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The regulation of neurotrophin (NT) secretion is critical for many aspects of NT-mediated neuronal plasticity. Neurons release NTs by activity-regulated secretion pathways, initiated either by neurotransmitters and/or by existing NTs by a positive-feedback mechanism. This process depends on calcium release from intracellular stores. Little is known, however, about potential pathways that down-regulate NT secretion. Here we demonstrate that nitric oxide (NO) induces a rapid down-regulation of brain-derived neurotrophic factor (BDNF) secretion in cultured hippocampal neurons. Similar effects occur by activating a downstream target of intracellular NO, the soluble guanylyl cyclase, or by increasing the levels of its product, cGMP. Furthermore, down-regulation of BDNF secretion is mediated by cGMP-activated protein kinase G, which prevents calcium release from inositol 1,4,5-trisphosphate-sensitive stores. Our data indicate that the NO/cGMP/protein kinase G pathway represents a signaling mechanism by which neurons can rapidly down-regulate BDNF secretion and suggest that, in hippocampal neurons, NT secretion is finely tuned by both stimulatory and inhibitory signals.

Axonal retrograde transport is essential for neuronal growth and survival. However, the nature and dynamics of the membrane compartments involved in this process are poorly characterized. To shed light on this pathway, we established an experimental system for the visualization and the quantitative study of retrograde transport in living motor neurons based on a fluorescent fragment of tetanus toxin (TENT [H.sub.c]). Morphological and kinetic analysis of TeNT [H.sub.c] retrograde carriers reveals two major groups of organelles: round vesicles and fast tubular structures. TeNT [H.sub.c] carriers lack markers of the classical endocytic pathway and are not acidified during axonal transport. Importantly, TeNT [H.sub.c] and NGF share the same retrograde transport organelles, which are characterized by the presence of the neurotrophin receptor [p7.sup.5NTR]. Our results provide the first direct visualization of retrograde transport in living motor neurons, and reveal a novel retrograde route that could be used both by physiological ligands (i.e., neurotrophins) and TeNT to enter the central nervous system.

Neurotrophins have been shown to be involved in functional strengthening of central nervous system synapses. Although their general importance in this process is undisputed, it remains unresolved whether neurotrophins are truly mediators of synaptic strengthening or merely important cofactors. To address this question, we have devised a method to inactivate endogenous brain-derived neurotrophic factor (BDNF) with high time resolution by 'caging' a function-blocking mAb against BDNF with a photosensitive protecting compound. Different assays were used to show that this inactivation of the Ab is reversible by UV light. Synaptic potentiation after [tau]-burst stimulation in the CA1 region of acute hippocampal slices was significantly less when applying the unmodified Ab compared with the caged Ab. Importantly, photoactivation of the caged Ab during the time of induction of synaptic enhancement led to a marked decrease in potentiation. Our experiments therefore strengthen the view that endogenous BDNF has fast effects during induction of synaptic plasticity. The results additionally show that caged Abs can provide a tool for precise spatiotemporal control over endogenous protein levels.

This review presents recent research on critical period or unique time and its influence on visual systems of vertebrates in vertebrate brain development. Molecular mechanisms regulating critical periods are discussed in relation to visual systems of cat and mouse and their responses to monocular deprivation.

The ever-unfolding biology of NGF is consistent with a target-derived retrograde mode of action in peripheral and central neurons. However, another member of the neurotrophin family, brain-derived neurotrophic factor (BDNF), is present within nerve terminals in certain regions of the brain and PNS that do not contain the corresponding mRNA. Recent studies have shown that the endogenous neurotrophins, BDNF and neurotrophin-3 (NT-3), are transported anterogradely by central and peripheral neurons. The supply of BDNF by afferents is consistent with their presynaptic synthesis, vesicular storage, release and postsynaptic actions. Anterograde axonal transport provides an 'afferent supply' of BDNF and NT-3 to neurons and target tissues,where they function as trophic factors and as neurotransmitters.

