronal differentiation are determined by extracellular signals. We report that basic fibroblast growth factor is mitogenic for stem cells and is a differentiation factor for calbindin-expressing hippocampal neurons. The neurotrophin NT-3 is a differentiation factor for the same neurons but does not affect proliferation. NT-3 and brain-derived neurotrophic factor promote the maturation of neurons derived from stem cells that have been grown in vitro. These results define functions for basic fibroblast growth factor and neurotrophins in the differentiation processes that direct a multipotential stem cell to a specific neuronal fate.