Transduction sites of vagal mechanoreceptors in the guinea pig esophagus.

Extrinsic afferent neurons play an essential role in both sensation and reflex control of visceral organs, but their specialized morphological peripheral endings have never been functionally identified. Extracellular recordings were made from fine nerve trunks running between the vagus nerve and esophagus of the guinea pig. Mechanoreceptors, which responded to esophageal distension, fired spontaneously, had low thresholds to circumferential stretch, and were slowly adapting. Calibrated von Frey hairs (0.12 mN) were used to probe the serosal surface at 100-200 sites, which were mapped on a video image of the live preparation. Each stretch-sensitive unit had one to three highly localized receptive fields ("hot spots"), which were marked with Indian ink applied on the tip of the von Frey hair. Recorded nerve trunks were then filled anterogradely, using biotinamide in an artificial intracellular solution. Receptive fields were consistently associated with intraganglionic laminar endings (IGLEs) in myenteric ganglia, but not with other filled neuronal structures. The average distance of receptive fields to IGLEs was 73 +/- 14 microm (24 receptive fields, from 12 units; n = 5), compared to 374 +/- 17 microm for 240 randomly generated sites (n = 5; p < 0.001). After maintained probing on a single receptive field, spontaneous discharge of units was inhibited, as were responses to distension. During adapted discharge to maintained distension, interspike intervals were distributed in a narrow range. This indicates that multiple receptive fields interact to encode mechanical distortion in a graded manner. IGLEs are specialized transduction sites of mechanosensitive vagal afferent neurons in the guinea pig esophagus.     

Changes in NT-3 and TrkC in the primary visual cortex of developing macaques

In the present study, we measured the levels of neurotrophin-3 (NT-3) protein and mRNA by ELISA and quantitative RT-PCR, and examined TrkC-immunoreactive structures using immunohistochemical method. In the nervous system of the adult monkey, higher levels of NT-3 were found in the hippocampus and cerebellum. During the development of the primary visual cortex, detected amounts of NT-3 protein peaked at embryonic day 140 and then gradually decreased. TrkC imunoreactivity was observed in the neurons in layer VI of the primary visual cortex of an embryonic monkey. These results support the hypothesis that NT-3 is involved in the specification of axon targeting from layer VI to layer IV during the late embryonic stages.     

Mice lacking NT-3, and its receptor TrkC, exhibit profound deficiencies in CNS glial cells.

Neurotrophin-3 (NT-3) and its receptor TrkC are known to be important for neuronal survival. More recently, NT-3 has been implicated as playing a role in oligodendrocyte (OL) proliferation and survival in vitro. Examination of NT-3 and TrkC knockout mice revealed a reduction in NT-3-dependent neurons. To date, no study has examined alterations in glial cell populations in these knockout mice. In this report, we demonstrate a decline in OL progenitor cell numbers within the CNS of NT-3 and TrkC knockout mice. We also observed that immature and mature OL-specific markers were attenuated in the NT-3 and TrkC knockout animals. Deficiencies in other CNS glial cells, including astrocytes and ameboid microglia, were also observed. The subventricular zone (SVZ), a highly proliferative region for progenitor glial cells, was reduced in size. Furthermore, a nuclear-specific stain revealed a decline in the numbers of pyknotic nuclei in and around the SVZ of the knockout mice. These data will support an in vivo NT-3-dependent mechanism for the normal development of CNS glial cells.     