Neurotrophic factors control the survival, differentiation and maintenance of neurons in the peripheral and central nervous systems. Their discovery and characterization have been instrumental to our understanding of a wide range of phenomena in the development, plasticity and repair of the nervous system. Their potential importance in the development of therapeutic agents against neurodegenerative disorders and nerve injury has led to a flurry of activity towards understanding their structure, function and signaling mechanisms. This knowledge has increased dramatically in recent years, in particular due to the elucidation of three-dimensional structures, the discovery of families of structurally related neurotrophic factors and the characterization of receptors and downstream signaling components. Common themes are emerging from these recent studies that allow us to make new insights and predictions as to the function and possible clinical utility of these molecules.

Recent studies have established that neurotrophin synthesis and secretion are regulated by activity and that these factors are involved in activity-dependent processes in the nervous system. Neurotrophins also are known to induce increases in intracellular calcium, a trigger for regulated secretion. This finding raises the possibility that neurotrophins themselves may stimulate regulated secretion of neurotrophins. To address this question, we studied the release of neurotrophins from transfected PC12 cells, a widely used model for neuronal secretion and neurotrophin signal transduction. We found that neurotrophins induced the regulated secretion of brain-derived neurotrophic factor, neurotrophin-3 (NT-3), and neurotrophin-4/5. The effect of brain-derived neurotrophic factor on release of NT-3 could be abolished by REX, a p75 blocking antibody, but not by K252a, an inhibitor of neurotrophin tyrosine kinase receptor (Trk) signaling. The nerve growth factor effect on release of NT-3 could be blocked only by simultaneous application of REX and K252a, suggesting that they are mediated by TrkA as well as p75. Our data show that neurotrophins are able to induce the regulated secretion of neurotrophins and suggest a signal-transducing role for both TrkA and p75 in this process. The neurotrophin-induced release of neurotrophins may be relevant for activity-dependent processes such as synaptic plasticity and memory formation.

The role of the p75 neurotrophin receptor in the developmental apoptosis of sympathetic neurons was investigated. Specifically, the experiment focused on sympathetic neurons in postnatal rats that express both the p75 neurotrophin and TrkA tyrosine kinase receptors. Results showed that activation of the p75 neurotrophin receptor led to cell death and that the p75 receptor inhibited developmental apoptosis. The TrkA receptor, on the other hand, mediated the survival of sympathetic neurons in rats.

The translocation of specific mRNAs to dendrites and their potential for locally regulated translation are likely to serve as an effector in neuronal plasticity. Whether translation in dendrites is regulated by delivery of the RNA to sites of plasticity or a stationary pool of localized RNA undergoes enhanced translational efficiency is not clear. We show that RNA can translocate into dendrites in response to NT-3. RNA granules were visualized in cultured rat cortical neurons using the dye SYTO 14, which labels poly-ribosome complexes. Long before the morphological effects of NT-3 appeared, there was increased distal translocation of labeled complexes. This effect was blocked by K252a, a potent inhibitor of tyrosine kinase receptors. Therefore, neurons can utilize extracellular signals to alter the distribution of protein synthetic machinery via the active transport of RNA granules.

We have generated null mutant mice that lack expression of all isoforms encoded by the trkC locus. These mice display a behavioral phenotype characterized by a loss of proprioceptive neurons. Neuronal counts of sensory ganglia in the trkC mutant mice reveal less severe losses than those in NT-3 null mutant mice, strongly suggesting that NT-3, in vivo, may signal through receptors other than trkC. Mice lacking either NT-3 or all trkC receptor isoforms die in the early postnatal period. Histological examination of trkC-deficient mice reveals severe cardiac defects such as atrial and ventricular septal defects, and valvular defects including pulmonic stenosis. Formation of these structures during development is dependent on cardiac neural crest function. The similarities in cardiac defects observed in the trkC and NT-3 null mutant mice indicate that the trkC receptor mediates most NT-3 effects on the cardiac neural crest.

