Substance P-like immunoreactivity in cultured spinal ganglia from chick embryos

Summary The localization of substance P (SP) or a SP-like peptide in cultured spinal ganglia from chick embryos was studied by the indirect immunofluorescence technique. Ganglia from 8–16 days old chick embryos and from newly hatched chickens were cultured in a control medium or in the presence of nerve growth factor (NGF). Addition of colchicine and exposure to different explanted peripheral tissues were also tried. Ganglia from the younger embryos (8–12 days) cultured for 24 h with added NGF showed a weak SP-like immunoreactivity (SPLI) in some cell bodies and strong specific immunofluorescence in nerve fibres growing out from the ganglia. In spinal ganglia of the older embryos (14 and 16 days) and newly hatched chickens cultured with and without NGF the concentration of SPLI in the cell bodies was considerably higher. Addition of colchicine to spinal ganglia cultured 12 h in NGF-medium, resulted in retraction of nerve fibres and strongly fluorescent, expanded nerve fibres were observed in peripheral parts of the ganglia. Explants of skin placed near the spinal ganglia stimulated the outgrowth of fibres, some of them containing SPLI. A few fluorescent fibres were also seen within the skin explants. Also heart tissue explants stimulated outgrowth of nerve fibres, but innervation of these explants with SPLI-containing nerves could not be observed. Nerve fibre-extension from the spinal ganglia was not stimulated by spinal cord explants. The present results support the existence of SP-containing primary sensory neurons in chickens.     

Neural crest-derived proprioceptive neurons express nerve growth factor receptors but are not supported by nerve growth factor in culture

The neural crest-derived, first-order, sensory neurons of the embryonic chick trigeminal mesencephalic nucleus were grown in dissociated, glia-free culture. Whereas brain-derived neurotrophic factor promoted the survival and growth of the majority of these neurons (over 70% after 48 h incubation), nerve growth factor had no effect on their survival. The percentage survival in cultures supplemented with nerve growth factor at concentrations ranging from 0.2 to 625 ng/ml was only 2%, the same percentage survival as in control cultures. Furthermore, nerve growth factor did not change the dose-response of these neurons to brain-derived neurotrophic factor. Although nerve growth factor did not influence the survival of trigeminal mesencephalic neurons in culture, nerve growth factor specifically bound to the great majority of neurons growing in the presence of brain-derived neurotrophic factor. Autoradiographs of cultures incubated with iodinated nerve growth factor showed that the perikarya and processes of neurons were heavily labelled with silver grains. These findings demonstrate the existence of a population of neural crest-derived sensory neurons which express nerve growth factor receptors but are not supported by nerve growth factor in culture.     

The chick embryo nodose ganglion: Effects of nerve growth factor in culture

Summary Nodose ganglia from 8-day-old chick embryos were cultured in collagen gels for 2 days, with or without added nerve growth factor (NGF), in order to discover whether the nodose neurons, derived from an epidermal placode, are susceptible to this trophic factor. Neuronal survival and neurite outgrowth were stimulated only after addition of NGF, and the enhancing effects could be blocked by introducing antibodies to NGF. Stereological analysis of ganglia sectioned for light microscopy showed that the NGF-treated neurons increased their volume by about 50%, as did the nodose neurons in ovo from the eighth to the tenth day of incubation. The volume density, however, was significantly lowerin vitro indicating a limited cell death during culture despite the presence of exogenously supplied NGF. The number of neurofilaments and microtubules increased in the cell centre of neurons treated with NGF; this region also showed numerous dense bodies and an extensive Golgi complex by electron microscopy. Ultrastructural similarities between neurons responding to NGF and neurons undergoing the axon reaction which follows axotomy are indicated. A role for a trophic factor resembling NGF in the normal development of placode-derived neurons of the sensory cranial ganglia is suggested.     

