Local neurotrophin effects on central trigeminal axon growth patterns

In dissociated cell and wholemount explant cultures of the embryonic trigeminal pathway NGF promotes exuberant elongation of trigeminal ganglion (TG) axons, whereas NT-3 leads to precocious arborization [J. Comp. Neurol. 425 (2000) 202]. In the present study, we investigated the axonal effects of local applications of NGF and NT-3. We placed small sepharose beads loaded with either NGF or NT-3 along the lateral edge of the central trigeminal tract in TG-brainstem intact wholemount explant cultures prepared from embryonic day 15 rats. Labeling of the TG with carbocyanine dye, DiI, revealed that NGF induces local defasciculation and diversion of trigeminal axons. Numerous axons leave the tract, grow towards the bead and engulf it, while some axons grow away from the neurotrophin source. NT-3, on the other hand, induced localized interstitial branching and formation of neuritic tangles in the vicinity of the neurotrophin source. Double immunocytochemistry showed that axons responding to NGF were predominantly TrkA-positive, whereas both TrkA and TrkC-positive axons responded to NT-3. Our results indicate that localized neurotrophin sources along the routes of embryonic sensory axons in the central nervous system, far away from their parent cell bodies, can alter restricted axonal pathways and induce elongation, arborization responses.     

Quantitative effects of NGF on the growth of embryonic frog axons

Sparse, dissociated cultures of embryonic Xenopus CNS neurons were grown with and without NGF. Under both conditions the same number of neurons survived and extended neurites, and under both conditions the neurites moved at approximately the same overall rates and with the same degree of straightness. On the other hand, neurons in the NGF-supplemented cultures had more neurites and these neurites branched 64% more often. Detailed measurements showed that the axons elongated 44% faster in NGF and that this increase could be ascribed to a selective increase in the stepping rate of axonal elongation. These observations raise the possibility that NGF may selectively modulate the rate of movement of the core cytoskeleton of the axon.     

Dynamic patterns of BDNF expression in injured sensory neurons: differential modulation by NGF and NT-3

It has been suggested that altered retrograde neurotrophin support contributes to the phenotypic switch observed in BDNF expression in injured sensory neurons. Thus, modulatory influences of NGF and NT-3 on BDNF expression in injured adult rat DRG neurons were examined using in situ hybridization and immunohistochemical approaches. Quantitative analysis reveals a biphasic response to sciatic nerve injury, whereby in the first day following injury, BDNF expression is up-regulated in approximately 83% of injured neurons including all small neurons, and a larger pool of trkB expressing neurons than in intact. By 1 week and up to 3 weeks later expression is still seen in approximately 66% of injured neurons, but the characteristic phenotypic switch in the subpopulations expressing BDNF occurs, whereby expression in the trkA population is reduced and expression in trkB- and in trkC-positive neurons is elevated. NGF infusion results in elevated levels of BDNF expression in both intact and injured trkA-positive neurons, accompanied by reduced trkB expression. NT-3 acts in an opposite fashion effecting a down-regulation in BDNF expression in intact neurons and preventing/reducing the injury-associated increases in BDNF expression in both trkC- and nontrkC-expressing subpopulations of injured neurons. These effects suggest NGF can regulate BDNF expression in trkA-expressing neurons regardless of the axonal state and that elevated levels of BDNF may contribute to the down-regulation in trkB expression associated with these states. Furthermore, the findings demonstrate that NT-3 can act in an antagonistic fashion to NGF in the regulation of BDNF expression in intact neurons, and mitigate BDNF's expression in injured neurons.     

The effects of nerve growth factor (NGF) and antiserum to NGF on the development of embryonic sympathetic neurons in vivo

The role of nerve growth factor (NGF) in the development of embryonic sympathetic neurons was examined in vivo. Individual mouse embryos received transuterine injections of NGF or antiserum to NGF (anti-NGF), and the effects on the superior cervical ganglion (SCG) were studied. Treatment with NGF at any gestational stage, from the time of ganglion aggregation to birth, increased ganglion tyrosine hydroxylase (T-OH) activity. Both the number of catecholaminergic neurons and T-OH activity per neutron were increased. Choline acetyltransferase (ChAc) activity was increased by NGF at early gestational stages, but not at later stages. These observations suggest that perikarya containing ChAc are responsive to NGF, whereas preganglionic nerve terminals are not. Treatment with anti-NGF rapidly and permanently decreased ganglion T-OH activity. The effects of anti-NGF were more pronounced at later gestational stages, suggesting that ganglia become increasingly dependent on NGF during development. Alteration of maternal levels of NGF had no effect on development of the embryonic SCG, suggesting that local embryonic concentrations of NGF are responsible for modulating sympathetic ontogeny.     

