CEP-1347 promotes survival of NGF responsive neurones in primary DRG explants

CEP-1347 inhibits the signalling pathway of c-jun-N-terminal kinase, and is neuroprotective in vivo and in vitro. Embryonic chick dorsal root ganglion neurones are dependent on NGF for survival and neurite outgrowth; NGF withdrawal results in apoptotic cell death. We examined the neuroprotective and neurite outgrowth promoting activity of CEP-1347 in dissociated DRG neurones and in primary DRG explants. CEP-1347 was as effective as NGF in promoting survival of dissociated DRG neurones, but caused only limited neurite outgrowth from DRG explants. When NGF was subsequently added to CEP-1347 treated explants, the outgrowth increased to a similar level to explants which had received NGF throughout. CEP-1347 may be a useful tool to maintain viable DRG explants to allow evaluation of neurite outgrowth promoting compounds and dissection of survival and neurite outgrowth signalling pathways.     

The cytokine/neurotrophin axis in peripheral axon outgrowth

Inflammation is part of the physiological wound healing response following mechanical lesioning of the peripheral nervous system. However, cytokine effects on axonal regeneration are still poorly understood. Because cytokines influence the expression of neurotrophins and their receptors, which play a major role in axonal outgrowth after lesioning, we investigated the hypothesis that cytokines influence specifically neurotrophin-dependent axon elongation. Therefore, we have characterized neurotrophin-dependent neurite outgrowth of murine dorsal root ganglia (DRG) in vitro and investigated the influence of pro- and anti-inflammatory cytokines on these outgrowth patterns. Embryonic day 13 (E13) DRG were cultured in Matrigel for 2 days and axonal morphology, density and elongation were determined using an image analysis system. Nerve growth factor (NGF), neurotrophin-3 (NT-3) and -4 (NT-4) were applied alone (50 ng/mL), in double or in triple combinations. NT-3, NT-4 and NT-3 + NT-4 combined induced a moderate increase in axonal outgrowth (P < 0.001) compared with controls, while NGF and all combinations including NGF induced an even more pronounced increase in axonal outgrowth (P < 0.001). After characterizing these outgrowth patterns, interleukin (IL)-1beta, IL-4, IL-6, interferon-gamma (IFNgamma) and tumour necrosis factor-alpha (TNFalpha) (50 or 500 ng/mL) were added to the different neurotrophin combinations. Low doses of TNFalpha and IL-6 influenced neurite extension induced by endogenous neurotrophins. IL-4 increased NT-4-induced outgrowth. IL-6 stimulated NT-3 + NT-4-induced outgrowth. IFNgamma stimulated neurite extension in the presence of NT-3 + NT-4 and NT-3 + NGF. TNFalpha inhibited NT-3-, NT-3 + NGF-, NT-4 + NGF- and NT-3 + NT-4 + NGF-induced outgrowth. These data suggest that inflammation following nerve injury modulates re-innervation via a cytokine/neurotrophin axis.     

Inhibition of axonal growth from sensory neurons by excess nerve growth factor

Fifteen-day embryonic rat dorsal root ganglion (DRG) neurons were exposed to 1 to 200 ng/ml nerve growth factor (NFG). Maximal neurite outgrowth was obtained with 10 to 20 ng/ml. Neurite outgrowth was reduced to 89% of maximal by increasing NGF to 50 ng/ml, to 66% by 100 ng/ml, and to 18% by 200 ng/ml NGF. Identical effects were seen with mouse 2.5S NGF and recombinant human NGF. Neuron cell counts demonstrated that significant cell death did not occur. In time course experiments, significant inhibition, compared with control, began within 1 hour of adding 200 ng/ml and 3 hours of adding 50 ng/ml NGF. The inhibitory effect of NGF on neurite outgrowth was reversed within 3 hours when DRG were incubated with 5 ng/ml NGF after treatment with 50 or 200 ng/ml NGF medium for 12 hours. The inhibition demonstrated for neurons did not occur in PC12 cells; axonal growth was not inhibited by up to 1,000 ng/ml NGF. Excess brain-derived neurotrophic factor or neurotrophin-3 did not inhibit neurite outgrowth. We conclude that high concentrations of NGF produces specific and reversible arrest of neurite outgrowth from sensory neurons. This observation has important clinical implications, because these inhibitory concentrations have been exceeded when NGF has been administered into the central nervous system of humans and animals.     