Adenovirus vector-directed expression of the neurotrophin-3 receptor (TrkC) in mouse astrocytes

In the present article we report the generation of a neurovirological reagent, an adenovirus vector that efficiently delivers the gene for the Neurotrophin-3 (NT-3) receptor, TrkC. Using this AdTrkC vector, we examined the induction of the expression of the above neurotrophin receptor in pure cultures of mouse astrocytes, a glial cell type that does not constitutively express this gene. Infection of astrocytes at an optimal dose of 100-200 plaque forming units (p.f.u.) per cell, induced expression of specific mRNA, as demonstrated by RT-PCR and Northern blot. This mRNA was translated to produce a mean of 20,157 biologically active receptor molecules per astrocyte with a Kd of 4.1 x 10(-11) M, as demonstrated by 125I-NT-3 binding. After 2D electrophoresis, the mature glycoprotein and some precursors were recognised by antibodies raised against the carboxy-terminal peptide of Trk. Binding of the ligand induced autophosphorylation ofTrkC and 3H-thymidine incorporation in transduced cells. These results demonstrate that our AdTrkC vector efficiently mediates the expression of high-levels of biologically active NT-3 receptors.     

Postnatal development of NT3 and TrkC in mouse ventral cochlear nucleus

In the developing nervous system, neurotrophin 3 (NT3) and brain‐derived neurotrophic factor (BDNF) have been shown to interact with each other and with different parts of a neuron or glia and over considerable distances in time and space. The auditory system provides a useful model for analyzing these events, insofar as it is subdivided into well‐defined groups of specific neuronal types that are readily related to each other at each stage of development. Previous work in our laboratory suggested that NT3 and its receptor TrkC in the mouse cochlear nucleus (CN) may be involved in directing neuronal migration and initial targeting of inputs from cochlear nerve axons in the embryo. NT3 is hard to detect soon after birth, but TrkC lingers longer. Here we found NT3 and TrkC around P8 and the peak around P30. Prominent in ventral CN, associated with globular bushy cells and stellate cells, they were localized to different subcellular sites. The TrkC immunostain was cytoplasmic, and that of NT3 was axonal and perisomatic. TrkC may be made by CN neurons, whereas NT3 has a cochlear origin. The temporal pattern of their development and the likelihood of activity‐dependent release of NT3 from cochlear axons suggest that it may not be critical in early synaptogenesis; it may provide long‐term trophic effects, including stabilization of synapses once established. Activity‐related regulation could coordinate the supply of NT3 with inner ear activity. This may require interaction with other neurotrophins, such as BDNF. © 2009 Wiley‐Liss, Inc.     

The origin and development of the vagal and spinal innervation of the external muscle of the mouse esophagus

Retrograde and anterograde tracing and immunohistochemical techniques were used to examine the origin of the extrinsic innervation, and the development of the vagal innervation to the mouse esophagus. Cholinergic nerve terminals were localised using an antiserum to the vesicular acetylcholine transporter and cholinergic cell bodies were localised using an antiserum to choline acetyltransferase. Cholinergic nerve terminals, which also contained calcitonin gene-related peptide, were present at the motor end plates in the external (striated) muscle of the esophagus. Following injection of Fast Blue into subdiaphragmatic or cervical levels of the esophagus, the only retrogradely-labelled cholinergic nerve cell bodies that also contained calcitonin gene-related peptide were found in the nucleus ambiguus. Neurons in the dorsal motor nucleus of the vagus, the nodose ganglia and dorsal root ganglia gave rise to a number of different types of nerve terminals within the myenteric plexus. Retrogradely-labelled neurons in the dorsal motor nucleus of vagus contained cholinergic markers only, nitric oxide synthase only or cholinergic markers plus nitric oxide synthase, retrogradely-labelled neurons in the dorsal root ganglia contained calcitonin gene-related peptide only, and a small number of retrogradely-labelled neurons in the nodose ganglia contained tyrosine hydroxylase. The development of the vagal innervation to the esophagus was examined following application of DiI to the vagus nerve of fixed mouse embryos. Anterogradely-labelled nerve fibres, which arose from both nodose ganglia and the medulla, were already present in the esophagus of embryonic day 12 (E12) mice. Some of the DiI-labelled vagal nerve fibres were present in among the smooth muscle cells of the external muscle layer prior to their transdifferentiation to striated muscle. We conclude that the neurons in the nucleus ambiguus that project to the esophagus differ from other extrinsic neurons in their chemistry as well as their targets within the esophagus. The development of the extrinsic innervation precedes the transdifferentiation of the external muscle to striated muscle, raising the possibility that, during development, smooth muscle of the esophagus is innervated transiently by vagal neurons.     