Trk receptor activation by neurotrophins is often considered to have a defined set of actions on target neurons, including supporting neuronal survival, inducing morphological differentiation, and regulating a host of target genes that specify neuronal phenotype. It is not known if all such regulatory effects are obligatory, or if some may vary depending on the cell context in which the receptors are expressed. We have examined this issue by comparing neurotrophin effects on the regulation of electrical excitability and morphological differentiation in two strains of PC12 cells. We found that while neurotrophins induced neurite extension and increased calcium currents in both PC12 cell types, sodium current levels were regulated in only one of these strains. Moreover, we found little correlation between calcium current levels and the extent of morphological differentiation when compared in individual cells of a single strain. Thus, the regulatory effects of neurotrophins on cell phenotype are not fully determined by the Trk receptors that they activate; rather, they can vary with differences in cell context that arise not only between different cell lineages, but also between individual cells of clonal relation.

Neurotrophins regulate the survival and growth of neurons in the cerebellum of hypothyroid rats. Thyroid hormone deficiency increases the mRNA levels of nerve growth factor, neurotrophin (NT)-4 and NT-5, and decreases that of brain-derived neurotrophic factor (BDNF) and NT-3. The administration of NT-3 or BDNF prevents neuronal death and reverses the changes induced by hypothyroidism in the cerebellum. Granule and Purkinje neurons require appropriate levels of NT-3 for normal development in vivo.

The brain-derived neurotrophic factor (BDNF) is essential for the extension and remodelling of the axonal branches and spikes in the central nervous system. Optic axon branching in Xenopus laevis tadpoles in vivo decreases at lower levels of BDNF in the optic tectum. Specific neutralizing antibodies of BDNF deplete the axon elaboration. The in vivo retinal ganglion cell response is related to BDNF. BDNF plays an important role in the interaction between the axon terminals and their targets.

The intraventricular application of a synthetic peptide, which behaves like the nerve growth factor, suppresses kindling epileptic seizures and formation of mossy fibers by decreasing the binding of neurotropins with their receptors. Kindling seizures, caused by electrical stimuli, cause an increase in the concentration of neurotropins and mossy-fiber formation in the hippocampus. Although neurotropins play an important role in the central nervous system, in some conditions they are not required and need to be inhibited.

The transfer of nerve growth factor (NGF) dependence requires extrinsic signals that act on embryonic mouse ganglion. Researchers cultured trigeminal neurons with brain-derived neuronal factor (BDNF), neurotropin-3 (NT3), neurotropin 4/5 (NT4/5) before the NGF dependence period. The neurons survived well after the NGF dependence period would have begun. Portions of the neurons were moved to NGF cultures at various times and died. Neurons started in the BDNF-NT3-NT4/5 culture medium and moved to NGF during the switchover period had a longevity that increased with age.

An in vivo study conducted on mouse embryos shows that the expression of NGF receptors p75 and TrkA, transmembrane glycoproteins, and sensitivity of neurons is independent of NGF, a neurotropin. Mutants lacking the NGF gene show normal expression of p75 and TrkA in the trigeminal ganglia during the E11 to E13 stage of development. The survival of neurons induced by NGF is also similar in both the mutant and the wild-type embryos.

The neurotrophin family of survival factors is distinguished by a unique receptor-signaling system that is composed of two transmembrane receptor proteins. Nerve growth factor (NGF), brain-derived neurotrophic factor, neurotrophin-3 (NT-3) and NT-4/5 share similar protein structures and biological functions and interact with two different types of cell-surface proteins, the Trk family of receptor tyrosine kinases, and the p75, or low-affinity neurotrophin receptor. An important question is whether a dual receptor system is necessary for neurotrophin action. Evidence indicates that co-expression of the two genes for the p75 receptor and the Trk NGF receptor can potentially lead to greater responsiveness to NGF, and suggests additional levels of regulation for the family of neurotrophin factors.