Differential effects of TrkC isoforms on sensory axon outgrowth

Neurotrophins are powerful regulators of neuronal morphology. Several lines of evidence are consistent with the idea that characteristic axonal and dendritic morphologies throughout the nervous system may be determined by local patterns of neurotrophin and neurotrophin receptor expression. Neurotrophin receptor tryosine kinases (Trks) exist in both tyrosine-containing (TK+) and tyrosine-lacking (TK-) isoforms, both of which are expressed in many neuronal populations. However, ratios of TK+ to TK- isoforms may vary at different stages of development and may be differentially distributed to cellular compartments. To test whether these isoforms have different functions related to axon outgrowth, full-length or tyrosine kinase-lacking TrkC receptors were overexpressed in embryonic dorsal root ganglion neurons maintained in explant cultures in neurotrophin-3 (NT-3)-containing media. Neurons were transfected with plasmid DNA encoding enhanced yellow fluorescent protein (EYFP) and TrkC receptor isoforms by particle-mediated gene transfer. Control neurons possessed 3.7 +/- 1.3 primary processes and 113.8 +/- 46 branch points. About 80% of the branches were located along the distal part of the axon. Transfection with the trkC TK+ increased the number of primary processes (6.5 +/- 2.8), whereas transfection with trkC TK- reduced the formation of primary processes (3.0 +/- 1.3). Surprisingly, the distribution of branch points was shifted to the proximal region of axons in neurons transfected with trkC TK-. These observations are consistent with the idea that differential expression of Trk isoforms during development may sculpt axonal morphology.     

NGF and NT-3 exert differential effects on the expression of neuropeptides in the suprachiasmatic nucleus of rats withdrawn from ethanol treatment

Some neurotrophins have the capability of enhancing neuropeptide expression in several regions of the brain. It was also recently shown that NGF, infused over 1 month, offsets the decreased synthesis and expression of vasopressin (VP) and vasoactive intestinal polypeptide (VIP) in the suprachiasmatic nucleus (SCN) of rats submitted to chronic ethanol treatment and withdrawal. In the present study we examined the effectiveness of neutrotrophin-3 (NT-3) in promoting such effects, given that SCN neurons express both the high and the low affinity receptors for this neurotrophin. NT-3 was intraventricularly infused during 10 days to rats withdrawn from prolonged ethanol treatment. The total number, and the mean somatic volume, of VP- and VIP-immunoreactive neurons was compared with the estimates obtained from control rats and withdrawn rats treated with either NGF or cerebrospinal fluid during the same period. The infusion of cerebrospinal fluid and of NT-3 did not prevent the reduction in the number of peptide-producing neurons induced by withdrawal from ethanol treatment. Conversely, NGF infusion increased their number to control levels and led to neuronal hypertrophy. Our results show that, unlike NGF, NT-3 does not display the capacity of enhancing neuropeptide expression in the SCN. Because SCN neurons express the low affinity p75(NTR), which is equally activated by both neurotrophins, our results additionally indicate that the effects of NGF upon SCN neurons are not receptor-mediated. Taken together, our data suggest that indirect mechanisms, rather than direct neutrophin signaling, are likely to mediate the trophic effects exerted by NGF upon SCN neurons.     

Differential effects of NT-4, NGF and BDNF on development of neurochemical architecture and cell size regulation in rat visual cortex during the critical period

Development of inhibition is a crucial determinant of the time course of visual cortical plasticity. BDNF strongly affects interneuron development and the onset and closure of the critical period for ocular dominance plasticity. Less is known on the effects of NT-4 despite a clear involvement in ocular dominance plasticity. We have investigated the effects of NT-4 on interneuron development by supplying NT-4 with osmotic minipumps during two time windows overlapping the onset (P12-20) and the peak (P20-28) of the critical period. We assessed the expression of interneuronal markers and soma size maturation either after the end of the infusion periods or at the end of the critical period (P45). We found that NT-4 was very effective in regulating interneuron development. NPY, SOM and PARV neuron somata grew faster during both infusion periods whereas CR neurons only responded during the early infusion period. The effects of soma size elicited during the earlier infusion period were still present at P45. In PARV neurons, NT-4 caused a long-lasting stabilization of CB and NPY expression. Furthermore, NT-4 accelerated the expression of GAD-65 mRNA in a subset of non-PARV neurons of layer V, which normally up-regulate GAD-65 towards the end of the critical period. Most of these effects were shared by NT-4 and BDNF. Some were unexpectedly also shared by NGF, which promoted growth of layer V PARV neurons, stabilized the CB expression and accelerated the GAD-65 expression. The results suggest that neurotrophins act on critical period plasticity by strengthening inhibition.     

