Retinoic acid induces NGF-dependent survival response and high-affinity NGF receptors in immature chick sympathetic neurons.

An important step in the development of peripheral sensory and sympathetic neurons is the onset of the survival response and dependence on the presence of nerve growth factor (NGF) or other neurotrophic factors. We have recently observed that immature sympathetic neurons from 7-day-old chick embryos are unable to become NGF-responsive in vitro and we have now used these cells to identify molecules that induce NGF-dependent neuronal survival. We found that retinoic acid (RA) induces the ability of these cells to survive in the presence of NGF. At RA concentrations of 10(-9)-10(-8)M virtually all neurons survived in the presence of NGF. RA was found to also induce the biologically active, high-affinity NGF receptor: high-affinity receptors were undetectable on dissociated E7 sympathetic neurons and were observed in vitro only in RA-treated neurons. These findings suggest that the induction of high-affinity NGF receptors may be sufficient to activate the survival response in sympathetic neurons and imply an important role for RA during neuron differentiation in the peripheral nervous system.     

Cellular retinoic acid-binding protein and the role of retinoic acid in the development of the chick embryo

The distribution of cellular retinoic acid-binding protein (CRABP) in four stages of chick development is described using an affinity-purified antibody against rat CRABP. CRABP is the protein to which retinoic acid (RA) binds when it enters cells and may reflect the requirement of those cells for RA. We found several discrete cell populations which showed high levels of immunoreactivity. Some were in the neural tube such as the commissural neurons and the dorsal roof plate. Some were of neural crest origin such as the dorsal root ganglia, sensory axons, sympathetic ganglia, and enteric ganglia. The remaining populations were certain connective tissue cells, limb bud cells, and the myotome. These results suggest that certain organ systems, particularly the nervous system, have a requirement for RA during development and they may further our understanding of the teratogenic effects of retinoids on the embryo.     

Localization of neuropeptides in efferent terminals of the eye in the marine snail,Bulla gouldiana

Summary Retinoic acid (RA), a naturally occurring metabolite of vitamin A, increased the number of receptors for nerve growth factor (NGF) in cultured human neuroblastoma cells (LA-N-1), as indicated by an immunofluorescence assay of cell surface receptors and by specific binding of ¹²⁵I-NGF to solubilized receptors. Analysis of ¹²⁵I-NGF binding showed that RA increased the number of both high affinity and low affinity receptors for NGF without affecting the equilibrium dissociation constants. Neurite outgrowth similar to that produced by NGF occurred following RA-treatment in LA-N-1 cells, in the SY5Y subclone of SK-N-SH human neuroblastoma cells and in explanted chick dorsal root ganglia (DRG). Whether morphological changes following RA treatment are directly related to the increase in NGF receptors is unknown. Data presented here are consistent with literature reports that RA modifies cell surface glycoproteins, including those that act as cell surface receptors for epidermal growth factor and insulin.Abbreviations DRG dorsal root ganglia - NGF nerve growth factor - RA retinoic acid     

Identification of neurons that express ghrelin receptors in autonomic pathways originating from the spinal cord