Study of the nervous system of mice reveals that the absence of neurotrophin-3, which maintains nervous system through neuronal survival and differentiation in mice, leads to chronic sensory and sympathetic deficits. These mice show absence of muscle spindles and exhibit extraordinary limb position. Sympathetic and sensory neurons which require nerve growth factor or neurotrophin factor for survival at a later stage require neurotrophin-3 which acts as a receptor binder.

Fibroblasts secreted by brain-derived neurotrophic factor (BDNF) inhibits dopaminergic neuronal degeneration of adult brain cells that are 1-methyl-4-phenylpyridinium (MPP) induced. Fibroblasts increase the lifetime of nigral dopaminergic neuronal structures more than control fibroblasts implants do and are used in the treatment of neurodegenerative diseases such as Parkinson's disease and mitochondrial malfunctions.

The germline mutation in the tyrosine kinase catalytic domain of the trkB gene of mice eliminates the expression of gp145trkB, the signaling receptor for two constituents of the nerve growth factor group of neurotrophins, neurotrophin-4 and brain-derived neurotrophic factor. The mice homozygous for the trkBTK mutation develops to birth. These defective animals do not display feeding activity and die by P1. They also have neuronal deficiencies in the central and peripheral nervous systems.

The stimulation of monoamine-containing neurone growth and function through exogenous delivery of brain-derived neurotrophic factors or neurotrophin-3 from antidepressants has been studied. The treatment of depression has made significant progress with the discovery of drugs that prevent the inactivation of the neurotransmitters 5-HT and noradrenaline. Monoamine oxidase inhibitors slows the enzymatic degradation of these neurotransmitters. Monoamine reuptake blockers stop the normal recapture of the neurotransmitters by transporting them back into the presynaptic nerver terminal.

Glial cell line-derived neurotrophic factors (GDNF) promote the survival of motor-neurons that contain dopamine and noradrenaline. The c-Ret signaling partner of the neurotrophin is dependent to the binding of GNDF to a Ralpha GNDF auxilliary-binding protein that is connected by glycosylphosphatidylinositol to the membrane of the superior cervical ganglion (SCG). Furthermore, the survival of SCG in cell cultures was reduced by the addition of neurotrophic growth factors (NGF) which activates the p75 NGF low-affinity receptor.

The TrkB ligand NT-4, a neurotrophin, protects the lateral geniculate neurons (LGN) from the dystrophic influence of monocular deprivation (MD). LGN competes for binding NT-4. MD shrinks the neurons in the closed-eye layer of the LGN but neurons with neurotrophin-coated microspheres are protected from atrophy. The fact that the other TrkB ligand brain-derived neurotrophic factor (BDNF) cannot protect the neurons from MD proves that NT-4 and BDNF play different roles in neural development.

Some experts believe gene expression contributes to the changes caused by repetitive brain seizures in the anatomy and excitability of brain networks. New research suggests the existence of previously unknown mechanisms whereby patterns of physiological activity associated with behavior promote survival and plasticity in brain systems. Activity-dependent changes in neurotropin expression are likely to play a major role in seizure-induced changes in the brain.

Results indicate that calcineurin/NFAT is essential for axon growth, which is stimulated by growth factors such as neurotrophins and netrins. Data indicate that neutrotrophins and netrins stimulate nuclear localization of NFATc4 and activation of NFAT-mediated gene transcription in neurons, in a calcineurin-dependent manner.

