Immunization of adult rats against 2.5 S NGF: Effects on the peripheral sympathetic nervous system

The biochemical and morphological changes effected by immunization of adult rats with 2.5 S mouse nerve growth factor (NGF) were studied in sympathetic ganglia and in representative target organs. This immunization procedure maintains high levels of circulating anti NGF-antibody for periods of months. Morphological analysis revealed a general reduction in the size of the adrenergic neurons in the superior cervical ganglion (SCG) which was also reflected at the biochemical level by a 30% decrease in total protein content and a 50--60% reduction in the total activities of all norepinephrine-synthesizing enzymes. However, there was no change in total choline acetyltransferase activity. The biochemical and morphological changes observed in the SCG seem to be confined to the neuronal cell body, since at any stage of immunization target organs (the submandibular and the pineal gland) remained unaffected. All sympathetic ganglia investigated--except the superior mesenteric ganglion--responded in a similar way to the immunization against 2.5 S NGF. These changes in the adrenergic cell bodies were largely reversible. The recovery of normal enzyme activities followed closely the decrease of the antibody titer after cessation of immunization boosting. This indicates that cell death is not caused by anti NGF-antibodies in ganglia of adult animals. Thus, in contrast to adrenergic neurons from newborn animals, which depend on NGF or a crossreacting NGF-like material for survival, differentiated adrenergic neurons need this factor for the maintenance of their normal function but not for survival.     

Effects of long-term nerve growth factor deprivation on the nervous system of the adult rat: An experimental autoimmune approach

Adult rats immunized with 2.5S mouse nerve growth factor (NGF) produced antibodies which cross-reacted with rat NGF. By the criterion of ammonium sulfate precipitation followed by Sephadex G-200 chromatography, all serum anti-NGF activity was retained in the IgG fraction. Animals which developed and maintained chronic (5–6 months) serum titers of anti-NGF demonstrated a pronounced biochemical and morphological atrophy of the superior cervical ganglion which was accompanied by a 35–40% reduction in neuronal number. Norepinephrine was reduced by approximately 90% in heart and brown fat. The extent of biochemical atrophy correlated well with serum titers of anti-NGF. No effects were observed on the short adrenergic neurons of the vas deferens, adrenal medullary chromaffin cells, central adrenergic neurons, or peripheral sensory neurons. These results strongly suggest that mature peripheral sympathetic neurons remain dependent on NGF for survival as well as for maintenance.     

Histochemistry of sympathetic neurons allotransplanted from young and aged mice to the submandibular gland.

Sympathetic ganglion tissue of young (3 months) and aged (24 months) NMRI mice was allotransplanted into the submandibular gland to study the influence of aging on the survival of grafted neurons. The submandibular gland (SMG) was chosen as a host tissue because of its high concentration of NGF and good blood supply. Four weeks postgrafting the viability of transplants was evaluated using the formaldehyde-induced fluorescence technique, tyrosine hydroxylase (TH) immunohistochemistry, and morphometry. The density of neurons, catecholamine fluorescence and TH immunoreactivity (TH-IR) appeared to be almost unchanged when the transplant was completely surrounded by the SMG tissue, whereas transplants located within the interlobular septum and capsule, or even outside the capsule, showed significantly reduced neuronal survival. The shape of most of the transplanted neurons was not different from those in the intact ganglia. The average diameter of the transplanted young neurons was significantly decreased; this was not the case with the aged neurons. The histograms of grouped diameter values showed a shift to smaller cells in ganglion transplants in both age groups. The transplants in mice treated with 6-OH-dopamine showed considerable regrowth of adrenergic nerve fibers. There seemed to be no marked difference in the survival of transplanted neurons between aged and young animals. The results indicate that the sympathetic neurons from both young adult and aged animals survive the allotransplantation procedure. The neurotrophic factors together with dense vascularization present in the mouse submandibular gland may be beneficial for the restoration of the integrity of mature and aged adrenergic neurons.     