Functional studies have shown that subsets of autonomic preganglionic neurons respond to ghrelin and ghrelin mimetics and in situ hybridisation has revealed receptor gene expression in the cell bodies of some preganglionic neurons. Our present goal has been to determine which preganglionic neurons express ghrelin receptors by using mice expressing enhanced green fluorescent protein (EGFP) under the control of the promoter for the ghrelin receptor (also called growth hormone secretagogue receptor). The retrograde tracer Fast Blue was injected into target organs of reporter mice under anaesthesia to identify specific functional subsets of postganglionic sympathetic neurons. Cryo-sections were immunohistochemically stained by using anti-EGFP and antibodies to neuronal markers. EGFP was detected in nerve terminal varicosities in all sympathetic chain, prevertebral and pelvic ganglia and in the adrenal medulla. Non-varicose fibres associated with the ganglia were also immunoreactive. No postganglionic cell bodies contained EGFP. In sympathetic chain ganglia, most neurons were surrounded by EGFP-positive terminals. In the stellate ganglion, neurons with choline acetyltransferase immunoreactivity, some being sudomotor neurons, lacked surrounding ghrelin-receptor-expressing terminals, although these terminals were found around other neurons. In the superior cervical ganglion, the ghrelin receptor terminals innervated subgroups of neurons including neuropeptide Y (NPY)-immunoreactive neurons that projected to the anterior chamber of the eye. However, large NPY-negative neurons projecting to the acini of the submaxillary gland were not innervated by EGFP-positive varicosities. In the celiaco-superior mesenteric ganglion, almost all neurons were surrounded by positive terminals but the VIP-immunoreactive terminals of intestinofugal neurons were EGFP-negative. The pelvic ganglia contained groups of neurons without ghrelin receptor terminal innervation and other groups with positive terminals around them. Ghrelin receptors are therefore expressed by subgroups of preganglionic neurons, including those of vasoconstrictor pathways and of pathways controlling gut function, but are absent from some other neurons, including those innervating sweat glands and the secretomotor neurons that supply the submaxillary salivary glands.     

Oxytocin modulation of intrathoracic sympathetic ganglionic neurons regulating the canine heart

Substance P and vasoactive intestinal peptide are known to activate intrathoracic sympathetic neurons which regulate the heart. In the present series of experiments, when 1 I.U. of oxytocin in 0.1 cc of normal saline was administered into the cranial poles of stellate or the middle of middle cervical ganglia cardiac rate and force were augmented. The locations of ganglionic loci which, when injected, resulted in cardiac changes varied between animals. Twenty active sites were identified in the 12 dogs investigated. When the vehicle (0.1 cc of normal saline) was injected into these active sites minimal cardiac responses were induced in one instance. When 1 or 2 I.U. of oxytocin was administered into the superior vena cave of seven animals slight systemic hypotension occurred in two of these animals. Cardiac responses were induced when oxytocin was reinjected into active intrathoracic ganglionic sites after whole body administration of hexamethonium (10 mg/kg IV), but not after local administration of timolol into the ganglia. Thus, it appears that oxytocin can activate intrathoracic ganglionic neurons involved in efferent sympathetic cardiac regulation. That such responses persist in the presence of nicotinic blockade, but not following beta-adrenergic blockade of ganglionic neurons, indicates that oxytocin modifies beta-adrenergic and not nicotinic receptors on neurons in these ganglia.     

Distributions of estrogen receptors alpha and beta in sympathetic neurons of female rats: enriched expression by uterine innervation

Estrogen modulates many features of the sympathetic nervous system, including cell numbers and ganglion synapses, and can induce uterine sympathetic nerve degeneration. However, distributions of estrogen receptors alpha and beta within sympathetic neurons have not been described, and their regulation by target tissue or estrogen levels has not been explored. We used immunofluorescence and retrograde tracing to define estrogen receptor expression in sympathetic neurons at large in pre- and paravertebral ganglia and in those projecting to the uterine horns. Estrogen receptor alpha immunoreactivity was present in 29 +/- 1%, while estrogen receptor beta was expressed by 92 +/- 1% of sympathetic neurons at large. The proportions of neurons expressing these receptors were comparable in the superior cervical and thoraco-lumbar paravertebral ganglia from T11 through L5, and in the suprarenal, celiac, and superior mesenteric prevertebral ganglia. Injections of FluoroGold into the uterine horns resulted in labeled neurons, with peak occurrences in T13, L1, and the suprarenal ganglion. Uterine-projecting neurons showed small but significantly greater incidence of estrogen receptor beta expression relative to the neuronal population at large, whereas the proportion of uterine-projecting neurons with estrogen receptor alpha-immunoreactivity was nearly threefold greater. Numbers of estrogen receptor-expressing neurons were not altered by acute estrogen administration. We conclude that the vast majority of sympathetic neurons express estrogen receptor beta immunoreactive protein, whereas a smaller, presumably overlapping subset expresses the estrogen receptor alpha. Expression of the latter apparently can be enhanced by target-mediated mechanisms.     

