Target-derived Molecules Research Articles

Rab10 regulates the sorting of internalised TrkB for retrograde axonal transport.

Neurons process real-time information from axon terminals to coordinate gene expression, growth, and plasticity. Inputs from distal axons are encoded as a stream of endocytic organelles, termed signalling endosomes, targeted to the soma. Formation of these organelles depends on target-derived molecules, such as brain-derived neurotrophic factor (BDNF), which is recognised by TrkB receptors on the plasma membrane, endocytosed, and transported to the cell body along the microtubules network. Notwithstanding its physiological and neuropathological importance, the mechanism controlling the sorting of TrkB to signalling endosomes is currently unknown. In this work, we use primary mouse neurons to uncover the small GTPase Rab10 as critical for TrkB sorting and propagation of BDNF signalling from axon terminals to the soma. Our data demonstrate that Rab10 defines a novel membrane compartment that is rapidly mobilised towards the axon terminal upon BDNF stimulation, enabling the axon to fine-tune retrograde signalling depending on BDNF availability at the synapse. These results help clarifying the neuroprotective phenotype recently associated to Rab10 polymorphisms in Alzheimer's disease and provide a new therapeutic target to halt neurodegeneration.

Synapse maturation by activity-dependent ectodomain shedding of SIRPα

Formation of appropriate synaptic connections is critical for proper functioning of the brain. After initial synaptic differentiation, active synapses are stabilized by neural activity-dependent signals to establish functional synaptic connections. However, the molecular mechanisms underlying activity-dependent synapse maturation remain to be elucidated. Here we show that activity-dependent ectodomain shedding of SIRPα mediates presynaptic maturation. Two target-derived molecules, FGF22 and SIRPα, sequentially organize the glutamatergic presynaptic terminals during the initial synaptic differentiation and synapse maturation stages, respectively, in the mouse hippocampus. SIRPα drives presynaptic maturation in an activity-dependent fashion. Remarkably, neural activity cleaves the extracellular domain of SIRPα, and the shed ectodomain, in turn, promotes the maturation of the presynaptic terminal. This process involves CaM kinase, matrix metalloproteinases, and the presynaptic receptor CD47. Finally, SIRPα-dependent synapse maturation has significant impacts on synaptic function and plasticity. Thus, ectodomain shedding of SIRPα is an activity-dependent trans-synaptic mechanism for the maturation of functional synapses.