Damon, Deborah H. NGF-independent survival of postganglionic sympathetic neurons in neuronal-vascular smooth muscle cocultures. Am J Physiol Heart Circ Physiol 280: H1722-H1728, 2001.--The present study tests the hypothesis that vascular cells promote the survival of postganglionic sympathetic neurons in the absence of nerve growth factor (NGF). To test this hypothesis, neurons isolated from superior cervical ganglia of 2- to 4-day-old rat pups were grown in the absence of NGF and in the absence and presence of vascular smooth muscle cells (VSM). Neuronal survival was assessed as a function of time in culture. At all time points studied, VSM promoted the survival of the neurons. After 5 days in the absence of NGF, 7 [+ or -] 2% of neurons survived in the absence and 28 [+ or -] 7% survived in the presence of VSM. An endothelin receptor antagonist reduced neuronal survival in cocultures grown in the absence of NGF. These data indicate that VSM produce factors other than NGF that promote the survival of cultured postganglionic sympathetic neurons. The data also indicate that endothelin contributes to this effect and suggest that endothelin as well as other VSM-derived factors may play a role in the development of sympathetic innervation to the vasculature. neurotrophins; endothelin; neuronal development

The NGF family of neurotrophins has a crucial role in regulating neuron numbers during vertebrate development. Six years ago the prediction was made that invertebrates with simple nervous systems, such as Caenorhabditis elegans, would lack neurotrophins. Surprisingly, it now appears that not only C. elegans but also Drosophila melanogaster, lack homologs of the neurotrophins or their trk receptors. Furthermore, functional studies indicate that control of neuronal numbers in Drosophila is primarily dependent on steroids. By contrast, a recognizable trk homolog exists in molluscs, a phylum that includes species with the most complex nervous systems in the invertebrate kingdom. This suggests that neurotrophic signaling mechanisms might be one of the prerequisites for evolution of complex nervous systems. Expansion of the genome projects to other invertebrates, such as molluscs and coelenterates, should provide new insights on the molecular correlates of building complex brains.

To identify potential functions for neurotrophins during sensory neuron genesis and differentiation, we determined the temporal and spatial protein expression patterns of neurotrophin receptors throughout the process of sensory neurogenesis in the dorsal root ganglia (DRG). We show that neurotrophin receptors are expressed early, being first detected on subsets of migrating neural crest cells, and that trkC is among the earliest markers of neural lineage specification. In the immature DRG, we find that both trkC and [p75.sup.NTR] are expressed on subsets of dividing progenitor cells in vivo. Furthermore, our data directly reveal distinct patterns of trk receptor expression by individual sensory neurons from the time of their inception with all early arising cells initially being trk[C.sup.+], some subsets of whom also coexpress either trkA or trkB or both. As sensory neurons innervate their targets and establish their mature identities, the spectrum of trk receptors expressed by individual neurons is altered. The stereotyped trk receptor expression profiles identified here may potentially correspond to distinct lineages of sensory neurons. These data, in conjunction with other studies, argue for multiple functions for neurotrophins during the process of sensory neuron differentiation, including effects on both neural crest and DRG mitotically active progenitor cells, in addition to possibly influencing the establishment of sensory neuron identity. [C] 2000 Academic Press Key Words: sensory neuron; neurotrophin receptors; avian; dorsal root ganglion; trk; neuronal differentiation.

Neurotrophin-3 (NT-3) is a member of the neurotrophin family of growth factors, best characterized by its survival- and differentiation-inducing effects on developing neurons bearing the trk C receptor tyrosine kinase. Through analysis of NT-3 and trk C gene-targeted mice we have identified NT-3 as critically regulating cardiac septation, valvulogenesis, and conotruncal formation. Although these defects could reflect cardiac neural crest dysfunction, the expression of NT-3 and trk C by cardiac myocytes prior to neural crest migration prompted analysis of cell-autonomous actions of NT-3 on cardiac myocytes. Retroviral-mediated overexpression of truncated trk C receptor lacking kinase activity was used to inhibit activation of trk C by endogenous NT-3, during early heart development in ovo. During the first week of chicken development, expression of truncated trk C reduced myocyte clone size by more than 60% of control clones. Direct mitogenic actions of NT-3 on embryonic cardiac myocytes were demonstrated by analysis of BrdU incorporation or PCNA immunoreactivity in control and truncated trk C-expressing clones. Inhibition of trk C signaling reduced cardiac myocyte proliferation during the first week of development, but had no effect at later times. These studies demonstrate that endogenous NT-3:trk C signaling regulates cardiac myocyte proliferation during cardiac looping and the establishment of ventricular trabeculation but that myocyte proliferation becomes NT-3 independent during the second week of embryogenesis. [c] 2000 Academic Press Key Words: cardiac myocyte proliferation; neurotrophin-3; trk C receptor; cardiogenesis.