Distribution of BDNF, NT-3 and NT-4 in the developing auditory brainstem

This study describes the developmental expression of three neurotrophins, brain derived neurotrophic factor (BDNF), neurotrophin 3 (NT-3) and neurotrophin (NT-4) in the rat auditory brain-stem using immunohistochemistry. At postnatal day 0 (PND 0), neurotrophins expression was virtually absent from all auditory nuclei in the brainstem, even though some positive neurons were observed in the mesencephalic trigeminal nucleus at this age. However, BDNF, NT-3 and NT-4 positive neurons were observed in most brainstem auditory nuclei by PND 6. At the following stages, there was a general increase in the intensity of the neurotrophins immunoreactivity and BDNF labeling was particularly prominent in most cochlear nucleus neurons. A differential pattern of staining emerged in cochlear nucleus subdivisions, with more intense staining present in the ventral part. The superior olivary complex nuclei followed a similar pattern of BDNF staining compared to the cochlear nucleus. In the adult, BDNF heavily labeled most neurons of the superior olivary nuclei and moderately labeled neurons of the inferior colliculus (IC). NT-3 and NT-4 showed a similar pattern of staining in most auditory brainstem nuclei. The first staining was observed by PND 6 in some neuronal cell bodies. NT-3 and NT-4 immunoreactivity increased in the following stages and in the adult moderate labelings were observed in most neurons of the cochlear nucleus, the superior olivary nuclei and the IC. These results show that neurotrophins are expressed 1 week before the onset of hearing and the increase of their expressions correlate with the appearance of sound-evoked activity in the system. The temporal distribution of neurotrophins does not correlate with neuronal birth, axonal outgrowth or the formation of connection in the auditory structures, suggesting a role primarily in the maintenance and/ or modulation of postnatal and adult functions.     

Physiological and morphological plasticity induced by chronic treatment with NT-3 or NT-4/5 in hippocampal slice cultures

Expression of neurotrophins (NTs) and their receptors is elevated in the adult CNS under several neuropathological conditions. We have investigated the anatomical and electrophysiological consequences of chronic NT-3 or NT-4/5 treatment on established organotypic hippocampal slice cultures maintained in vitro for > 14 days. Both NT-3 and NT-4/5 increased spontaneous, action potential-dependent excitatory synaptic activity (sEPSCs), but only NT-3 increased inhibitory synaptic activity (sIPSCs) in CA3 pyramidal cells. Both NTs strongly promoted spontaneous synaptic bursting activity. Spontaneous bursts of EPSCs were observed after either NT treatment but only NT-3-treated cultures exhibited an increase in spontaneous bursts of IPSCs. In addition, sIPSC bursts were eliminated by blocking glutamatergic excitation. The frequency of miniature inhibitory postsynaptic currents, but not miniature excitatory postsynaptic currents, was also increased by both NT-3 and NT-4/5. Furthermore, NT-3 and NT-4/5 induced an up-regulation of the growth-associated protein GAP-43, suggesting that neurotrophins may be able to induce axonal reorganization in established neuronal networks. CA1 pyramidal cells exhibited slight alterations in dendritic branching after NT-4/5, but not NT-3 treatment. We conclude that chronic treatment with NT-3 or NT-4/5 can affect an established hippocampal network by elevating spontaneous inhibitory and excitatory synaptic activity and inducing coordinated pre- and postsynaptic structural changes.     

Neural crest stem cell and cardiac endothelium defects in the TrkC null mouse

TrkC null mice have multiple cardiac malformations. Since neural crest cells participate in cardiac outflow tract septation, the aim of this study was to determine at the cellular level the putative neural crest defect. We have identified three types of progenitor cells: stem cells that undergo self-renewal and can generate many cell types, cells that are restricted in their developmental potentials, and cells that are committed to the smooth muscle cell lineage. In TrkC null mice, there is a greater than 50% decrease in stem cell numbers and an equivalent increase in fate-restricted cells. The outflow tract wall is thickened and the endothelial tube is disorganized. We conclude that deletion of the TrkC gene causes precocious fate restrictions of the neural crest stem cell and a defect of the outflow tract endothelium, both of which may contribute to the outflow tract malformations that occur in TrkC null mice.     