NGF/BDNF chimeric proteins: analysis of neurotrophin specificity by homolog-scanning mutagenesis.

Despite their extensive sequence identities at the amino acid level (approximately 55%), NGF and brain-derived neurotrophic factor (BDNF) display distinct neuronal specificity toward neurons of both the PNS and CNS. To explore which region(s) within these neurotrophic factors might determine their differential actions on various subpopulations of peripheral neurons, a systematic series (homolog-scanning mutagenesis) of chimeric NGF/BDNF molecules was prepared using PCR overlap-extension techniques. After expression in COS-7 cells, the chimeric proteins were tested for their biological activities in neurite outgrowth and neuronal survival assays. This approach led to the functional expression of 12 NGF/BDNF chimeras. Surprisingly, despite replacing successive amino acid segments throughout the entire length of NGF with the corresponding parts of BDNF, all chimeras displayed full NGF-like activity in bioassays carried out with PC12 cells, embryonic chick dorsal root ganglion explants, sympathetic ganglion explants, and dissociated cultures of dorsal root ganglion neurons. Most of the chimeras additionally showed BDNF-like activity as defined by neurite outgrowth on chick nodose ganglion explants. However, none of the chimeras supported the survival of dissociated nodose ganglion neurons. Our results suggest that NGF and BDNF must share very similar higher-order protein structures, and we propose that the overall structure or conformation of NGF, in contrast to short amino acid "active-site" segments, may determine its exact neuronal specificity.     

Cloning and expression of a novel neurotrophin,NT-7, from carp

Neurotrophins have been demonstrated to play important roles in the development and functioning of the nervous system. This family of proteins consists of four homologous members in mammals: NGF, BDNF, NT-3, and NT-4/5. A new member, called NT-6, was recently cloned from the platyfish Xiphophorus maculatus. This protein shares closer structural relationship to NGF than the other neurotrophins, but contains a characteristic insertion of 22 amino acids that constituted the heparin-binding domain. Here we report the cloning of a novel neurotrophin from the fish Cyprinus carpio (carp), which shared about 66% amino acid identity to Xiphophorus NGF and NT-6. The neurotrophin, designated NT-7, possesses structural characteristics common to all known neurotrophins, such as the presence of six conserved cysteine residues and the flanking conserved sequences. In addition, there is an insertion of 15 amino acids at the position corresponding to that observed for NT-6. The neurotrophic activity of NT-7 was demonstrated by its ability to promote neurite outgrowth and neuronal survival of chick dorsal root ganglia. Phosphorylation assay of various Trk receptors overexpressed in fibroblasts suggested that NT-7 could activate TrkA but not TrkB or TrkC. Northern blot analysis revealed that NT-7 was predominantly expressed in peripheral tissues, though weak expression was also detected in the brain. Like NT-6, this novel neurotrophin might represent yet another NGF-like neurotrophin in lower vertebrates.     

Neural crest-derived spinal and cranial sensory neurones are equally sensitive to NGF but differ in their response to tissue extracts

The response of two distinct populations of neural crest-derived sensory neurones to nerve growth factor (NGF) and other neurotrophic activities present in extracts of chick tissues has been studied in vitro. Dorsal root ganglia (DRG) and the dorso-medial part of the trigeminal ganglion (DM-TG) from embryonic chicks of 6-11 days of incubation (E6-E11) were grown as either explant or dissociated neurone-enriched cultures. Over the age range studied NGF promoted survival and pronounced neurite outgrowth from both DRG and DM-TG neurones. Whilst extracts of chick eye, liver and spinal cord also elicited a marked response from E8 and older DRG neurones, DM-TG neurones were almost entirely unresponsive to the neurotrophic activity of these extracts.     