A study of the de- and regenerative changes in the sympathetic nervous system of the adult mouse after treatment with the antiserum to nerve growth factor.

In the adult mouse, the antiserum to nerve growth factor (NGF) induced marked atrophic changes of the ganglionic cell bodies in the superior cervical ganglion (SCG) and a disappearance of adrenergic nerve terminals in several peripheral tissues. By fluorescence histochemistry a lower-than-normal content of the noradrenaline (NA) transmitter was observed within the entire adrenergic neurone only 1 day after a single injection of NGF-antiserum (0.1 ml/g body weight). An atrophy of adrenergic nerve cell bodies and a disappearance of adrenergic nerve terminals were observed after 3 days, but the antiserum-induced effects did not appear maximally developed until 7 days after treatment. These fluorescence histochemical findings were paralleled by a gradual decrease of the endogenous NA levels in peripheral tissues and also of the weight of the SCG. A gradually proceeding restoration towards normal of the adrenergic innervation apparatus was observed fluorescence histochemically following a 5-day treatment with NGF-antiserum (0.1 ml/g body weight each dose), and after 6 weeks to 3 months a normal or close to normal fluorescence microscopical appearance was regained in the peripheral tissues and also in the SCG. These findings were parelleled by the results of the determinations of endogenous NA in peripheral tissues and by the results of weighing the SCG. We discuss some important differences between NGF-antiserum and 6-hydroxydopamine with respect to their mode of action on the mature sympathetic nervous system. Finally, we suggest that a decreased availability of NGF in a terminal area, due to an interference with endogenous NGF by NGF-antibodies, may temporarily result in an impaired function of the supplying adrenergic neurone, including a degeneration of nerve terminals.     

Nerve growth factor synthesis by mouse submandibular gland cells in culture

Mouse submandibular gland (SMG) cells in culture rarely retian functional properties of SMG cells in vivo. We demonstrate that both primary SMG cells and the mouse SMG cell line SCA-9 secrete biologically active nerve growth factor (NGF). However, primary cells secrete 40-fold more NGF than SCA-9 cells, demonstrating that SCA-9 cells cannot substitute for primary SMG cells for the study of SMG NGF in vitro.     

Transplantation of male mouse submaxillary gland increases survival of axotomized basal forebrain neurons

Transection of the fimbria-fornix results in a loss of magnocellular neurons in the medial septum and vertical limb of the diagonal band (MS/VDB), possibly due to the deprivation of a retrogradely transported trophic substance, such as nerve growth factor (NGF), derived from the hippocampal formation. We have utilized a transplantation model in which grafts of NGF-rich male mouse submaxillary gland were placed in the lateral ventricle adjacent to the MS/VDB of rats with transections of the fimbria-fornix. At 2-4 weeks following transection, animals with grafted submaxillary glands exhibited enhanced survival of MS/VDB neurons, which stained positive for acetylcholinesterase and were immunoreactive for the NGF receptor. These experiments demonstrate that grafts of male mouse submaxillary gland can facilitate the survival of axotomized MS/VDB cholinergic neurons and may therefore prove beneficial in promoting regeneration of damaged neural systems.     

Biological importance of retrograde axonal transport of nerve growth factor in adrenergic neurons.