A network map of BDNF/TRKB and BDNF/p75NTR signaling system

Neurotrophic factors are growth factors, which play an essential role in the development and maintenance of nervous system. These include members of the nerve growth factor (NGF) family, known as neurotrophins. Nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin 3 (NT-3), neurotrophin 4 (NT4/5) and neurotrophin 6 (NT-6) constitute the family of neurotrophins (Barbacid 1995). The identification of neurotrophic factors began from the search for target-derived molecules, which act on neurons and promote their survival (Lindsay 1996). BDNF is one such essential survival factor. BDNF was first isolated and purified from pig brain (Barde et al. 1982). BDNF, encoded by the BDNF gene, is the second member of the neurotrophin family after NGF (Timmusk et al. 1993). BDNF, like other neurotrophins, exists as a homodimer. It is synthesized as a precursor molecule, proBDNF (32 kDa), which is cleaved to form the mature and biologically active form of BDNF (∼13 kDa) (Negro et al. 1994). It is expressed throughout the central nervous system (Leibrock et al. 1989) and also in peripheral tissues including muscle, liver, pancreas, colon, duodenum, lung, kidney and bladder (Lommatzsch et al. 1999). The dimerized BDNF binds to the tyrosine kinase receptor, tropomyosin-related kinase B (TrkB), leading to activation of various signaling modules. Trk was first identified as an oncoprotein in human tumors (Martin-Zanca et al. 1986). The Trk family of receptors includes TrkA, which binds to NGF (Kaplan et al. 1991); TrkB, which is the receptor for BDNF and NT-4 (Klein et al. 1989); and TrkC, the receptor for NT-3 (Lamballe et al. 1991). TrkB is an 821 amino acid long glycoprotein, encoded by the NTRK2 gene. It comprises of a signal peptide followed by an extracellular domain, a transmembrane domain and a cytoplasmic region consisting of the tyrosine kinase domain (Nakagawara et al. 1995). TrkB is expressed in the central and peripheral nervous system (Allen et al. 1994). Like other neurotrophins, BDNF also binds to nerve growth factor receptor (NGFR), also known as p75 neurotrophin receptor (p75NTR), albeit with a low-affinity (Chao et al. 1986). Binding of BDNF to TrkB results in the dimerization of the receptor and activation of its cytoplasmic kinase domain (Ohira et al. 2001; Wu et al. 1996), which results in the recruitment of adaptor proteins to the receptor, which in turn leads to the activation of intracellular signaling cascades including RAS/ERK pathway, PI3K/AKT pathway, PLC/PKC pathway and NFκB pathway. BDNF/TrkB signaling is essential for growth, differentiation and survival of neurons. It is also important for neuronal morphogenesis and synaptic plasticity (Binder and Scharfman 2004). BDNF also plays a major role in other processes such as energy metabolism (Matthews et al. 2009), behavior, learning, memory (Hall et al. 2000), pain (Pezet et al. 2002) and apoptosis (Yeiser et al. 2004). BDNF is implicated in Alzheimer’s disease (Ferrer et al. 1999), Huntington’s disease (Zuccato et al. 2001), epilepsy (Takahashi et al. 1999) and bipolar disorder (Neves-Pereira et al. 2002). However, despite its biological significance, molecular events induced by either BDNF/TrkB or BDNF/p75NTR interactions, were not organized into a signaling network. As a part of the ongoing NetPath project, which aims at the development of a centralized resource for human signaling pathways (Kandasamy et al. 2010; Raju et al. 2011b), we have documented molecular reactions induced by several signaling systems by systematically reviewing published reports and assembled them in the form of signaling pathways. We have previously published molecular networks of signaling induced by prolactin (Radhakrishnan et al. 2012), oncostatin M (Dey et al. 2012); corticotropin releasing hormone (Subbannayya et al. 2013) and advanced glycation end-products (Soman et al. 2013). Similarly, as BDNF signaling pathway is of immense biomedical interest, we analyzed literature pertaining to BDNF-induced signaling events and developed a graphical network map of BDNF/TrkB and BDNF/p75NTR signaling pathways.

Retrograde signaling in the optic nerve is necessary for electrical responsiveness of retinal ganglion cells.

We investigated the role of retrograde signaling in the optic nerve on retinal ganglion cell (RGC) electrical responsiveness in the mouse model. Electrical response of RGC was measured by pattern electroretinogram (PERG) in 43 C57BL/6J mice 4 to 6 months old under ketamine/xylazine anesthesia. PERGs were recorded before and at different times after blockade of axon transport with lidocaine at either the retrobulbar level (2 μL, 40 μg/μL) or at level of the superior colliculus (SC, 1 μL, 40 μg/μL). PERGs also were recorded before and at different times after optic nerve crush 1.5 mm behind the eye, followed by TUJ1-positive RGC counts of excised retinas. As controls, PERGs also were recorded after either saline injections or sham optic nerve surgery. The photopic flash electroretinogram (FERG) and visual evoked potential (FVEP) also were recorded before lidocaine and at relevant times afterwards. Lidocaine injection caused rapid (retrobulbar ~10 minutes, SC 1 hour), reversible reduction of PERG amplitude (≥50%). Optic nerve crush caused rapid (10-20 minutes), irreversible reduction of PERG amplitude (70-75%), increase of PERG latency (>25%), as well as RGC loss (88%) 1 month after crush. FVEP was unaltered by lidocaine. For all procedures, the FERG was unaltered. As experimental interventions were made at postretinal level(s), PERG changes were likely associated with altered supply of retrogradely-delivered material from the SC. This implies that retrograde transport of target-derived molecules is necessary for normal RGC electrical responsiveness. The time course of early PERG changes is consistent with the speed of fast retrograde axon transport.