Age-dependent changes of the autonomic neurons expressing vanilloid TRPV1 receptors

Expression of vanilloid receptors in sympathetic and afferent ganglionic neurons was studied in rats of different ages (newborn, 10-day old, 20-day old, 30-day old, 60-, 180-day old) using immunohistochemical methods. The results obtained indicate that the majority of the afferent neurons in the nodose ganglion of vagus nerve (GNVN) and in the spinal ganglia (SG) were TRPV1-positive from birth onwards. The percentage of neurons containing TRPVT receptors in SG slightly increased with age up to 30 days postnatally. In the GNVN, the percentage of TRPV1-positive neurons was higher in comparison with the SG in all age groups. The vast majority of the sympathetic neurons were TRPV1-positive from birth onwards, and the percentage of TRPV1-immunoreactive neurons substantially decreased during further development. In 20-day old and older animals, we observed only few TRPV1-immunoreactive neurons in sympathetic ganglia. Finally, the percentage of neurons containing these types of neurons, become similar to adult animals to the end of the first month of life.     

The role of GDNF family ligand signalling in the differentiation of sympathetic and dorsal root ganglion neurons

The diversity of neurons in sympathetic ganglia and dorsal root ganglia (DRG) provides intriguing systems for the analysis of neuronal differentiation. Cell surface receptors for the GDNF family ligands (GFLs) glial cell-line-derived neurotrophic factor (GDNF), neurturin and artemin, are expressed in subpopulations of these neurons prompting the question regarding their involvement in neuronal subtype specification. Mutational analysis in mice has demonstrated the requirement for GFL signalling during embryonic development of cholinergic sympathetic neurons as shown by the loss of expression from the cholinergic gene locus in ganglia from mice deficient for ret, the signal transducing subunit of the GFL receptor complex. Analysis in mutant animals and transgenic mice overexpressing GFLs demonstrates an effect on sensitivity to thermal and mechanical stimuli in DRG neurons correlating at least partially with the altered expression of transient receptor potential ion channels and acid-sensitive cation channels. Persistence of targeted cells in mutant ganglia suggests that the alterations are caused by differentiation effects and not by cell loss. Because of the massive effect of GFLs on neurite outgrowth, it remains to be determined whether GFL signalling acts directly on neuronal specification or indirectly via altered target innervation and access to other growth factors. The data show that GFL signalling is required for the specification of subpopulations of sensory and autonomic neurons. In order to comprehend this process fully, the role of individual GFLs, the transduction of the GFL signals, and the interplay of GFL signalling with other regulatory pathways need to be deciphered.     

Synthesis, insertion into the plasma membrane, and turnover of alpha- bungarotoxin receptors in chick sympathetic neurons

alpha-Bungarotoxin was used to identify an integral membrane protein in the plasma membrane of chick sympathetic neurons. The synthesis, insertion into the plasma membrane, and turnover of the alpha-bungarotoxin receptor were studied using isotopically labeled amino acids (2H, 13C, 15N) to directly label receptor molecules. Neurons incubated in medium containing dense amino acids continued to insert unlabeled receptors from a pool of previously synthesized molecules for 2 h. Density-labeled receptors began to appear in the plasma membrane after this 2-h period. Synthesis of receptors, but not insertion into the surface, was blocked by cycloheximide (100 microgram/ml). Neither colchicine (0.05 microgram/ml) of actinomycin D (5 microgram/ml) has any effect on alpha-bungarotoxin receptor synthesis or insertion. Autoradiographic studied revealed that receptors occur on growth cones, axons, and cell bodies of single neurons and explanted ganglia. The rate of insertion of newly synthesized receptors into the plasma membrane of axons extending from explanted sympathetic ganglia was approximately the same as that into the cell body portion of the ganglion. Cytochalasin B (2 microgram/ml) rapidly distrupted growth cones but had no effect on receptor insertion. These experiments suggested that the growth cone is not the sole or even the primary site for insertion of this membrane protein. The kinetics of turnover of the alpha-bungarotoxin receptor were a first-order exponential with t 1/2 = 11 h. Neurons that had their surface receptors labeled with 125I-alpha-bungarotoxin produced [125I]iodotyrosine. This process was inhibited by low temperature (23 degrees C) and also by a metabolic inhibitor. This is interpreted as evidence that receptors turn over by a mechanism in which they are internalized and then proteolytically degraded.     