Previous studies have shown that nerve growth factor (NGF) produces a selective induction of tyrosine hydroxylase (TH) in peripheral adrenergic neurons and that NGF is transported retrogradely with a high selectivity from the adrenergic nerve terminals to the perikaryon. In order to investigate the biological importance of retrograde NGF transport, the following experiments have been performed; (a) effect of NGF on TH activity in superior cervical ganglia (SCG) after unilateral injection into the anterior eye chamber and the submaxillary gland; and (b) effect of systemic injection of NGF on TH activity in SCG after blockage of retrograde axonal transport by axotomy. After unilateral injection of NGF into the anterior eye chamber and submaxillary gland of both 8-10-day-old rats and adult mice, the increase in TH activity in the SCG was considerably larger on the injected than on the non-injected side although the adrenergic neurons supplying the two organs do not account for more than 25% of the total number of adrenergic neurons in the SCG. A direct diffusion mechanism could be excluded by the fact that unilateral local injection of [125 I] produced no significant side difference in the accumulation of radioactivity in the SCG 2 after injection whereas after 14 h there was a several-fold difference between the injected and non-injected side. Moreover, the nodose ganglia which are located very close to the SCG exhibited no statistically significant difference in the accumulation of radioactivity at any time. Forty-eight hours after subcutaneous injections of 10 mg/kg of NGF the increase in TH activity of the SCG amounted to 154% on the intact side and to 92% on the axotomized side. However, these experiments do not permit decisions about the extent the axotomy, as such, impaired the response to NGF. It is concluded that the biological effect of NGF results to a considerable extent, from the moiety which reaches the cell body by retrograde transport from the nerve terminals.     

Monoamine oxidase B inhibitor selegiline protects young and aged rat peripheral sympathetic neurons against 6-hydroxydopamine-induced neurotoxicity

Selegiline is a selective and irreversible monoamine B inhibitor with the capacity to increase the level of several antioxidative enzymes in rat brain. It can protect adrenergic neurons against injury induced by neurotoxins such as MPTP, DSP-4 and AF64A in animal studies. In addition, the protective action is not limited to catecholaminergic cells, as selegiline can also minimize the loss of developing motoneurons after axotomy. The aim of this study was to determine whether selegiline can protect peripheral catecholaminergic neurons against the neurotoxic effect of 6-OHDA. This kind of protective effect against 6-OHDA neurotoxicity has not been reported before. Wistar albino male rats aged 4 or 24 months were treated with selegiline or saline solution 1 h before 6-OHDA injection. At 2 weeks after the 6-OHDA injection, the superior cervical ganglia (SCG) and submandibular glands (SMG) were studied using catecholamine histofluorescence and immunohistochemistry for tyrosine hydroxylase (TH). The number of TH-positive cells in the SCG and the length and number of adrenergic nerve fibers in the SMG were quantified. Our findings showed that 6-OHDA caused a reduction of TH immunoreactivity and catecholamine histofluorescence in neuronal somata, as well as a decrease in the number and length of adrenergic nerve fibers in the submandibular gland. Selegiline pretreatment protected SCG neurons and their postganglionic nerve fibers in SMG against these changes in a dose-dependent manner. The mechanism through which selegiline exerts its neuroprotective effect is as yet unknown. Received: 5 September 1995 / Revised, accepted: 13 November 1995     

Selective uptake and retrograde axonal transport of dopamine-beta-hydroxylase antibodies in peripheral adrenergic neurons.

In the present experiments the uptake and retrograde axonal transport of antibodies to dopamine beta-hydroxylase (DBH) in adrenergic neurons was studied. When partially purified labelled antibodies to DBH were injected unilaterally into the vicinity of the adrenergic nerve terminals in the iris, radioactive substances accumulated preferentially in the superior cervical ganglia of the injected. By SDS (sodium dodecyl sulfate) gel electrophoresis and immunoprecipitation it could be shown that the accumulated radioactivity in the superior cervical ganglion represented antibodies to DBH. This retrograde accumulation was greatly reduced by colchicine, axotomy or destruction of the adrenergic nerve terminals by 6-hydroxydopamine. The rate of retrograde transport was the same as that of nerve growth factor (NGF) and tetanus toxin in sympathetic neurons. The retrograde transport of antibodies was confined to sympathetic neurons and could not be detect in either sensory or motor neurons.     