Neurite outgrowth in normal and injured primary sensory neurons reveals different regulation by nerve growth factor (NGF) and artemin

Neurotrophic factors have been intensively studied as potential therapeutic agents for promoting neural regeneration and functional recovery after nerve injury. Artemin is a member of the glial cell line-derived neurotrophic factor (GDNF) family of ligands (GFLs) that forms a signalling complex with GFRα3 and the tyrosine kinase Ret. Systemic administration of artemin in rodents is reported to facilitate regeneration of primary sensory neurons following axotomy, improve recovery of sensory function, and reduce sensory hypersensitivity that is a cause of pain. However, the biological mechanisms that underlie these effects are mostly unknown. This study has investigated the biological significance of the colocalisation of GFRα3 with TrkA (neurotrophin receptor for nerve growth factor [NGF]) in the peptidergic type of unmyelinated (C-fibre) sensory neurons in rat dorsal root ganglia (DRG). In vitro neurite outgrowth assays were used to study the effects of artemin and NGF by comparing DRG neurons that were previously uninjured, or were axotomised in vivo by transecting a visceral or somatic peripheral nerve. We found that artemin could facilitate neurite initiation but in comparison to NGF had low efficacy for facilitating neurite elongation and branching. This low efficacy was not increased when a preconditioning in vivo nerve injury was used to induce a pro-regenerative state. Neurite initiation was unaffected by artemin when PI3 kinase and Src family kinase signalling were blocked, but NGF had a reduced effect.     

Repeated,intermittent treatment with amphetamine induces neurite outgrowth in rat pheochromocytoma cells (PC12 cells)

Repeated, intermittent treatment with amphetamine (AMPH) leads to long-term neurobiological adaptations in rat brain including an increased number and branching of dendritic spines. This effect depends upon several different cell types in the intact brain. Here we demonstrate that repeated, intermittent AMPH treatment induces neurite outgrowth in cultured PC12 cells without the requirement for integrated synaptic pathways. PC12 cells were treated with 1 micro M AMPH for 5 min a day, for 5 days. After 10 days of withdrawal, there was an increase in the percentage of cells with neurites ( approximately 30%) and the length of neurites as well as an increase in the level of GAP-43 and neurofilament-M. Neurite outgrowth was enhanced as withdrawal time was increased. Neurite outgrowth was much greater following repeated, intermittent treatment with AMPH compared to continuous or single treatment with AMPH. Pretreatment with cocaine, a monoamine transporter blocker, inhibited the AMPH-mediated increase in neurite outgrowth. Neither NGF antibody nor DA receptor antagonists blocked AMPH-induced neurite outgrowth, demonstrating that AMPH-induced neurite outgrowth is not dependent on endogenous NGF release or DA receptors. Thus we have demonstrated that repeated, intermittent treatment with AMPH has a neurotrophic effect in PC12 cells. The effect requires the action of AMPH on the norepinephrine transporter, and shares characteristics in its development with other forms of sensitization but does not require an intact neuroanatomy.     

BDNF and NT-4 differentially modulate neurite outgrowth in developing retinal ganglion cells.

We show here that neurite outgrowth of ganglion cells (RGCs) was selectively enhanced following treatment with BDNF or NT-4 in short-term cultures of dissociated cells derived from the neuroretina of postnatal rats. NT-4 was more effective than BDNF. The effect of NT-3 was variable, whereas NGF and CNTF had no effects upon neurite elongation. The neuritogenic responses of RGCs to both BDNF and NT-4 were prevented by competition with soluble TrkB receptor, and abolished by K252a, a selective inhibitor of the tyrosine kinase activity of Trks. These results indicate that the differentiating effects of BDNF and NT-4 are mediated by TrkB receptors, naturally expressed by RGCs. Developing RGCs treated with these TrkB ligands displayed distinct, albeit partially overlapping, patterns of neurite morphology. BDNF supported predominantly polarized outgrowth, whereas NT-4 induced the appearance of intensely branched symmetrical arbors. The lack of RGCs showing combined morphologies (e.g., highly arborized unipolar cells) suggests distinct mechanisms underlying either elongation or branching, and implicates distinct responses of RGC subsets. We conclude that neurite growth in vitro is extensively promoted by neurotrophins in developing RGCs. Moreover, highly homologous neurotrophins such as BDNF and NT-4, presumably activating via TrkB receptors, selectively control the differentiation of distinct ganglion cell neuritic morphologies.     