Retinoic acid and neurotrophins collaborate to regulate neurogenesis in adult‐derived neural stem cell cultures

The adult rat hippocampus contains fibroblast growth factor 2–responsive stem cells that are self‐renewing and have the ability to generate both neurons and glia in vitro, but little is known about the molecular events that regulate stem cell differentiation. Hippocampus‐derived stem cell clones were used to examine the effects of retinoic acid (RA) on neuronal differentiation. Exposure to RA caused an immediate up‐regulation of NeuroD, increased p21 expression, and concurrent exit from cell cycle. These changes were accompanied by a threefold increase in the number of cells differentiating into immature neurons. An accompanying effect of RA was to sustain or up‐regulate trkA, trkB, trkC, and p75NGFR expression. Without RA treatment, cells were minimally responsive to neurotrophins (NTs), whereas the sequential application of RA followed by brain‐derived neurotrophic factor or NT‐3 led to a significant increase in neurons displaying mature γ‐a‐minobutyric acid, acetylcholinesterase, tyrosine hydroxylase, or calbindin phenotypes. Although NTs promoted maturation, they had little effect on the total number of neurons generated, suggesting that RA and neurotrophins acted at distinct stages in neurogenesis. RA first promoted the acquisition of a neuronal fate, and NTs subsequently enhanced maturation by way of RA‐dependent expression of the Trk receptors. In combination, these sequential effects were sufficient to stimulate stem cell–derived progenitors to differentiate into neurons displaying a variety of transmitter phenotypes. © 1999 John Wiley & Sons, Inc. J Neurobiol 38: 65–81, 1999     

The early expression of VAChT and VIP in mouse sympathetic ganglia is not induced by cytokines acting through LIFRbeta or CNTFRalpha

Sympathetic ganglia consist of noradrenergic and cholinergic neurons. The cholinergic marker protein vesicular acetylcholine transporter (VAChT) and the neuropeptide vasoactive intestinal peptide (VIP), co-expressed in mature cholinergic sympathetic neurons, are first detectable during embryonic development of rat sympathetic ganglia. However, the subpopulation of cholinergic sympathetic neurons which innervates sweat glands in mammalian footpads starts to express VAChT and VIP during the first postnatal weeks, under the influence of sweat gland-derived signals. In vitro evidence suggests that the sweat gland-derived cholinergic differentiation factor belongs to a group of neuropoietic cytokines, including LIF, CNTF and CT-1, that act through a LIFRbeta-containing cytokine receptor. To investigate whether the embryonic expression of cholinergic properties is elicited by a related cytokine, the expression of VAChT and VIP was analyzed in stellate ganglia of mice deficient for the cytokine receptor subunits LIFRbeta or CNTFRalpha. The density of VAChT- and VIP-immunoreactive cells in stellate ganglia of new-born animals was not different in LIFRbeta(-/-) and CNTFRalpha(-/-) ganglia as compared to ganglia from wild-type mice. These results demonstrate that the early, embryonic expression of VAChT and VIP is not induced by cytokines acting through LIFRbeta- or CNTFRalpha-containing receptors.     

Galanin is more common than NPY in vascular sympathetic neurons of the brush-tailed possum.