Somatostatin-, vasoactive intestinal polypeptide- and neuropeptide Y-like immunoreactivity in eye- and submandibular gland-projecting sympathetic neurons

Studies combine the use of the retrograde tracer, fluorogold, and immunocytochemical staining to determine whether superior cervical ganglion (SCG) neurons projecting to the iris or submandibular gland (SMG) in adult male and female rats show distinctive immunoreactivity to somatostatin (SS), vasoactive intestinal polypeptide (VIP), or neuropeptide Y. Overall, more SMG-projecting neurons than eye-projecting neurons contain VIP-like immunoreactivity (VIP-LI), and more eye-projecting neurons than SMG-projecting neurons contain SS-LI and VIP-LI. Thus, postganglionic neurons of the SCG that project to specific target tissues are heterogeneous in their peptide content, and there are differences in the pattern of peptide-immunoreactivity between neurons projecting to these two target tissues. In addition, the results indicate that there may be gender differences in the expression of these neuropeptides.     

A new low-molecular-weight component promoting adrenergic development in cultured chick sympathetic neurons

A low-molecular-weight component present in medium conditioned by cultured chick liver cells (LCM) enhances the adrenergic properties of dissociated chick superior cervical ganglion (SCG) neurons in culture (Zurn and Mudry, 1986). This substance cannot replace NGF as a survival, growth, or differentiation factor. However, in the presence of NGF, it stimulates neuronal metabolism and catecholamine (CA), but not ACh production by the SCG neurons. The effect on transmitter production is greater than that on neuronal metabolism. Yet this is not due to an increase in the specific activity of tyrosine hydroxylase (TH), the rate-limiting enzyme in CA synthesis. Interestingly, the effect of LCM on CA and ACh production, but not on neuronal metabolism, is potentiated in the presence of a large excess of NGF. The active component(s) present in LCM has a molecular weight lower than 500 Da and is not inactivated by heat or pronase treatment. So far, none of the small molecules tested (ascorbic acid, pyruvate, glucose, L-glutamic acid, glutathione, etc.) were able to mimic the effects of LCM on the SCG neurons. Thus this report describes a novel low-molecular-weight component different from NGF that promotes metabolism and adrenergic development in cultured chick sympathetic neurons.     

Treatment with 6-hydroxydopamine and colchicine decreases nerve growth factor levels in sympathetic ganglia and increases them in the corresponding target tissues

A two-site enzyme immunoassay was used to determine the nerve growth factor (NGF) contents of sympathetic ganglia and their corresponding target tissues in adult rats. The destruction of sympathetic nerve terminals by 6-hydroxydopamine (6-OHDA) and the blockade of axonal transport by colchicine resulted in a rapid increase in the NGF levels of sympathetically innervated organs and a rapid decrease in the sympathetic ganglia. NGF levels in heart atrium, heart ventricle, submandibular gland, and iris increased 2- to 4-fold 12 hr after injection of 6-OHDA, whereas the NGF contents of stellate and superior cervical ganglia dropped to a minimal level of 3 to 4% of control 24 hr after injection. Twelve hours after treatment with colchicine the NGF levels in sympathetically innervated organs increased 2- to 3-fold, whereas the NGF contents of sympathetic ganglia fell to one-third of control values. The half-lives of NGF in the superior cervical and stellate sympathetic ganglia were 4.5 and 4.8 hr, respectively, as determined by the decrease of NGF content after treatment with 6-OHDA. These results indicate that the synthesis of NGF is normally confined to the innervated target organs with no significant contribution of ganglionic cells. This is consistent with the concept that NGF acts as a retrograde messenger between target organs and innervating sympathetic neurons.     

Immunohistochemical localization of nerve growth factor in the rat pineal gland

Sympathetic nerve fibers arising from the superior cervical ganglia are the main innervation of the rat pineal gland. Since most organs innervated by these ganglia contain nerve growth factor (NGF), the hypothetical existence of NGF in the pineal gland was investigated. The peroxidase anti-peroxidase technique was applied for the immunohistochemical demonstration of NGF using a polyclonal antiserum on Bouin-fixed, paraffin-embedded pineal glands from adult, young and 6-hydroxydopamine (6-OHDA)-treated rats. Few immunopositive cells were observed in the adult pineal gland. A more conspicuous population of immunoreactive cells was noted in young animals (20-45 days old), especially in those chemically denervated with 6-OHDA. NGF immunoreactive cells displayed a stellate shape resembling the interstitial or glial cells previously described in the rat pineal gland. Since NGF plays a trophic effect on sympathetic neurons during development and adulthood, we postulate that its presence in the pineal gland may exert a trophic role on its sympathetic innervation.     