The effect of nerve growth factor on developing primary sensory neurons of the trigeminal nerve in chick embryos

Summary The response to nerve growth factor (NGF) of two sensory neuron populations of the trigeminal nerve was studied in chick embryos. NGF promoted neuronal survival and cellular hypertrophy in the Gasserian ganglia with minimal effect on the neuron population of the mesencephalic trigeminal nucleus. NGF induced prolific neurite outgrowth from cultured Gasserian ganglia, in contrast, cultured mesencephalic trigeminal neurons remained refractory to NGF treatment.The apparent lack of response of mesencephalic trigeminal neurons to NGF may be explained either by their derivation from placodal material rather than from the neural crest, or their lost sensitivity to NGF due to interaction with the local environment in the central nervous system.     

Developmentally regulated induction of neurite outgrowth from immature chick sensory neurons (DRG) by homogenates of avian or mammalian heart, liver and brain

Neurite outgrowth is elicited from whole explants or dissociated neurons of 8--10-day-old chick embryo sensory, dorsal root ganglia when cultured in the presence of a high speed supernatant fraction (105,000 g) from homogenates of chick or rat heart, liver or brain. The neurite promoting activity is not identical to mouse nerve growth factor (NGF) and is non-dialyzable. Expression of this neurogenic factor would appear to be developmentally regulated as its activity is barely detectable in organs from 6--9-day-old embryos but specific activity rises dramatically in homogenates of organs from embryos of greater than 11 days incubation. Greatest activity is found in chick heart and rat brain with only trace levels in lung or kidney and none in spinal cord.     

Peripheral nerve regeneration and neurotrophic factors

The role of neurotrophic factors in the maintenance and survival of peripheral neuronal cells has been the subject of numerous studies. Administration of exogenous neurotrophic factors after nerve injury has been shown to mimic the effect of target organ-derived trophic factors on neuronal cells. After axotomy and during peripheral nerve regeneration, the neurotrophins NGF, NT-3 and BDNF show a well defined and selective beneficial effect on the survival and phenotypic expression of primary sensory neurons in dorsal root ganglia and of motoneurons in spinal cord. Other neurotrophic factors such as CNTF, GDNF and LIF also exert a variety of actions on neuronal cells, which appear to overlap and complement those of the neurotrophins. In addition, there is an indirect contribution of GGF to nerve regeneration. GGF is produced by neurons and stimulates proliferation of Schwann cells, underlining the close interaction between neuronal and glial cells during peripheral nerve regeneration. Different possibilities have been investigated for the delivery of growth factors to the injured neurons, in search of a suitable system for clinical applications. The studies reviewed in this article show the therapeutic potential of neurotrophic factors for the treatment of peripheral nerve injury and for neuropathies.     

Injured primary sensory neurons switch phenotype for brain-derived neurotrophic factor in the rat.

Peripheral nerve injury results in plastic changes in the dorsal root ganglia and spinal cord, and is often complicated with neuropathic pain. The mechanisms underlying these changes are not known. We have now investigated the expression of brain-derived neurotrophic factor in the dorsal root ganglia with histochemical and biochemical methods following sciatic nerve lesion in the rat. The percentage of neurons immunoreactive for brain-derived neurotrophic factor in the ipsilateral dorsal root ganglia was significantly increased as early as 24 h after the nerve lesion and the increase lasted for at least two weeks. The level of brain-derived neurotrophic factor messenger RNA was also significantly increased in the ipsibut not contralateral dorsal root ganglia. Both neurons and satellite cells in the lesioned dorsal root ganglia synthesized brain-derived neurotrophic factor messenger RNA after the nerve lesion. There was a dramatic shift in size distribution of positive neurons towards large sizes seven days after sciatic nerve lesion. Morphometric analysis and retrograde tracing studies showed that no injured neurons smaller than 600 microm2 were immunoreactive for brain-derived neurotrophic factor, whereas the majority of large injured neurons were immunoreactive in the ipsilateral dorsal root ganglia seven days postlesion. The brain-derived neurotrophic factor-immunoreactive nerve terminals in the ipsilateral spinal cord were reduced in the central region of lamina II, but increased in more medial regions or deeper into laminae III/IV. These studies indicate that sciatic nerve injury results in a differential regulation of brain-derived neurotrophic factor in different subpopulations of sensory neurons in the dorsal root ganglia. Small neurons switched off their normal synthesis of brain-derived neurotrophic factor, whereas larger ones switched to a brain-derived neurotrophic factor phenotype. The phenotypic switch may have functional implications in neuronal plasticity and generation of neuropathic pain after nerve injury.     