The distribution of galanin (Gal) in sympathetic vascular neurons of adult and juvenile brush-tailed possums (Trichosurus vulpecula), was examined using double-labelling immunohistochemistry. This was compared with the distribution of neuropeptide Y (NPY) in the same tissues. Immunoreactivity (IR) to galanin was present in the majority (64-99%) of nerve cell bodies in paravertebral sympathetic ganglia, where it mostly co-existed with IR to the catecholamine-synthesizing enzyme, tyrosine hydroxylase (TH). Gal-IR also was present in most, if not all, TH-IR perivascular axons supplying systemic arteries and veins. NPY-IR was less common than Gal-IR in all sympathetic ganglia and perivascular axons examined. Some sympathetic, TH-IR axons supplying the abdominal aorta and renal artery contained both Gal-IR and NPY-IR, while TH-IR axons supplying cephalic and thoracic vessels contained Gal-IR but not NPY-IR. Limited observations on sympathetic neurons in two species of wallabies indicated that Gal-IR also was more common than NPY-IR in other marsupial species, but the incidence of NPY-IR was higher in these wallabies than in the brush-tailed possum. Together with previous studies, this work suggests that the coexistence of galanin and NPY may be the primitive condition for sympathetic neurons in tetrapods. The differential expression of these peptides in specific populations of sympathetic neurons may have important functional consequences in the autonomic control of the circulation.     

Retinoic acid and neurotrophins collaborate to regulate neurogenesis in adult-derived neural stem cell cultures

ABSTRACT: The adult rat hippocampus contains fibroblast growth factor 2-responsive stem cells that are self-renewing and have the ability to generate both neurons and glia in vitro, but little is known about the molecular events that regulate stem cell differentiation. Hippocampus-derived stem cell clones were used to examine the effects of retinoic acid (RA) on neuronal differentiation. Exposure to RA caused an immediate up-regulation of NeuroD, increased p21 expression, and concurrent exit from cell cycle. These changes were accompanied by a threefold increase in the number of cells differentiating into immature neurons. An accompanying effect of RA was to sustain or up-regulate trkA, trkB, trkC, and p75NGFR expression. Without RA treatment, cells were minimally responsive to neurotrophins (NTs), whereas the sequential application of RA followed by brain-derived neurotrophic factor or NT-3 led to a significant increase in neurons displaying mature y-a-minobutyric acid, acetylcholinesterase, tyrosine hydroxylase, or calbindin phenotypes. Although NTs promoted maturation, they had little effect on the total number of neurons generated, suggesting that RA and neurotrophins acted at distinct stages in neurogenesis. RA first promoted the acquisition of a neuronal fate, and NTs subsequently enhanced maturation by way of RA-dependent expression of the Trk receptors. In combination, these sequential effects were sufficient to stimulate stem cell-derived progenitors to differentiate into neurons displaying a variety of transmitter phenotypes.     

Electrophysiological effects of GABA on cat pancreatic neurons

In mammalian peripheral sympathetic ganglia GABA acts presynaptically to facilitate cholinergic transmission and postsynaptically to depolarize membrane potential. The GABA effect on parasympathetic pancreatic ganglia is unknown. We aimed to determine the effect of locally applied GABA on cat pancreatic ganglion neurons. Ganglia with attached nerve trunks were isolated from cat pancreata. Conventional intracellular recording techniques were used to record electrical responses from ganglion neurons. GABA pressure microejection depolarized membrane potential with an amplitude of 17.4 +/- 0.7 mV. Electrically evoked fast excitatory postsynaptic potentials were significantly inhibited (5.4 +/- 0.3 to 2.9 +/- 0.2 mV) after GABA application. GABA-evoked depolarizations were mimicked by the GABA(A) receptor agonist muscimol and abolished by the GABA(A) receptor antagonist bicuculline and the Cl(-) channel blocker picrotoxin. GABA was taken up and stored in ganglia during preincubation with 1 mM GABA; beta-aminobutyric acid application after GABA loading significantly (P < 0.05) increased depolarizing response to GABA (15.6 +/- 1.0 vs. 7.8 +/- 0.8 mV without GABA preincubation). Immunolabeling with antibodies to GABA, glial cell fibrillary acidic protein, protein gene product 9.5, and glutamic acid decarboxylase (GAD) immunoreactivity showed that GABA was present in glial cells, but not in neurons, and that glial cells did not contain GAD, whereas islet cells did. The data suggest that endogenous GABA released from ganglionic glial cells acts on pancreatic ganglion neurons through GABA(A) receptors.     