NGF, BDNF and NT-5, but not NT-3 protect against MPP toxicity and oxidative stress in neonatal animals

A growing body of evidence suggests that neurotrophic factors can protect neurons against neuronal death. In the present study we examined whether systemic administration of members of the neurotrophin family, nerve growth factor (NGF), brain derived neurotrophic factor (BDNF), neurotrophin 3 (NT-3) and neurotrophin 5 (NT-5) and basic fibroblast growth factor (bFGF) could protect against 1-methyl-4-phenylpyridinium (MPP⁺) induced striatal damage in neonatal rats. Systemic administration of NGF, BDNF and NT-5 produced significant neuroprotective effects, whereas NT-3 was ineffective. Systemic administration of bFGF had significant neuroprotective effects as assessed by T₂-weighted magnetic resonance imaging and measurements of n-acetylaspartate and lactate using chemical shift magnetic resonance imaging. Systemic administration of NGF, BDNF and bFGF, but not NT-3 attenuated MPP⁺ induced increases in hydroxyl radical generation as assessed by the conversion of salicylate to 2,3- or 2,5-dihydroxybenzoic acid (DHBA). These results show that systemic administration of several neurotrophins and bFGF can attenuate neuronal damage induced by chemical hypoxia in vivo by a mechanism which may involve attenuation of oxidative stress.     

人参皂甙对NGF引导的鼠胚脊髓神经节细胞轴突生长的影响

通过NGF引导下鼠胚脊神经节体外培养模型的建立,观察9种主要人参皂甙单体以对感觉神经元轴突生长的影响。植块法鼠胚脊神经节体外培养,确定轴突生长所需NGF的最低维持浓度,建立NGF引导下鼠胚脊神经节体外培养模型,研究9种主要人参皂甙单体（Rb1,Rb3,Rd,Re,Rf,Rg1,Rh1,Rh2）对脊神经节轴突生长的作用。结果提示人参皂甙Rg1,Rb1,Re,Rf,Rh1具有促进周围神经轴突生长作用。     

人参皂甙对NGF引导的鼠胚脊髓神经节细胞轴突生长的影响

通过NGF引导下鼠胚脊神经节体外培养模型的建立,观察9种主要人参皂甙单体对感觉神经元轴突生长的影响.植块法鼠胚脊神经节体外培养,确定轴突生长所需NGF的最低维持浓度,建立NGF引导下鼠胚脊神经节体外培养模型,研究9种主要人参皂甙单体(Rb1,Rb3,Rd,Re,Rf,Rg1,Rg2,Rh1,Rh2)对脊神经节轴突生长的作用.结果提示人参皂甙Rg1,Rb1,Re,Rf,Rh1具有促进周围神经轴突生长作用.     

Differential effects of trigeminal tractotomy on Adelta- and C-fiber-mediated nociceptive responses

In this study we have tested in the rat, whether trigeminal tractotomy, which deprives the spinal trigeminal nucleus caudalis (Sp5C) of its trigeminal inputs, affected differentially nociceptive responses mediated by C- vs. Adelta-nociceptors from oral and perioral regions. Tractotomy had no effect on the threshold of the jaw opening reflex, induced by incisive pulp stimulation (Adelta-fiber-mediated), but blocked the formalin response (mainly C-fiber-mediated). These results suggest that nociceptive responses mediated by trigeminal C-fibers completely depend on the integrity of the Sp5C, while intraoral sensations triggered Adelta-fibers (especially of dental origin) are primarily processed in the rostral part of the spinal trigeminal nucleus.     

Differential regulation of NPY mRNA expression in embryonic sympathetic and chromaffin cultures by NGF

RT-PCR analysis of NPY mRNA expression in chick embryonic sympathoadrenal cells in culture showed that NGF increases sympathetic but not adrenal NPY mRNA content. These results show that the previously reported differential effect at the protein level can also be detected at the mRNA level, suggesting a pre-translational point of regulation. The differential NGF effect in such closely related phenotypes is particularly relevant to studies of plasticity and differentiation.     

Differential effects of trigeminal tractotomy on Aδ- and C-fiber-mediated nociceptive responses

In this study we have tested in the rat, whether trigeminal tractotomy, which deprives the spinal trigeminal nucleus caudalis (Sp5C) of its trigeminal inputs, affected differentially nociceptive responses mediated by C- vs. Aδ-nociceptors from oral and perioral regions. Tractotomy had no effect on the threshold of the jaw opening reflex, induced by incisive pulp stimulation (Aδ-fiber-mediated), but blocked the formalin response (mainly C-fiber-mediated). These results suggest that nociceptive responses mediated by trigeminal C-fibers completely depend on the integrity of the Sp5C, while intraoral sensations triggered Aδ-fibers (especially of dental origin) are primarily processed in the rostral part of the spinal trigeminal nucleus.     