Pharmacological effects of nerve growth factor and fibroblast growth factor applied to the transectioned sciatic nerve on neuron death in adult rat dorsal root ganglia

Sciatic nerve transection performed on adult rats caused neuronal losses after 4 weeks in the L4-6 dorsal root ganglia (DRG) of 35% as compared to the unlesioned side. Nerve growth factor (NGF) administered at a single dose of 6000 Biological Units in silicone tubes fixed to the proximal nerve stump completely prevented these cell losses. Basic fibroblast growth factor (bFGF) also protected DRG neurons, but at the concentrations applied (6000 Trophic Units, tested on embryonic chick ciliary ganglion neurons), failed to maintain cell numbers identical to unoperated side. Our data indicate that NGF and bFGF protect adult sensory neurons from lesion-induced death. Preliminary results suggest that local accumulation of neurotrophic activities at the proximal nerve stump elicited by NGF and, possibly bFGF, might be involved in the beneficial effects of these proteins on the maintenance of axotomized sensory neurons.     

Downregulation of TrkA expression in primary sensory neurons after unilateral lumbar spinal nerve transection and some rescuing effects of nerve growth factor infusion

Peripheral nerve injury results in sprouting of sympathetic and sensory nerve terminals around large diameter neurons in the dorsal root ganglia (DRG), but the underlying mechanism is not clear. Current study sought to examine changes of the nerve growth factor (NGF) receptor TrkA in DRG and spinal cord after a spinal nerve transection by an immunohistochemical technique and to investigate effects of NGF on the expression of TrkA protein in the same animal model. In the control rat, TrkA immunoreactivity was localized to about 55 +/ -1% of total neurons in DRG and to laminae I and II of the spinal cord. The percentage of TrkA immunoreactive neurons in DRG and TrkA staining intensity of spinal cord were reduced 1 week after the nerve lesion. The changes became maximal 2 weeks, but recovered partially 4 weeks after the lesion. The size of TrkA immunoreactive neurons dramatically shifted to smaller sizes, becoming more remarkable 4 weeks after the lesion. In the contralateral DRG, the percentage of TrkA immunoreactive neurons also decreased significantly. Exogenous NGF delivered to DRG for 2 weeks partially reversed the reduction of TrkA expression as well as atrophy of TrkA immunoreactive neurons. No TrkA immunoreactive basket was found around neuronal somata. Our data show that unilateral peripheral nerve injury results in dynamic downregulation of TrkA in sensory neurons in bilateral DRG and spinal cord, and that TrkA expression in sensory neurons is partially regulated by target-derived NGF.     

鸡胚背根神经节感觉神经元体外培养研究

本文对背根神经节感觉神经元的体外培养条件进行了研究。结果表明利用差速贴壁法可使神经元纯度达到95％以上;在血清和适宜的细胞外基质存在条件下，神经元分散良好。本研究还通过外源性脑源性神经营养因子对神经元纤维再生的作用进一步证实了该方法的可靠性。     

Ultrastructural localization of brain-derived neurotrophic factor in rat primary sensory neurons