Nerve Terminal Growth Remodels Neuromuscular Synapses in Mice following Regeneration of the Postsynaptic Muscle Fiber

Muscle fibers degenerate and regenerate in response to contractile damage, during aging, and in various muscle diseases that weaken the fibers. It is known that degeneration and regeneration of the segment of the postsynaptic fiber produces dramatic alterations in the neuromuscular junction (NMJ) that forms on the regenerated fiber, but the mechanisms here are incompletely understood. We have used a laser microbeam to damage the postsynaptic fibers at individual NMJs in the sternomastoid muscle of living young adult mice and then followed the synapses vitally over time using fluorescent proteins expressed in motor neurons and glial cells and staining of postsynaptic acetylcholine receptors. We find, in contrast to previous reports, that the mouse nerve terminal retains contact with the synaptic basal lamina marked by cholinesterase staining even in the absence of the target, showing that this terminal does not require a continuous supply of target-derived molecules for its maintenance. Thus, remodeling of the nerve terminal during the period of target absence does not explain the subsequent changes in the new NMJ. Rather, we see that the synapse becomes altered as the new fiber segment regenerates. Mechanisms for remodeling the synapse include failure of the regenerating muscle fiber to contact the old basal lamina and nerve terminal, growth of the nerve terminal and its glia toward the regenerating fiber, and remodeling of the initial contact as the nerve terminal becomes varicose.

Somatic gene therapy for nervous system disease.

Neurotrophic factors are target-derived molecules that prevent neuronal degeneration during development and, in some cases, during adulthood. They offer substantial promise as therapeutic agents in neurological disease by preventing cell loss and promoting axonal regeneration. However, the optimal means of delivering neurotrophic factors to the nervous system, and the CNS in particular, is an unresolved issue. Neurotrophic factors rarely influence only a single target neuronal population, hence broad delivery of neurotrophic factors to the nervous system may results in effects on multiple non-targeted neuronal populations. Ideally, neurotrophin delivery to the nervous system should be target-specific, regionally restricted, chronic, safe, well-tolerated and of sufficient concentration to elicit responses from target neurons. In this paper we discuss the use of somatic gene transfer methods to deliver neurotrophic factors to the CNS in a manner that seeks to meet the above criteria.

Thyroid hormone and retinoids affect motoneuron phenotype and reaction after axotomy in the spinal cord of adult rats

Motoneuron phenotype in the spinal cord is regulated by an intrinsic genetic program, extrinsic environmental signals and target-derived molecules. Axonal lesions trigger a phenotype switch to foster repair phenomena and axonal re-growth. We have investigated the influence of the long-term treatment with thyroid hormone and all trans retinol palmitate (RA) on motoneuron phenotype and spinal cord reaction to axotomy in adult male rats. Neurochemical markers, investigated by in situ hybridization and immunocytochemistry, included choline acetyltransferase (ChAT), calcitonin gene-related peptide (CGRP) and neurotrophin low affinity receptor p75. Treatment was administered for 56 days and then mid-thigh sciatic axotomy was performed on a number of animals from each experimental groups; the rats were examined 9 days after surgery. The results indicate that: (1) Number and size of ChAT-immunoreactive neurons in the lumbar tract of the spinal cord was reduced in hypothyroid compared to control rats, whereas steady-state level of ChAT mRNA in labelled motoneurons failed to be modified by hypo and hyperthyroidism, but was increased by RA administration; (2) none of the administered treatments did alter CGRP mRNA level, whereas all of them influenced the axotomy-induced changes of motoneuron phenotype; (3) in hyperthyroid rats ChAT mRNA level of lumbar motoneurons not reduced homolateral to lesion while the number of ChAT-IR profiles was pronouncedly reduced; (4) up-regulation of p75 induced by peripheral nerve lesion was reduced in RA-treated rats. These data indicate that the motoneuron phenotype is regulated by transcription factors, which also play a role in phenotype switch regulation after axotomy.

Sorting of Regenerating Rat Sciatic Nerve Fibers with Target-Derived Molecules

The functional outcome of microsurgical repair of divided nerves is disappointing since many regenerating axons fail to reach appropriate targets. Sorting of regenerating axons according to target tissue might be used to improve functional regeneration. The aim of the present study is to see if regenerating axons can be sorted into functionally different bundles with target-derived molecules. The proximal stump of the adult rat sciatic nerve was sutured into the inlet of a silicon Y-tube. The two branches of the Y-tube were filled with agarose primed with filtrates prepared from skin and muscle homogenates from the operated rat. The tibial and sural nerves were inserted in the two branches of the Y-tube. Six weeks later the sciatic nerve axons showed vigorous regeneration into both branches. Electron microscopic examination of regenerated nerve segments showed numerous myelinated and unmyelinated axons. The proportion of myelinated axons was significantly larger in the muscle-gel branch than in the skin-gel branch. Retrograde tracing from the nerve regenerates with Fast Blue and Fluoro-Ruby showed that ventral horn neurons at L4–L5 segmental levels were preferentially labeled from the muscle-gel branch. Neurons in corresponding dorsal root ganglia were labeled from both Y-tube branches (no significant numerical difference). A few neurons of both types contained both tracers. Measurements revealed that sensory neurons labeled from the muscle-gel branch were significantly larger (mean perikaryal area 870 μm2) than neurons labeled from the skin-gel branch (mean area 580 μm2). We conclude that regenerating motor and sensory axons can be sorted with target-derived molecules.