Monoamine-activated α2-macroglobulin inhibits choline acetyltransferase of embryonic basal forebrain neurons and reversal of the inhibition by NGF and BDNF but not NT-3

Monoamine-activated α₂-macroglobulin (α₂M) has recently been shown to inhibit the growth and survival of cholinergic neurons of the basal forebrain (Liebl and Koo: J Neurosci Res 35:170–182, 1993). The mechanism of this inhibitory effect is believed to involve the regulation of growth factor activities by α₂M. The objectives of this study are to determine whether monoamine-activated α₂M can inhibit choline acetyltransferase (ChAT) activity of cholinergic basal forebrain neurons, and whether some common neurotrophins in the CNS can reverse the inhibition. This study demonstrates that both methylamine-activated α₂M (MA-α₂M) and serotonin-activated α₂M (5HT-α₂M) can dose-dependently suppress the expression of normal basal levels of ChAT activity in embryonic rat basal forebrain cells in vitro, while normal α₂M has little or no effect. As little as 0.35 μM monoamine-activated α₂M can suppress the ChAT activity, whereas either nerve growth factor (NGF) or brain-derived neurotrophic factor (BDNF), but not neurotrophin-3 (NT-3), stimulates ChAT expression of these cells. The addition of either NGF or BDNF to the α₂M-suppressed cells can increase ChAT activity back to its normal levels, while NT-3 can not. These results demonstrate that (1) monoamine-activated α₂M is a potent non-cytotoxic inhibitor of the ChAT activity in cholinergic basal forebrain neurons, and (2) NGF and BDNF are capable of not only stimulating the ChAT activity but can also specifically reverse the α₂M inhibition. The potential physiological role of monoamine-activated α₂M and neurotrophins in the degeneration and regeneration of cholinergic neurons is discussed. In addition, we propose that α₂M may serve as an important tool for evaluating the roles of growth factors in the nervous system. © 1994 Wiley-Liss, Inc.     

Peripheral target-specific influences on embryonic neurite growth vigor and patterns

We examined axon–target interactions in cocultures of embryonic rat trigeminal, dorsal root, nodose, superior cervical ganglia or retina with a variety of native or foreign peripheral targets such as the whisker pad, forepaw, and heart explants. Axon growth into these peripheral target tissues was analyzed by the use of lipophilic tracer DiI. Embryonic day 15 dorsal root and trigeminal axons grew into isochronic normal and foreign cutaneous targets. Both axon populations avoided the same age heart tissue, but grew profusely into younger (embryonic day 13) or older (postnatal) heart explants. In contrast, embryonic day 15 superior cervical or nodose ganglion axons grew heavily into the same age heart and forepaw explants and to a lesser extent into the whisker pad explants. Embryonic day 15 retinal axons grew into all three peripheral targets used in this study. Primary sensory and sympathetic axons, but not retinal axons, formed target-specific patterns in the whisker pad and forepaw explants. DiI-labeling and immunostaining of primary sensory neurons in coculture revealed that these neurons retain their bipolar characteristics, and express class-specific markers such as parvalbumin, calcitonin gene-related peptide and TrkA receptors. In the whisker pad explants, axons positive for all three markers were seen to form patterns around the follicles. Our results indicate that developing peripheral targets can attract and support axon growth from a variety of sources. Whereas neurotrophins play a major role in attracting and supporting survival of subpopulations of sensory neurons, other substrate-bound or locally released molecules must regulate sensory neurite growth into specific peripheral and central targets. J. Comp. Neurol. 399:427–439, 1998. © 1998 Wiley-Liss, Inc.     