In a previous study we have shown that brain-derived neurotrophic factor (BDNF) is present in a subpopulation of small- to medium-sized sensory neurons in the dorsal root ganglia (DRG) and is anterogradely transported in both the peripheral and central processes. Within the spinal cord, BDNF is localized to varicosities of sensory nerve terminals in laminae I and II of the dorsal horn. This study raised the question of whether BDNF is localized in synaptic vesicles of the afferent nerve terminals. Using immunohistochemical and immunocytochemical techniques we have now investigated the ultrastructural localization of BDNF in the spinal cord of the rat. In addition, its colocalization with the low affinity neurotrophin receptor, p75, and calcitonin gene related peptide (CGRP) was also investigated. In lamina II of the spinal cord, BDNF immunoreactivity was restricted to nerve terminals. The reaction product appeared associated with dense-cored and clear vesicles of terminals superficial laminae. Double labelling experiments at the light microscopic level showed that 55% of BDNF immunoreactive neurons in DRG are colocalized with CGRP and many nerve terminals in laminae I and II of the spinal cord contained both BDNF and CGRP immunoreactivities. The results of double labelling at the ultrastructural level showed that most BDNF-ir (immunoreactive) nerve terminals contained CGRP or the low affinity neurotrophin receptor, p75, but not vice versa. These results point to the conclusion that BDNF may be released in parallel with neurotransmitters from nerve terminals in the spinal cord from a subpopulation of nociceptive primary afferents.     

Peripheral projections of the chick primary sensory neurons expressing gamma-aminobutyric acid immunoreactivity.

The expression of gamma-aminobutyric acid was studied in sensory neurons and peripheral target tissues of the chick dorsal root ganglia by combining immunocytochemistry and electron microscopy. In the chick embryos, the first immunoreaction was observed at embryonic day 12 in 1.4% of ganglion cell bodies. The intensity of immunostaining gradually increased during development and the percentage of immunostained neurons reached an average of 7.3% after hatching. These immunostained cell bodies could be identified as sensory neurons belonging either to some large neurons of the A1 subclass or to a few small neurons of the B1 subclass. The other neuronal cell bodies, corresponding to the A2 and B2 subclasses, as well as the satellite and glial cells were apparently devoid of any gamma-aminobutyric acid immunostaining. Among the peripheral tissues innervated by the primary sensory neurons, the nerve endings of Achilles' tendon and the paravertebral autonomic ganglia appeared devoid of immunoreactivity. In contrast, immunoreactivity was found within nerve endings located in some neuromuscular spindles of the skeletal muscles and within some Herbst's corpuscles in the subcutaneous tissue of the skin. Thus, the present results provide evidence that gamma-aminobutyric acid may be expressed by neuronal cell bodies belonging to two subclasses of primary sensory neurons and could be a putative neurotransmitter involved in the peripheral sensory innervation of, at least in part, skin and skeletal muscles.     

Ontogeny of galanin‐immunoreactive elements in chicken embryo autonomic nervous system

To elucidate the main ontogenetic steps of galanin immunoreactivity within the extrinsic nerve supply of the alimentary tract, we undertook an immunohistochemical study of chicken embryo specimens. Fluorescence and streptavidin‐biotin‐peroxidase protocols were combined, using a galanin polyclonal antiserum, on transverse serial sections obtained from chicken embryos from embryonic Day 3 (E3) to hatching, and from 9‐day‐old newborn chicks. Galanin‐immunoreactive cells were first detected at E3.5 within the pharyngeal pouch region, the nodose ganglion, the primary sympathetic chain, primitive splanchnic branches and the caudal portion of the Remak ganglion. At E5.5 galanin‐immunoreactive cells and fibers appeared in the secondary (paravertebral) sympathetic chain, splanchnic nerves, peri‐ and preaortic plexuses, adrenal gland anlage and visceral nerves. Galanin‐immunoreactive cells also lay scattered along the vagus nerve, and in the intermediate zone of the thoracolumbar spinal cord. At E18, galanin‐immunoreactive cells and fibers were found along the entire Remak ganglion and around the gastrointestinal blood vessels. In post‐hatching‐9‐day old chicks, the para‐ and prevertebral ganglia, but not the intermediate zone of the spinal cord, contained galanin‐immunoreactive cells. Data indicate the presence of a consistent “galaninergic” nerve system supplying the chick embryonal gut wall. Whether this system has growth or differentiating role remains to be demonstrated. Its presence and distribution pattern in the later stages clearly support its well known role as a visceral neuromodulator of gut function. Anat Rec 262:266–278, 2001. © 2001 Wiley‐Liss, Inc.     