Neurotrophins influence the epigenetic shaping of the vertebrate nervous system by regulating neuronal numbers during development and synaptic plasticity. Here we attempt to determine whether these growth factors can also regulate neurotransmitter plasticity. As a model system we used the selection between noradrenergic and cholinergic neurotransmission by paravertebral sympathetic neurons. Developing sympathetic neurons express the neurotrophin receptors TrkA and TrkC, two highly related receptor tyrosine kinases. Whereas the TrkA ligand nerve growth factor (NGF) has long been known to regulate both the survival and the expression of noradrenergic traits in sympathetic neurons, the role of TrkC and of its ligand neurotrophin-3 (NT3) has remained unclear. We found that TrkC expression in the avian sympathetic chain overlaps substantially with that of choline acetyltransferase. In sympathetic chain explants, transcripts of the cholinergic marker genes choline acetyltransferase and vasoactive intestinal polypeptide were strongly enriched in the presence of NT3 compared with NGF, whereas the noradrenergic markers tyrosine hydroxylase and norepinephrine transporter were reduced. The transcription factor chicken achaete scute homolog 1 was coexpressed with cholinergic markers. The effects of NT3 are reversed and antagonized by NGF. They are independent of neuronal survival and developmentally regulated. These results suggest a role for NT3 as a differentiation factor for cholinergic neurons and establish a link between neurotrophins and neurotransmitter plasticity.

Multipotent neural progenitor species present within developing and adult periventricular generative zones can give rise to all of the major cellular elements of the brain. Although lineage specification during development has been thought to be restricted to these generative zones, we have utilized quantitative immunoselection techniques to isolate an enriched population of multipotent neural progenitor cells that express polysialylated neural cell adhesion molecule (PSA-NCAM) from postnatal day 2 cerebral cortex independent of generative zones. This population of cerebral cortical progenitor cells exhibited robust proliferation in response to epidermal growth factor and subsequently gave rise to clonally derived neurons, astrocytes, and oligodendrocytes. Quantitative regional analysis further demonstrated that while the multipotent cells derived from the cerebral cortex uniformly expressed PSA-NCAM, multipotent cells derived from generative zones contained equal proportions of PSA-NCAM-positive and -negative multipotent progenitor cells. The generation of individual cellular lineages from cortical multipotent progenitors could be enhanced by specific cytokines that are expressed within the cerebral cortex. Further, while oligodendroglial progenitor cells derived from cortical multipotent progenitors exhibited responsiveness to platelet-derived growth factor (PDGF) and neurotrophin-3 (NT-3), primary cultures of cortical oligodendroglial progenitors were responsive to PDGF but not to NT-3. These observations suggest that in addition to glial progenitors that commit to a specific lineage prior to migration from generative zones, there is within the cerebral cortex a separate pool of multipotent cells that are capable of generating mature glial progeny in response to specific environmental cues. Therapeutic interventions aimed at differentiation of endogenous cerebral pools of multipotent progenitors may provide a novel strategy for amelioration of the sequelae of environmental and genetic insults to the postnatal cerebrum. Key Words: multipotent progenitors; glial progenitors; cerebral cortex; epidermal growth factor; neurotrophin-3; platelet-derived growth factor; bone morphogenetic proteins.