N-cadherin regulates primary motor axon growth and branching during zebrafish embryonic development

N-cadherin is a classical type I cadherin that contributes to the formation of neural circuits by regulating growth cone migration and the formation of synaptic contacts. This study analyzed the role of N-cadherin in primary motor axons growth during development of the zebrafish (Danio rerio) embryo. After exiting the spinal cord, primary motor axons migrate ventrally through a common pathway and form the first neuromuscular junction with the muscle pioneer cells located at the horizontal myoseptum, which serves as a choice point for cell-type-specific pathway selection. Analysis of N-cadherin mutants (cdh2(hi3644Tg) ) and embryos injected with N-cadherin antisense morpholinos showed primary motor axons extending aberrant axonal branches at the choice point in ～40% of the somitic hemisegments and an ～150% increase in the number of branches per axon length within the ventral myotome. Analysis of individual axons trajectories showed that the caudal (CaP) and rostral (RoP) motor neurons axons formed aberrant branches at the choice point that abnormally extended in the rostrocaudal axis and ventrally to the horizontal myoseptum. Expression of a dominant-interfering N-cadherin cytoplasmic domain in primary motor neurons caused some axons to stall abnormally at the horizontal myoseptum and to impair their migration into the ventral myotome. However, in N-cadherin-depleted embryos, the majority of primary motor axons innervated their appropriate myotomal territories, indicating that N-cadherin regulates motor axon growth and branching without severely affecting the mechanisms that control axonal target selection.     

The Neurotrophins BDNF, NT-3 and NT-4/5, But Not NGF, Up-regulate the Cholinergic Phenotype of Developing Motor Neurons

Although developing motor neurons express low-affinity nerve growth factor (NGF) receptors, there is no known biological effect of NGF on developing or adult motor neurons. In this study, we found that, unlike NGF, brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3) and neurotrophin-4/5 (NT-4/5) stimulated cholinergic phenotype by increasing choline acetyltransferase (CAT) activity in cultures enriched with embryonic rat motor neurons. Ciliary neurotrophic factor (CNTF) also stimulated CAT activity. The effects of BDNF and NT-4/5 on CAT activity appeared to be synergistic with that of CNTF. Cotreatment with BDNF and NT-3 resulted in an additive effect, suggesting that signal transduction was mediated through different high-affinity receptors tyrosine kinases B and C (Trk B and Trk C). However, cotreatment with BDNF and NT-4/5 did not result in an increase in CAT activity greater than that of either BDNF or NT-4/5 alone, suggesting that their effects were mediated via the same receptor Trk B. Supporting our findings that spinal cholinergic neurons are responsive to trophic actions of members of the neurotrophin family, motor neuron-enriched cultures were found to express mRNA for Trk B and Trk C, which have been identified as high-affinity receptors for BDNF and NT-4/5, and NT-3, respectively.     

Synergistic effects of BDNF and NT-3 on postnatal spiral ganglion neurons

Neurotrophins have profound effects on the development and maintenance of neurons that compose the VIIIth cranial nerve. In the auditory division of the nerve, brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) have been localized to the sensory epithelium, and their respective high-affinity tyrosine kinase receptors (TrkB and TrkC) are expressed within the neuronal population. By using a culture methodology that allows evaluation of single neurons, we determined that BDNF and neurotrophin-4 (NT-4), which both bind to the TrkB high-affinity receptor, greatly enhanced neuron survival above control cultures. NT-3, which acts via the TrkC high-affinity receptor, also increased survival, but to a lesser extent. By testing a variety of neurotrophin concentrations and combinations, we observed that simultaneous activation of the TrkB and TrkC receptors synergistically promoted neuron survival compared to cultures that contained either neurotrophin alone at the same total concentration. Antibody labeling showed that the high-affinity Trk receptors were localized predominantly to the neurons and not to the surrounding satellite cells; furthermore, TrkB- and TrkC-specific antibodies each labeled 100% of the cultured neurons. These results suggest that synergistic interactions between BDNF and NT-3 may be crucial for spiral ganglion neuron survival during the final stages of development. J. Comp. Neurol. 386:529–539, 1997. © 1997 Wiley-Liss, Inc.     

BDNF,NT-3对体外培养的胚鼠脊髓AChE和NADPH-d阳性神经元生长发育的影响

利用AChE和NADPH-d酶组织化学染色法研究了脑源性神经营养因子(brain-derived neurotrophic factor,BDNF)和神经营养因子-3(neurotrophin-3,NT-3)对离体培养的胚胎大鼠脊髓胆碱能神经元和一氧化氮能神经元生长发育的影响.结果显示:BDNF处理组和NT-3处理组AChE阳性神经元数和NADPH-d阳性神经元数均显著高于对照组(P＜0.05).BDNF组AChE阳性神经元和NADPH-d阳性神经元胞体平均直径、每细胞突起数和最长突起长度均显著高于对照组(P＜0.05).NT-3组NADPH-d阳性神经元的生长发育与对照组无明显差异,仅AChE阳性神经元的每细胞突起数和最长突起长度显著高于对照组(P＜0.05),对胞体发育无影响.结果提示:BDNF,NT-3促进脊髓神经元的存活和生长发育,二者的作用具有选择性和特异性.