Nerve growth factor treatment does not prevent dorsal root ganglion cell death induced by target removal in chick embryos

Summary In chick embryos, on the 3rd day of incubation, the developing right wing bud was removed. One group of the operated embryos was treated with a daily dose of 20 g purified nerve growth factor (NGF) from the 5th day of incubation and sacrificed on the 12th day. The other group was sacrificed on the 12th day of incubation and served as control. NGF was also administered to intact, unoperated embryos for comparison. The size of the dorsal root ganglia in segments 13–16 innervating the wings, were estimated and the number of surviving dorsal root ganglion cells counted both on the right (operated) and left (intact) sides. Although NGF brought about an increase in the size of the ganglia and an increase in the number of dorsal root ganglion cells bilaterally, it was not able to prevent excessive cell death of dorsal root ganglion cells on the operated side. The number of surviving neurons in the dorsal root ganglia on the operated side in embryos with or without NGF administration was only about 30–50% of the number of the intact side.These results show that cell death induced by target removal cannot be offset by NGF administration. It is concluded that NGF may act as a growth promoting agent for developing sensory neurons but other peripheral trophic factor/s are also needed for the maintenance and survival of dorsal root ganglion cells.     

Cholinergic properties of embryonic chick sensory neurons

Experiments were carried out to determine the cholinergic properties of sensory neurons of the chick embryo by measuring the choline acetyltransferase activity (ChAT) and [3H]choline uptake. The choline acetyltransferase activity in the dorsal root ganglia of an 8-day-old chick embryo was 24.2 +/- 2.52, which increased to 45.4 +/- 9.69 pmol ACh/mg protein/min in the ganglia of 12-day-old embryos. Sensory neurons derived from dorsal root ganglia of 10-day-old embryos and maintained in a serum-free culture medium supplemented with insulin, transferrin and nerve growth factor (NGF) also contained significant amounts of ChAT (21.9 pmol ACh/mg protein/min). Omission of NGF resulted in neuronal death, and the enzyme activity could not be measured in these cultures. A specific inhibitor of ChAT, hydroxyethyl naphthylvinyl pyridine (NVP), when added to the assay mix produced a dose-dependent inhibition of ChAT from cultured neurons. Cultured sensory neurons incubated with [3H]choline followed by repeated washouts took up and retained [3H]choline. The uptake of [3H]choline was reduced by about 45% when NaCl, in the incubation medium, was replaced by LiCl. A specific inhibitor of choline uptake, hemicholinium-3, caused about 75% inhibition of [3H]choline uptake. It is implied that sensory neurons of the chick dorsal root ganglia express cholinergic properties during development.     

Influence of peripheral and central targets on subpopulations of sensory neurons expressing calbindin immunoreactivity in the dorsal root ganglion of the chick embryo

The influence of central and peripheral target tissues on the expression of calbindin D-28k by sensory neurons of the chick dorsal root ganglia was tested under various experimental conditions. Firstly, dorsal root ganglia of chick embryos were transplanted at two stages of development onto the chorioallantoic membrane of a host embryo for a period of 4 or 8 days. In dorsal root ganglia grafted at E12, 20% of the ganglion cell bodies were immunoreactive to calbindin 4 and 8 days later; the percentage of calbindin-immunostained neurons in grafted dorsal root ganglia was similar to that observed in control dorsal root ganglia of the same embryonic age (E16 or 20). In contrast, when grafted dorsal root ganglia were taken from a donor embryo at E8, no calbindin-immunoreactive neuron was found 4 or 8 days later. However, when dorsal root ganglia at E8 were cotransplanted with musculature cells, 14% of the grafted ganglion cell bodies were again immunoreactive to calbindin 4 or 8 days later. Secondly, peripheral targets of sensory neurons were suppressed by excision of one hindlimb. After excision at E6, virtually all the ipsilateral dorsal root ganglia cells were free of calbindin immunoreaction after 6 days of reincubation. In contrast, when the excision was performed at E11, calbindin was expressed in about 9% of the nerve cell bodies. Thirdly, central connections were destroyed by cauterization of the lumbosacral spinal cord at E6 or E11. Six days after deprivation of central connections, the percentage of calbindin-immunoreactive ganglion cells was the same as in control dorsal root ganglia of the same age.(ABSTRACT TRUNCATED AT 250 WORDS)     