Sympathetic axons surround nerve growth factor-immunoreactive trigeminal neurons: Observations in mice overexpressing nerve growth factor

It has been postulated that the aberrant projection of sympathetic axons to individual primary sensory neurons may provide the morphological basis for pain-related behaviors in rat models of chronic pain syndrome. Since nerve growth factor (NGF) can elicit the collateral sprouting of noradrenergic sympathetic terminals, it might be predicted that NGF plays a role in mediating the sprouting of sympathetic axons into sensory ganglia. Using a line of transgenic mice overexpressing NGF among glial cells, it was first found that trigeminal ganglia from adult transgenic mice possessed significantly higher levels of NGF protein in comparison to age-matched wild-type mice; as well, detectable levels of NGF mRNA transgene expression were present in both the ganglia and brain stem. Within the trigeminal ganglia, a small proportion of the sensory neuronal population stained immunohistochemically for NGF; a higher percentage of NGF-positive neurons was evident in transgenic mice. New sympathetic axons extended into the trigeminal ganglia of transgenic mice only and formed perineuronal plexuses surrounding only those neurons immunostained for NGF. In addition, such plexuses were accompanied by glial processes from nonmyelinating Schwann cells. From these data, we propose that accumulation of glial-derived NGF by adult sensory neurons and its putative release into the ganglionic environment induce the directional growth of sympathetic axons to the source of NGF, namely, the cell bodies of primary sensory neurons. © 1998 John Wiley & Sons, Inc. J Neurobiol 34: 347–360, 1998     

Role of neurotrophin signalling in the differentiation of neurons from dorsal root ganglia and sympathetic ganglia

Manipulation of neurotrophin (NT) signalling by administration or depletion of NTs, by transgenic overexpression or by deletion of genes coding for NTs and their receptors has demonstrated the importance of NT signalling for the survival and differentiation of neurons in sympathetic and dorsal root ganglia (DRG). Combination with mutation of the proapoptotic Bax gene allows the separation of survival and differentiation effects. These studies together with cell culture analysis suggest that NT signalling directly regulates the differentiation of neuron subpopulations and their integration into neural networks. The high-affinity NT receptors trkA, trkB and trkC are restricted to subpopulations of mature neurons, whereas their expression at early developmental stages largely overlaps. trkC is expressed throughout sympathetic ganglia and DRG early after ganglion formation but becomes restricted to small neuron subpopulations during embryogenesis when trkA is turned on. The temporal relationship between trkA and trkC expression is conserved between sympathetic ganglia and DRG. In DRG, NGF signalling is required not only for survival, but also for the differentiation of nociceptors. Expression of neuropeptides calcitonin gene-related peptide and substance P, which specify peptidergic nociceptors, depends on nerve growth factor (NGF) signalling. ret expression indicative of non-peptidergic nociceptors is also promoted by the NGF-signalling pathway. Regulation of TRP channels by NGF signalling might specify the temperature sensitivity of afferent neurons embryonically. The manipulation of NGF levels “tunes” heat sensitivity in nociceptors at postnatal and adult stages. Brain-derived neurotrophic factor signalling is required for subpopulations of DRG neurons that are not fully characterized; it affects mechanical sensitivity in slowly adapting, low-threshold mechanoreceptors and might involve the regulation of DEG/ENaC ion channels. NT3 signalling is required for the generation and survival of various DRG neuron classes, in particular proprioceptors. Its importance for peripheral projections and central connectivity of proprioceptors demonstrates the significance of NT signalling for integrating responsive neurons in neural networks. The molecular targets of NT3 signalling in proprioceptor differentiation remain to be characterized. In sympathetic ganglia, NGF signalling regulates dendritic development and axonal projections. Its role in the specification of other neuronal properties is less well analysed. In vitro analysis suggests the involvement of NT signalling in the choice between the noradrenergic and cholinergic transmitter phenotype, in the expression of various classes of ion channels and for target connectivity. In vivo analysis is required to show the degree to which NT signalling regulates these sympathetic neuron properties in developing embryos and postnatally. U.E. is supported by the DFG (Er145-4) and the Gemeinnützige Hertie-Stiftung.