Time and dose dependencies of effects of nerve growth factor on sympathetic and sensory neurons in neonatal rats

Nerve growth factor (NGF) plays a role in the development of several components of the sympathetic and sensory nervous systems. The objectives of this study were to examine the time and dose dependencies of some of the well known effects of NGF on sympathetic ganglia and to examine qualitatively and quantitatively the recently described effects on sensory ganglia of neonatal rats. Single doses of NGF as low as 0.1 mg/kg produce increases in tyrosine hydroxylase (TOH) activity in superior cervical ganglia (SCG), and doses of 3 mg/kg produce maximal effects. Larger doses and longer treatments are required to see increases in protein content of the SCG. Larger doses are also required to affect TOH activity in the adrenal gland. Increases in TOH activity in SCG can be observed within 18 h of injection. Chronic NGF treatment for three weeks produces no change in blood pressure or heart rate in neonatal rats. Chronic administration of NGF (1 or 3 mg/kg/day) results in dose-related increases in the protein content of dorsal root ganglia (DRG). The increase in protein content of the DRG was associated with an increase in the diameter of smaller neurons (those<30 μm in diameter), but NGF caused no change in the number of neurons.     

Cyclosporin A: effects on the secretory process and noradrenergic activity in the submandibular gland of the rat.

OBJECTIVES: The aim of the present work was to study the effect of long-term cyclosporine (CSA) administration on norepinephrine (NE) metabolism and adrenergic-evoked secretion in the rat submandibular gland (SMG). METHODS: Dose-response curves to adrenergic agonists (methoxamine, isoproterenol, NE) were performed in control and CSA (10 and 30 mg/kg every 2 days for 1 month)-treated rats after SMG duct cannulation. In SMG tissue neuronal NE uptake, release, synthesis and endogenous content were determined. In addition phosphoinositide intracellular signaling was also investigated. RESULTS: CSA administration caused an increase in salivary secretion evoked by methoxamine (alpha-adrenergic agonist) and NE but failed to modify salivation evoked by beta-adrenergic stimulation (isoproterenol). Long-term CSA administration decreased NE release and synthesis whereas it enhanced the amine uptake and phosphoinositide hydrolysis in the SMG. CONCLUSIONS: The administration of CSA for 30 days induced salivary gland sensitization likely mediated by diminished adrenergic input. Present results suggest that the decreased sympathetic activity evoked by long-term CSA administration in the rat SMG may lead to sensitization of the gland supported by increased phosphoinositide hydrolysis and enhanced adrenergic-evoked salivation.     

Selective trans-synaptic migration of tetanus toxin after retrograde axonal transport in peripheral sympathetic nerves: a comparison with nerve growth factor.

Adult rats were injected unilaterally into the anterior eye chamber and the submandibular gland with either [125I]tetanus toxin or [125I]nerve growth factor (NGF). Fourteen and 24 h later in electron microscopic autoradiographs of the superior cervical ganglia of the injected side the labeling was confined to a limited number (15-20%) of adrenergic ganglion cells and the silver grains were localized over axons, perikarya and dendrites providing evidence for a retrograde intraaxonal transport of the two macromolecules. Moreover, after injection of [125]tetanus toxin there was a very marked labeling of the presynaptic cholinergic nerve terminals. In contrast, after [125I]NGF these terminals were free of label. In both cases no specific labeling could be detected over glia and extracellular space. In the postganglionic axons the radioactivity seemed to be mainly associated with vesicles and smooth endoplasmic reticulum, in the perikarya and dendrites of the adrenergic neurons with secondary lysosomes, vesicles and smooth endoplasmic reticulum. The Golgi cisternae and the nuclei were free of radioactivity. The specific labeling of presynaptic terminals after injection of [125I]tetanus toxin together wirans-symaptic migration of [125I]tetanus toxin from the adrenergic ganglion cell to its innervating presynaptic terminals following retrograde intraaxonal transport.     