Accelerated regenerative neurite formation by a neuronal surface epitope reactive with the monoclonal antibody, Leu 7

A family of glycoproteins sharing an epitope with myelin associated glycoprotein as recognized by the monoclonal antibody Leu 7 (HNK-1) has been found to be present on neurons grown in culture from embryonic chicks and rats. Immunofluorescent staining demonstrates that, in vitro, 100% of the neurons from dorsal root ganglia and spinal cord from 7-8 day chick embryos react with Leu 7. Analysis of in vitro regenerative neurite formation by neurons on substrates enriched with Leu 7 showed accelerated regenerative process formation under limiting conditions. These results indicate that the Leu 7 epitope on neurons is appropriate for substrate adhesion and promotes rapid process extension.     

Trophic interactions between the cochleovestibular ganglion of the chick embryo and its synaptic targets in culture

The effect of the availability of synaptic targets on neuronal survival was tested by explanting the cochleovestibular ganglion from embryonic day 3-1/2 chick embryos and maintaining it in the presence or absence of appropriate synaptic target tissues for 14 days in culture. The targets were the inner ear, peripherally, and the myelencephalon, centrally. Light and electron microscopic observations showed that the ganglion cells in the explants with targets present had generally achieved a degree of differentiation comparable to that of their counterparts in embryonic day 14 embryos. The variety of cell types seen in the normal embryonic day 14 ganglia was also evident in vitro. In ganglia explanted without peripheral or central targets, few neurons survived. Ganglia explanted with either peripheral or central target intact showed considerably better survival than those explanted without any target. Ganglia explanted with only the peripheral target (the inner ear) survived equally as well as those with both central and peripheral targets. Ganglia cultured with the central target (myelencephalon) did not survive as well as those with peripheral targets. The effect of the peripheral target on the ganglion was less clear-cut when ganglia were first dissected from their targets and then recombined in culture. However, the results of such experiments in which nerve fascicles were traced in serial sections from ganglia to target areas, suggest that the actual innervation of target cells, as well as proximity of ganglia to target tissues, could influence neuronal survival. Establishment of innervation appeared to be selective, in that the closest available target area was not always the one contacted by the ganglionic fibers. The present findings are consistent with a role of neuron-target cell interactions in supporting neuronal survival in the cochleovestibular ganglion of the chick embryo. Both the central and the peripheral targets are implicated in trophic interactions with the sensory neurons.     

Nicotine upregulates nerve growth factor expression and prevents apoptosis of cultured spinal cord neurons

Modulation of neurotrophic factor expression may constitute an important part of neuroprotective effects of nicotine. Therefore, the effects of nicotine on expression of nerve growth factor (NGF) and its receptor, tyrosine receptor kinase A (trkA), were studied in cultured spinal cord neurons treated with arachidonic acid. Because injury to spinal cord is associated with elevated levels of arachidonic acid, this cell culture system has been developed in our laboratory as an in vitro model of neuronal injury in spinal cord trauma. Treatment with nicotine markedly upregulated NGF mRNA and protein expression in spinal cord neurons. In addition, a 12h treatment with nicotine increased mRNA levels of trkA. Both nicotine and exogenous NGF inhibited arachidonic acid induced apoptosis of spinal cord neurons. However, the blockage of the trkA receptor prevented nicotine-mediated anti-apoptotic effects. The present results indicate that increased expression of NGF may be an important element of the neuroprotective effects of nicotine in injured spinal cord neurons.