Effect of lens lesion on neurite outgrowth of retinal ganglion cells in vitro

Recent studies have shown that lens lesion promotes axonal regeneration in the optic nerve of adult rats. In the present investigations, dissociated retinal ganglion cells (RGC) from intact postnatal (P) 9-11 rats showed spontaneous neurite outgrowth on laminin-1, in contrast to RGC from intact P14-adult rats. Neurite outgrowth from P9-14 RGC on laminin-1 was promoted by prior lens lesion and also during coculture with lesioned lenses. Neurite outgrowth from adult RGC following prior lens lesion, or in cocultures with lesioned lenses, required the presence of laminin-2. In media conditioned by lesioned lenses, the stimulatory effect on neurite outgrowth was still observed in the presence of K252a (trk receptor blocker) and mAb 228 (which blocks the effects of leukemia inhibitory factor and ciliary neurotrophic factor). Together, these results suggest the existence of a neuritogenic factor(s) associated with the lesioned lens that belongs to neither the neurotrophin nor the gp130 cytokine family.     

Bone mesenchymal stromal cells stimulate neurite outgrowth of spinal neurons by secreting neurotrophic factors

It has been demonstrated that bone mesenchymal stromal cells (BMSCs) stimulate neurite outgrowth from dorsal root ganglion (DRG) neurons. The present in vitro study tested the hypothesis that BMSCs stimulate the neurite outgrowth from spinal neurons by secreting neurotrophic factors. Spinal neurons were cocultured with BMSCs, fibroblasts and control medium in a non-contact system. Neurite outgrowth of spinal neurons cocultured with BMSCs was significantly greater than the neurite outgrowth observed in neurons cultured with control medium or with fibroblasts. In addition, BMSC-conditioned medium increased the length of neurites from spinal neurons compared to those of neurons cultured in the control medium or in the fibroblasts-conditioned medium. BMSCs expressed brain-derived neurotrophic factor (BDNF) and glial cell line-derived neurotrophic factor (GDNF). The concentrations of BDNF and GDNF in BMSC-conditioned medium were 132±12 and 70±6 pg ml(-1), respectively. The addition of anti-BDNF and anti-GDNF antibodies to BMSC-conditioned medium partially blocked the neurite-promoting effect of the BMSC-conditioned medium. In conclusion, our results demonstrate that BMSCs promote neurite outgrowth in spinal neurons by secreting soluble factors. The neurite-promoting effect of BMSCs is partially mediated by BDNF and GDNF.     

Cooperation between nerve growth factor and laminin or fibronectin in promoting sensory neuron survival and neurite outgrowth

Chick embryo dorsal root ganglion (DRG) neurons were purified by differential adhesion to plastic. The purified neurons were used to study the cooperation between nerve growth factor (NGF) and laminin or fibronectin in promoting neuron survival and neurite outgrowth. NGF alone supported the survival of only 20% embryonic day 10 (E10) cells, of which only 40-50% had neurites. Treatment of the substrate with fibronectin or laminin increased survival in the presence of NGF up to 80% of the seeded neurons, all of which showed extensive neurite outgrowth. Survival and neurite outgrowth were also enhanced by the combined effects of elevated potassium and laminin. In contrast to E8-10 cells, 85% of E16 neurons survived in the basal culture conditions, i.e. without additional NGF, fibronectin or laminin, although neurite outgrowth was enhanced by all 3 proteins. Antisera to NGF, laminin and fibronectin, each independently decreased survival and neurite outgrowth of DRG neurons, totally with E9 and partially with E16 cells. The results suggest that the cooperative actions of extracellular matrix proteins and NGF are essential for survival and neurite outgrowth of embryonic DRG neurons and that these neuronal requirements change during development.     