Development of neuromuscular junctions in the mouse esophagus: morphology suggests a role for enteric coinnervation during maturation of vagal myoneural contacts

The time course of establishment of motor endplates and the subsequent developmental changes in their enteric and vagal innervation were examined in esophageal striated muscle of perinatal and adult C57/Bl6 mice by using immunocytochemistry and confocal laser scanning microscopy. Nicotinic acetylcholine receptors were visualized with alpha-bungarotoxin; vagal motor nerve terminals with antisera against vesicular acetylcholine transporter; and enteric nerve fibers with antisera against neuronal nitric oxide synthase, vasoactive intestinal peptide, and galanin. Because the various stages of esophageal striated myogenesis advance caudocranially, i.e., more mature stages are found cranial to immature stages, longitudinal cryosections through the esophagus were investigated. Synaptogenesis was divided into several distinct stages. 1) Mononucleated cells express acetylcholine receptors over their entire surface. 2) They start to cluster receptors without nerve fiber contacts. 3) The first nerve contact on a growing receptor cluster is made by a vagal nerve terminal, followed by an enteric terminal. 4) Vagal terminals grow until they match the size of endplate areas, and one to three enteric terminals intertwine with them on every receptor cluster. 5) After vagal terminals have covered the whole endplate area, enteric terminals are withdrawn from the majority of motor endplates. In a minority of endplates, enteric coinnervation persists through adulthood. The enteric innervation of all developing motor endplates, shortly after vagal terminals have contacted them, and the removal of enteric nerve fibers from the majority of mature motor endplates suggest a major role of enteric nerve fibers during maturation of esophageal neuromuscular junctions.     

Ultrastructural evidence for communication between intramuscular vagal mechanoreceptors and interstitial cells of Cajal in the rat fundus.

To assess whether afferent vagal intramuscular arrays (IMAs), putative gastrointestinal mechanoreceptors, form contacts with interstitial cells of Cajal of the intramuscular type (ICC-IM) and to describe any such contacts, electron microscopic analyses were performed on the external muscle layers of the fundus containing dextran-labelled diaminobenzidin (DAB)-stained IMAs. Special staining and embedding techniques were developed to preserve ultrastructural features. Within the muscle layers, IMA varicosities were observed in nerve bundles traversing major septa without contact with ICC-IM, contacting unlabelled neurites and glial cells. IMA varicosities were encountered in minor septa in contact with ICC-IM which were not necessarily in close contact with muscle cells. In addition, IMA varicosities were observed within muscle bundles in close contact with ICC-IM which were in gap junction contact with muscle cells. IMAs formed varicosities containing predominantly small agranular vesicles, occasionally large granular vesicles and prejunctional thickenings in apposition to ICC-IM processes, indicating communication between ICC and IMA via synapse-like contacts. Taken together, these different morphological features are consistent with a hypothesized mechanoreceptor role for IMA-ICC complexes. Intraganglionic laminar ending varicosities contacted neuronal somata and dendrites in the myenteric plexus of the fundus, but no contacts with ICC associated with Auerbach's plexus were encountered.     

人重组NT-3基因体外转染胚胎干细胞及其表达

目的 探讨神经营养素-3(NT-3)基因体外转染小鼠胚胎干细胞(ES)的可能性及其表达情况.方法 用脂质体介导的方法,将重组真核表达载体PCDNA-3.1(+).NT-3瞬时转染ES.用细胞免疫组化及RT-PCR检测转染细胞NT-3蛋白及mRNA表达.ELISA检测细胞分泌上清液中NT-3蛋白表达.结果 细胞免疫组化结果显示,转染NT-3基因的ES细胞胞浆呈红色染色;RT-PCR得到200 bp的基因片段;ELISA检测细胞分泌上清液中NT-3蛋白表达呈阳性,与对照组有显著差别(P＜0.05).结论 重组真核表达载体PCDNA-3.1(+).NT-3可成功转染ES,获得稳定表达NT-3的ES细胞株.     