Trophic dependencies of rodent corticospinal neurons.

According to the classical neurotrophin hypothesis, neuronal survival is regulated by limited access to target-derived neurotrophic substances. Recent studies have indicated that this regulation is more complex than originally thought. First, neurons are not only supported by target-derived molecules but also via anterograde, paracrine, and autocrine mechanisms. Second, phenotypes of neurotrophic factor-/receptor-mutant animals displayed fewer neuronal deficits than predicted, suggesting interactivity and redundancy of trophic support of neurons. Finally, certain neurotrophins, in addition to their survival-promoting action, are able to induce neuronal death. Observations in the corticospinal system support the general applicability of these concepts and provide additional insights into the integrative mode of neuronal survival regulation. CNTF and GDNF support developing corticospinal neurons (CSN) by direct mechanisms, while the effects of NT-4/5 require cell contacts of CSN with other cortical neurons in vitro. Thus, these effects do not merely reflect trophic redundancy but the ability of CSN to integrate survival signals of growth factors from different families via different pathways. CNTF and GDNF also promote survival of adult axotomized CSN in vivo. Virtually all adult CSN express mRNA coding for the NT-3-receptor TrkC and the BDNF-receptor TrkB, and after axotomy, CSN also express mRNA for the common neurotrophin-receptor p75NTR, suggesting a role of endogenous neurotrophins for survival regulation of CSN. Indeed, most axotomized CSN depend on endogenous BDNF for survival, and endogenous NT-3 promotes the death of BDNF-dependent CSN. NT-3-mediated death-induction requires co-signalling of TrkC- and p75NTR-receptors. With BDNF/TrkB promoting survival and NT-3/TrkC/p75NTR promoting death, CSN integrate at least three different neurotrophin/receptor-signals for death/survival decisions.

Activin A and follistatin influence expression of somatostatin in the ciliary ganglion in vivo.

An important developmental question concerns whether neurotransmitter phenotype is an inherent property of neurons or is influenced by target tissues. This issue can be addressed in the avian ciliary ganglion (CG) which contains two cholinergic populations, ciliary and choroid neurons, that differentially express the peptide cotransmitter, somatostatin. The present study tests the hypothesis that differences in the level of expression of activin A and its endogenous inhibitor follistatin in CG neuron target tissues are responsible for selective expression of somatostatin in choroid neurons. Intraocular injection of activin A or follistatin (300 ng injected at E10/E11) in cultured embryos resulted in a 39% increase or a 23% decrease, respectively, in somatostatin-positive neurons relative to controls. Chorioallantoic membrane application of follistatin (1 microgram daily from E7 to E13) reduced somatostatin positive neurons by 54%. Neuron number, size, and target tissue morphology were unaffected by these treatments. Together with our previous studies, these data suggest that activin A and follistatin are target-derived molecules that regulate neuropeptide phenotype in the ciliary ganglion.

A BRAIN-DERIVED NEUROTROPHIC FACTOR (BDNF) RELATED SYSTEM IS INVOLVED IN THE MAINTENANCE OF THE POLYINNERVATE TORPEDO ELECTRIC ORGAN

Target-derived molecules are essential for the maintenance of neuron survival. In the present work, we introduce the electric organ of Torpedo marmorata as a tool for the study of trophic interactions in a polyinnervate system. This electric organ maintains a large number of cholinergic terminals on the postsynaptic cell surface. We have observed that a soluble extract derived from the electric organ induces the maturation of Xenopus oocytes injected with presynaptic plasma membranes (PSPM), indicating that a trophic system may exist. Moreover, we have detected a p75NGFR related protein in PSPM by Western blot analysis. These results suggest the presence of a neurotrophin-related system maintaining the polyinnervate electric organ. Furthermore, molecular experiments showed that the brain-derived neurotrophic factor (BDNF) is the neurotrophin operating in our model. Using degenerate oligonucleotides which comprise a conserved fragment of all neurotrophins, we have only amplified by polymerase chain reaction a BDNF fragment. In a similar way, we have amplified and cloned a fragment of the TrkB/C high affinity BDNF receptor. The fact that degerate oligonucleotides only amplify BDNF allows us to conclude that the polyinnervation is maintained by this neurotrophin either alone or in combination with other trophic factors. © 1997 Elsevier Science Ltd