Quantitation of neurite growth parameters in explant cultures using a new image processing program

An interactive image processing program was developed to quantify the effects of various biochemical and physical factors on cultured explants of nerve tissue. We used this method to obtain a growth curve of chick embryo dorsal root ganglia (DRG) in media containing various concentrations of nerve growth factor (NGF). In the past, neurite lengths and numbers were measured manually using collages of 35 mm color photographs or made directly under the microscope. Our new program makes it possible to quantify the growth of whole live, unstained DRG's on photograph collages or digital images with respect to center area, neurite area, total explant area, and the number and length of neurites almost exclusive of background artifacts. After comparing the old and new methods, we conclude that our analysis algorithm correlates well with previously accepted protocols for assessing stimulation and inhibition of growth. It rapidly measures several biologically-relevant properties and provides a means to obtain information on six parameters (neurite area, neurite length, neurite number, center area, total area, neurite density) using a single quantitative method. Neurite area in the presence of 10 ng/ml or 20 ng/ml NGF was the most significantly increased parameter as was expected from previous studies since it includes both neurite length and number as well as any crossing fibers.     

Bone mesenchymal stromal cells stimulate neurite outgrowth of spinal neurons by secreting neurotrophic factors

Abstract

It has been demonstrated that bone mesenchymal stromal cells (BMSCs) stimulate neurite outgrowth from dorsal root ganglion (DRG) neurons. The present in vitro study tested the hypothesis that BMSCs stimulate the neurite outgrowth from spinal neurons by secreting neurotrophic factors. Spinal neurons were cocultured with BMSCs, fibroblasts and control medium in a non-contact system. Neurite outgrowth of spinal neurons cocultured with BMSCs was significantly greater than the neurite outgrowth observed in neurons cultured with control medium or with fibroblasts. In addition, BMSC-conditioned medium increased the length of neurites from spinal neurons compared to those of neurons cultured in the control medium or in the fibroblasts-conditioned medium. BMSCs expressed brain-derived neurotrophic factor (BDNF) and glial cell line-derived neurotrophic factor (GDNF). The concentrations of BDNF and GDNF in BMSC-conditioned medium were 132±12 and 70±6 pg ml^?1, respectively. The addition of anti-BDNF and anti-GDNF antibodies to BMSC-conditioned medium partially blocked the neurite-promoting effect of the BMSC-conditioned medium. In conclusion, our results demonstrate that BMSCs promote neurite outgrowth in spinal neurons by secreting soluble factors. The neurite-promoting effect of BMSCs is partially mediated by BDNF and GDNF.     

Neurotrophins and extracellular matrix molecules modulate sensory axon outgrowth

Neurotrophins have been known to play a pivotal role in axonal guidance. Recent research has implicated the role of extracelluar matrix molecules in co-ordinating axonal movement. In this study, we examined the influence of neurotrophins (nerve growth factor (NGF) and neurotrophin-3 (NT-3)) and extracellular matrix molecules (laminin, fibronectin, and poly-l-lysin) on sensory neurite outgrowth in thoracic dorsal root ganglia (DRG) dissected from rats at embryonic day 13. Adjacent DRG were embedded in a collagen gel matrix and supplemented with NGF or NT-3. Under NT-3 conditions, DRG axons extended towards each other and intermingled, while neurites from NGF-treated DRG demonstrated a strong repellent effect, resulting in turning responses and growth cone collapse. This effect was not observed on a collagen culture surface. Interestingly, the composition of the extracellular matrix strongly influenced the observed repellent effect. Sensory neurites from NGF-stimulated DRG again demonstrated a repellent effect when plated on a laminin surface, but showed intermingling behavior when plated on poly-l-lysin or fibronectin. This observation suggests that a factor secreted by NGF-treated DRG axons interacts with laminin, enabling repulsion. This factor and its interaction with the extracellular matrix play an important role in the mechanism of sensory axonal pathfinding.     