Expression of PTPRO during mouse development suggests involvement in axonogenesis and differentiation of NT-3 and NGF-dependent neurons

Competition and cooperation between type II and type III receptor protein tyrosine phosphatases (RPTPs) regulate axon extension and pathfinding in Drosophila. The first step to investigate whether RPTPs influence axon growth in the more complex vertebrate nervous system is to identify which neurons express a particular RPTP. We studied the expression of mouse PTPRO, a type III RPTP with an extracellular region containing eight fibronectin type III domains, during embryogenesis and after birth. Mouse PTPRO mRNA is expressed exclusively in two cell types: neurons and kidney podocytes. Maximal expression in the brain was coincident with mid to late gestation and axonogenesis in the brain. We cloned two cDNAs, including a splice variant without sequence coding of 28 amino acids within the juxtamembrane domain that was found mostly in kidney. In situ hybridization detected mPTPRO mRNA in the cerebral cortex, olfactory bulb and nucleus, hippocampus, motor neurons, and the spinal cord midline. In addition, mPTPRO mRNA was found throughout dorsal root, cranial, and sympathetic ganglia and within kidney glomeruli. Mouse PTPRO mRNA was observed in neuron populations expressing TrkA, the high-affinity nerve growth factor receptor, or TrkC, the neurotrophin-3 receptor, and immunoreactive mPTPRO and TrkC colocalized in large dorsal root ganglia proprioceptive neurons. Our results suggest that mPTPRO is involved in the differentiation and axonogenesis of central and peripheral nervous system neurons, where it is in a position to modulate intracellular responses to neurotrophin-3 and/or nerve growth factor.     

Genetic tracing of Nav1.8-expressing vagal afferents in the mouse

Nav1.8 is a tetrodotoxin-resistant sodium channel present in large subsets of peripheral sensory neurons, including both spinal and vagal afferents. In spinal afferents, Nav1.8 plays a key role in signaling different types of pain. Little is known, however, about the exact identity and role of Nav1.8-expressing vagal neurons. Here we generated mice with restricted expression of tdTomato fluorescent protein in all Nav1.8-expressing afferent neurons. As a result, intense fluorescence was visible in the cell bodies, central relays, and sensory endings of these neurons, revealing the full extent of their innervation sites in thoracic and abdominal viscera. For instance, vagal and spinal Nav1.8-expressing endings were seen clearly within the gastrointestinal mucosa and myenteric plexus, respectively. In the gastrointestinal muscle wall, labeled endings included a small subset of vagal tension receptors but not any stretch receptors. We also examined the detailed innervation of key metabolic tissues such as liver and pancreas and evaluated the anatomical relationship of Nav1.8-expressing vagal afferents with select enteroendocrine cells (i.e., ghrelin, glucagon, GLP-1). Specifically, our data revealed the presence of Nav1.8-expressing vagal afferents in several metabolic tissues and varying degrees of proximity between Nav1.8-expressing mucosal afferents and enteroendocrine cells, including apparent neuroendocrine apposition. In summary, this study demonstrates the power and versatility of the Cre-LoxP technology to trace identified visceral afferents, and our data suggest a previously unrecognized role for Nav1.8-expressing vagal neurons in gastrointestinal functions.     