CNTF and LIF Are Not Required for the Target-Directed Acquisition of Cholinergic and Peptidergic Properties by Sympathetic Neurons in Vivo

During development, the sympathetic innervation of two targets, sweat glands and periosteum, changes the neurotransmitters it expresses from noradrenaline to acetylcholine and vasoactive intestinal peptide (VIP). The target-derived molecules that induce these changes have not been identified. Neuropoietic cytokines, including ciliary neurotrophic factor (CNTF) and leukemia inhibitory factor (LIF), induce the same phenotypic changes in sympathetic neurons in vitroas sweat glands and periosteum do in vivo,raising the possibility that one of these factors mediates induction of cholinergic traits and VIP by these target tissues. Because CNTF and LIF have overlapping functions and signalling pathways, they could act interchangeably or in concert to influence neurotransmitter expression. To determine whether CNTF or CNTF and LIF together are responsible for the induction of cholinergic and peptidergic function in vivo,we analyzed the neurotransmitter properties of sweat gland innervation in mice lacking CNTF or CNTF and LIF. We find that, as in wild-type mice, gland innervation in mice lacking one or both molecules appropriately expresses cholinergic properties and VIP immunoreactivity. Furthermore, footpads of mice lacking one or both genes contain choline acetyltransferase activity comparable to that of wild-type mice, and CNTF- or CNTF/LIF-deficient mice possess the normal complement of active sweat glands. We analyzed the innervation of a second, recently identified cholinergic sympathetic target, the periosteum, which is the connective tissue surrounding bone. Periosteal innervation of mice lacking CNTF, LIF, or both, like that of wild-type mice, is immunoreactive for the vesicular acetylcholine transporter, a recently identified cholinergic marker, and VIP. These results provide evidence that neither CNTF, LIF, nor a combination of the two are required for the developmental change from noradrenergic to cholinergic function that occurs in sympathetic innervation of sweat glands and periosteum.

Molecular genetics of neuronal survival

The past year has witnessed remarkable progress towards understanding the molecular genetics of neuronal survival. Gene-targeting experiments in mice have confirmed the long-standing idea that the nerve growth factor model of neuronal survival — that is, neuronal dependence on target-derived molecules during a critical period in development — is broadly applicable. Furthermore, a variety of biochemical and genetic techniques applied to both mammals and invertebrates have identified new genes involved in regulating cell survival during development.

Target‐derived molecules that influence the development of neurons in the avian ciliary ganglion

The developing avian ciliary ganglion has been a particularly amenable system for the identification, isolation, and characterization of putative target-derived molecules that mediate retrograde interactions. To date a number of biochemically distinct activities that regulate neuronal survival, transmitter phenotype, and chemosensitivity of ciliary ganglion neurons have been identified. Of these, only two survival-promoting molecules have been purified to homogeneity: ciliary neurotrophic factor and a related molecule, growth-promoting activity. A somatostatin-inducing activity found in cultured choroid cells is very likely to be chick activin A. Other molecules that regulate acetylcholine and acetylcholine receptor expression comigrate on a gel filtration column at a molecular weight of 50-60 kD, but they have yet to be isolated. Once molecules that mimic retrograde influences are identified, a number of criteria must be met before their physiological significance can be established. These criteria are (1) availability of the molecule from the target at the appropriate time in development; (2) ability of the neurons to respond to the molecule at the appropriate time in development; (3) demonstration that blocking the activity or availability of the molecule is able to block the target-derived developmental change expressed in the neurons. Of the molecules that are thought to retrogradely influence ciliary neuron development, only growth-promoting activity is known to meet criteria 1 and 2, and experiments are currently underway to test whether inhibition of growth-promoting activity in vivo will exacerbate normal cell death.

Neurotrophic factors and diseases of the nervous system.