Relation of target encounter and neuronal death to nerve growth factor responsiveness in the developing mouse trigeminal ganglion

An aim of this study was to define precisely the period in the developmental history of primary sensory neurons during which nerve growth factor (NGF) exerts its growth-promoting effect. The mouse trigeminal ganglion and its peripheral projection were studied at closely staged intervals throughout development using light and electron microscopy, and the influence of NGF and anti-NGF antiserum on neurite outgrowth from ganglion explants was investigated at corresponding stages in culture. By embryonic day 9.5 (E9.5) peripheral fibers were first visible and increased in number until E13. Throughout this period in vitro neurites grew in the presence of anti-NGF. Peripheral fibers initially contacted the epithelium of the mandibular process by E10.5 and the maxillary process by E11. This coincided with the stage in vitro during which the magnitude of neurite outgrowth was significantly increased by NGF. The development of this response was independent of target encounter since it occurred in neurons which had not contacted their targets prior to explantation. There was an approximate one-to-one relationship between the number of neurons in the ganglion and peripheral fibers throughout development. A peak of some 44,400 fibers and 42,600 neurons was reached by E13 and fell to 20,800 and 19,000, respectively, by birth. Neurite outgrowth was elicited by NGF throughout the period of neuronal death. It is argued that the time course of the influence of NGF is consistent with a role as a selective maintenance factor but not as an agent directing initial outgrowth.     

Neurite outgrowth and GAP-43 mRNA expression in cultured adult rat dorsal root ganglion neurons: Effects of NGF or prior peripheral axotomy

Adult dorsal root ganglion (DRG) cells are capable of neurite outgrowth in vivo and in vitro after axotomy. We have investigated, in cultured adult rat DRG cells, the relative influence of nerve growth factor (NGF) or a prior peripheral nerve lesion on the capacity of these neurons to produce neurites. Since there is evidence suggesting that the growth-associated protein GAP-43 may play a crucial role in axon elongation during development and regeneration, we have also compared the effect of these treatments on GAP-43 mRNA expression. NGF increased the early neurite outgrowth in a subpopulation of DRG cells. This effect was substantially less, however, than that resulting from preaxotomy, which initiated an early and profuse neurite outgrowth in almost all cells. No difference in the expression of GAP-43 mRNA was found between neurons grown in the presence or absence of NGF over 1 week of culture, in spite of the increased growth produced by NGF. In contrast, cultures of neurons that had been preaxotomized showed substantial increase in GAP-43 mRNA and NGF had, as expected, a significant effect on substance P mRNA levels. Two forms of growth may be present in adult DRG neurons: an NGF-independent, peripheral nerve injury-provoked growth associated with substantial GAP-43 upregulation, and an NGF-dependent growth that may underlie branching or sprouting of NGF-sensitive neurons, but which is not associated with increased levels of GAP-43 mRNA. © 1994 Wiley-Liss, Inc.     

Reduced NGF secretion by Schwann cells under the high glucose condition decreases neurite outgrowth of DRG neurons

Background

Schwann cells (SCs) have been supposed to play prominent roles in axonal regeneration under various diseases. Here, to evaluate the direct interaction between SCs and dorsal root ganglion (DRG) neurons under a diabetic condition, the effects of Schwann cell-conditioned media on neurite outgrowth of DRG neurons were investigated.

Methods

Immortalized mouse Schwann cells (IMS) were cultured under 5.5 mM glucose (NG) or 30 mM glucose (HG) conditions for 4 days. IMS-conditioned media (IMS-media) were added to the culture media of neurons isolated from 8-week-old DDY mice. Neurons were cultured for 48 h with or without mouse recombinant NGF (mrNGF) or nerve growth factor (NGF) neutralizing antibody. The concentrations of NGF in IMS-media by ELISA and neurite outgrowth by a computed image analysis system were evaluated.