Neurotrophin-3 and TrkC in the frog visual system: changes after axotomy

Neurotrophins are potent regulators of the survival of different neuronal populations in the CNS. Little is known of the immunodistribution of neurotrophin-3 (NT-3) and tyrosine kinase C (TrkC) receptor in the frog visual system, which can successfully regenerate and recover vision after injury. In this study we show that both NT-3 and TrkC are present in the frog retina and tectum, and that their distribution changes after optic nerve transection. Both NT-3 and TrkC are present in the ganglion cell layer, inner nuclear layer, nerve fiber layer and outer plexiform layer, and in Müller cells of control retinas. Quantification of identified RGCs shows that there are only small changes in the proportion, or intensity, of NT-3 immunostained cells surviving after axotomy and regeneration. Müller cell staining, however, is increased. TrkC staining in the retina does not change after axotomy. In the tectum, NT-3 immunoreactivity is present in the retinorecipient layer 9, and in radial processes of neurons and ependymoglia. TrkC is present in ependymoglia and in tectal neurons. After axotomy or colchicine treatment fewer NT-3-immunoreactive processes are present in layer 9 and there is decreased staining of tectal neurons. These data are consistent with the hypothesis that NT-3 is synthesized in the retina and anterogradely transported to the tectum. TrkC immunostaining, on the other hand, increases in tectal cells after optic nerve transection, suggesting that it may be regulated by the supply of NT-3 from the retina.     

Voluntary exercise increases neurotrophin-3 and its receptor TrkC in the spinal cord

We have evaluated changes in the expression of neurotrophin-3 (NT-3) and its tyrosine kinase C (TrkC) receptor in the neuromuscular system as a result of voluntary physical activity. We assessed changes in the mRNAs and proteins for NT-3 and TrkC in the lumbar spinal cord and associated soleus muscle following 3 and 7 days of voluntary wheel running. We used quantitative Taqman RT-PCR to measure mRNA and ELISA to assess protein levels. NT-3 mRNA and protein levels increased in the spinal cord to reach statistical significance after 7 days of exercise compared to sedentary control rats. Immunohistochemical analyses localized the elevated NT-3 to the substantia gelatinosa (SG) and nucleus of the dorsal horn. TrkC mRNA levels were significantly elevated in the spinal cord after 3 and 7 days of running. In the soleus muscle, NT-3 mRNA levels and its receptor TrkC were elevated after 3 days, while NT-3 protein levels remained unaffected. The results demonstrate that voluntary exercise has a differential effect on NT-3 as well as its receptor TrkC in the neural and muscular components of the neuromuscular system, and emphasize the role of voluntary activity on the spinal cord and muscle.     

Phosphatidylinositol kinase enzymes regulate the retrograde axonal transport of NT-3 and NT-4 in sympathetic and sensory neurons

Phosphatidylinositol 3-kinase (PI3-kinase) and phosphatidylinositol 4-kinase (PI4-kinase) enzymes are an important family of signaling molecules that have been implicated in the regulation of intracellular vesicle trafficking. It has previously been shown that PI3-kinase and PI4-kinase enzymes regulate neuronal survival and the retrograde axonal transport of nerve growth factor in sympathetic and sensory neurons. We have extended these studies to examine the role these enzymes play in the regulation of the retrograde axonal transport of neurotrophin-3 (NT-3) and neurotrophin-4 (NT-4) in sympathetic and sensory neurons in vivo. Wortmannin (0.1 nmol/eye), a PI3-kinase and PI4-kinase antagonist, reduced the amount of (125)I-NT-3 retrograde transport in sympathetic neurons by approximately 50% and (125)I-NT-4 in sympathetic neurons by approximately 40% and sensory neurons by approximately 20%. The PI3-kinase antagonist LY294002 (100 nmol/eye) reduced the retrograde axonal transport of (125)I-NT-4 in sympathetic and sensory neurons, and (125)I-NT-3 in sympathetic neurons. Phenylarsine oxide (PAO), a PI4-kinase antagonist, significantly inhibited (125)I-NT-4 retrograde axonal transport in sympathetic and sensory neurons. These results show that wortmannin-sensitive PI3-kinases and PI4-kinases may be involved in NT-3 and NT-4 retrograde axonal transport. The retrograde axonal transport of neurotrophic factors in sympathetic and sensory neurons in vivo appears to depend upon the activation of different receptors and second messenger cascades at the nerve terminal.