Neuronal degeneration and death occur in mammals in the course of nervous system development, during senescence, and as a consequence of pathological events throughout life. Neurotrophic factors are natural target-derived molecules that prevent neuronal degeneration and death during development and promote neuronal recovery from injury and age-related atrophy. One neurotrophic factor--nerve growth factor-prevents the death of cells that are a target of varicella-zoster virus: dorsal root sensory neurons. Delivery of neurotrophic factors to dysfunctional neurons may be a useful therapeutic strategy for promoting neuronal recovery in a number of diseases, including varicella-zoster virus infection.

Choroid tissue supports the survival of ciliary ganglion neurons in vitro

It is well established that during in vivo development the neurons of the avian ciliary ganglion are dependent for their survival on structures in the eye. Separate neuron populations innervate intraocular smooth and striated muscle targets. All ciliary neurons survive when cocultured with striated muscle. We demonstrate that when ciliary ganglion neurons are plated on explants of the choroid coat (a smooth muscle-containing target tissue) using a defined medium (N2), the neurons survive and grow vigorously into the tissue, forming contacts between axons and target cells identified as smooth muscle. Conditioned medium from choroid explants also rescues all the neurons, as does coculturing ciliary ganglion neurons with dissociated choroid cells. However, the presence of horse serum and chick embryo extract in the medium inhibits the choroid's ability to support ciliary neurons. The effects of these additives on the phenotypic expression of the smooth muscle may explain the inability of previous investigators to demonstrate target-derived support from smooth muscle preparations. Because the choroid contains cell types other than smooth muscle (e.g., fibroblasts and endothelial cells), we could not identify smooth muscle as the only cell type responsible for the release of the soluble trophic factor present in the target tissue. However, indirect evidence using avian primary fibroblast cultures, a fibroblast cell line, and an anatomically simple smooth muscle preparation, the avian amnion, suggests that smooth muscle cells are sufficient to account for the observed trophic activity, and that similar target-derived molecules support the survival of both types of ciliary ganglion cells.

Rescue of both rapidly and slowly degenerating neurons in the dorsal lateral geniculate nucleus of adult rats by a cortically derived neuron survival factor

We investigated the death of dorsal lateral geniculate nucleus (dLGN) neurons after lesions to the visual cortex of adult rats and the effects of supplying target-derived neurotrophic molecules to the lesion cavity. The neurotrophic factor is retrieved from cocultures of the embryonic primordia of the geniculocortical pathway and its survival promoting properties for different populations of dLGN neurons (based on their time of orgin) have been documented in previous studies of neonatal rats with occipital cortex lesions. In the present study, rats were exposed to [ 3H]thymidine on E14 or E15 16 to label either earlier or later generated dLGN neurons. When animals were at least 45 days old we made discrete lesions to the principal projection zones in area 17 of these two dLGN populations. Counts of surviving labeled cells show a relatively rapid death of E15 16 dLGN neurons in control animals, with a maximal loss by 2 weeks postlesion. The death of E14 dLGN neurons is more protracted, with a maximal loss by 2 months postlesion. A 2-week infusion of the CM fraction rescues the majority of the neurons that would otherwise die in both populations compared to the controls which receive a similarly prepared fraction of unconditioned medium. Moreover, this CM fraction can sustain E14-generated dLGN neurons up to 6 weeks after the neurotrophic factor(s) is no longer being supplied exogenously. Thus the rescue of axotomized adult dLGN neurons appears to be permanent, at least for the early generated population. These findings are consistent with the idea that target-derived molecules have a role in the survival of mature neurons, as they are known to have for developing neurons. Comparison of these results with those obtained in neonatal rats suggests that intrinsic differences in the neurotrophic requirements of different dLGN neuron populations that are found during development are retained in maturity.

The neurotrophic theory and naturally occurring motoneuron death

There is increasing evidence that target-derived molecules play a crucial role in the regulation of neuronal survival during development. These molecules, termed neurotrophic factors, are thought to act in specific ways as defined by the neurotrophic theory. One central tenet of the neurotrophic theory is that some neurons in a population die because trophic molecules are available in only limited amounts during periods of naturally occurring cell death. Delivery of trophic factor to nerve terminals could be regulated by several mechanisms, including, for example, limited production (biosynthesis) by target cells, limited release by targets, or limited uptake by pre-synaptic terminals. An examination of recent studies of motoneuron development indicates that motoneurons compete, via axonal branching and synaptic contacts, for restricted sites on targets that provide access to trophic factors. According to this view, it is terminal branches and contact (‘synaptic’) sites that limit the regulation of neuronal survival, rather than trophic factor production.