Results

Neurite outgrowth was significantly enhanced by IMS-media (IMS-media (–): 177 ± 177 µm, IMS-media (+): 1648 ± 726). The neurite outgrowth cultured with IMS-media obtained under the HG condition was significantly reduced compared with that under the NG condition (NG: 1474 ± 652, HG: 734 ± 331). The NGF concentrations were significantly lower in IMS-media under the HG condition than in those under the NG condition. The accelerated neurite outgrowth by IMS-media was inhibited by NGF neutralizing antibody.

Conclusions

These results suggest that SCs play important roles in neurite outgrowth of DRG neurons, and that the decreased NGF secretion by SCs under the diabetic condition would cause a defect of axonal regeneration, resulting in the development of diabetic neuropathy.     

Neurotrophin-3 increases neurite outgrowth and apoptosis in explants of the chicken neural plate

This study examined the effect of neurotrophin-3 (NT-3) on neurite outgrowth and apoptosis of chicken neural plate explants prior to neural tube formation. In situ hybridization revealed that mRNA for the full-length (catalytic) NT-3 receptor, TrkC, was present in, and limited to, the neural plate (including the neural folds) coincident with its formation. Neural plate explants were maintained in vitro on a collagen gel under serum-free conditions in the presence or absence of exogenous NT-3 and/or an antibody to NT-3. In the absence of exogenous NT-3, explants exhibited neurite outgrowth after several days in vitro; apoptotic cells were also seen after 2 days in vitro. This does not appear to be due to endogenous NT-3, since the total number of neurites or apoptotic cells was unchanged if explants were exposed to an NT-3 antibody for the entire culture period. In the presence of exogenous NT-3, neural plate explants exhibited a dose-dependent statistically significant increase in the total number of neurites as compared to explants maintained under control conditions, as well as a statistically significant increase in apoptosis. These NT-3 effects were blocked by an NT-3 antibody. In contrast, NT-3 had no effect on the length of neurites. These findings suggest that NT-3 may play a role during early neural development in vivo.     

Oestrogen regulates sympathetic neurite outgrowth by modulating brain derived neurotrophic factor synthesis and release by the rodent uterus

Sympathetic innervation of the adult rodent uterus undergoes cyclic remodelling. Terminal sympathetic axons degenerate when oestrogen levels rise and regenerate when oestrogen levels decline. This study examined the role of neurotrophins in oestrogen-mediated uterine sympathetic nerve remodelling. Oestrogen injection of ovariectomized female rats did not affect uterine NT-3 levels 24 h postinjection, and increased endometrial NGF protein, indicating that reduced NGF or NT-3 is not responsible for the oestrogen-induced denervation. Oestrogen also raised BDNF protein and mRNA in myometrium and endometrium. To assess whether increased BDNF affects uterine receptivity to sympathetic outgrowth, sympathetic ganglion explants were co-cultured with myometrium. Myometrium from ovariectomized rats induced neuritogenesis in oestrogen-free conditions, and this was abolished when BDNF was added to the medium. Neuritogenesis induced by ovariectomized myometrium was suppressed by oestrogen, and restored by a BDNF function-blocking antibody. To determine if target BDNF synthesis is required for oestrogen to suppress sympathetic neurite outgrowth, uteri from wild-type mice and mice homozygous or heterozygous for recombinant mutations of the BDNF gene were cultured with rat sympathetic ganglia. Neuritogenesis induced by wild-type uteri was diminished by oestrogen. Neurite formation in the presence of homozygous BDNF mutant uteri was not affected by oestrogen, but was lower than that of wild-type mice. Uteri from mice heterozygous for the BDNF mutation, who have reduced BDNF synthesis, showed normal neuritogenic properties, but were not affected by oestrogen. These findings suggest that oestrogen alters neuritogenic properties of the rodent uterus by regulating BDNF synthesis, which inhibits sympathetic neurite outgrowth.