Rescue of NGF-deficient mice I: transgenic expression of NGF in skin rescues mice lacking endogenous NGF

Mice lacking a functional NGF gene (ngf-/- mice) have less than one third of the normal complement of sensory neurons, few sympathetic postganglionic neurons and die shortly after birth. We report here that transgenic expression of NGF under control of the K14 keratin promoter can rescue some elements of the peripheral nervous system and restore normal growth and viability to ngf-/- mice. While hybrid transgenic-ngf-/- mice (ngfTKOs) displayed marginal rescue of trigeminal ganglion neurons, the percentage of CGRP-positive neurons was restored to normal. Restoration of CGRP-positive terminals in skin and spinal cord was also found and accompanied by recovery of behavioral responses to noxious stimuli. ngfTKO mice displayed a normal number of superior cervical ganglion neurons and recovery of sympathetic innervation of skin. These results demonstrate that substitution of a functional NGF locus by a transgene directing expression largely to skin can result in normal growth and viability. Thus, the most vital functions of NGF are not dependent on faithful recapitulation of the normal spatiotemporal pattern of gene expression.     

BDNF activated TrkB/IRR receptor chimera promotes survival of sympathetic neurons through Ras and PI-3 kinase signaling

Insulin receptor-related receptor (IRR) expression is tightly coupled to the nerve growth factor (NGF) receptor, TrkA, throughout development. Expression of both receptors is primarily localized to neural crest derived sensory and sympathetic neurons. In contrast to TrkA, however, the physiological ligand for IRR is unknown. To analyze the intracellular signaling and potential function of the orphan IRR in neurons, an adenovirus expressing a TrkB/IRR chimeric receptor was used to infect cultured mouse superior cervical ganglion neurons that normally require NGF for survival. Brain derived neurotrophic factor (BDNF)-activated TrkB/IRR induced neuronal survival. We utilized numerous receptor mutants in order to identify the intracellular domains of IRR necessary for signaling and neuron survival. Finally, we employed adenovirus encoding dominant negative forms of the extracellular signal-regulated kinase (ERK) signaling cascade to demonstrate that IRR, like TrkA, requires ras activation to promote neuron survival. Therefore, by use of the chimeric TrkB/IRR receptor, we have demonstrated the ability of IRR to elicit activation of signaling cascades resulting in a biological response in superior cervical ganglion (SCG) neurons.     

The retrograde axonal transport of nerve growth factor

A retrograde axonal transport of nerve growth factor (NGF) from the adrenergic nerve terminals in the mouse iris to the cell bodies of postganglionic sympathetic neurones in the superior cervical ganglion has been demonstrated. After injection of iodinated nerve growth factor (125I-NGF) into the anterior eye-chamber there was a relatively rapid accumulation of radioactivity in the superior cervical ganglia on both injected and non-injected sides, as was the case after subcutaneous injection. However, 4 h after intraocular injection a preferential accumulation of radioactivity became apparent in the superior cervical ganglion on the injected side, and this difference between the ganglia on injected and non-injected sides gradually increased to a maximum at 16 h. Transection of the postganglionic adrenergic fibres as well as the prior intraocular injection of colchicine abolished the preferential accumulation of 125I-NGF in the superior cervical ganglion of the injected side, whereas the destruction of adrenergic nerve terminals by 6-hydroxydopamine did not impair the preferential accumulation. It is concluded that the retrograde axonal transport of NGF, which was estimated to take place at a rate of about 2.5 mm/h, depends on a colchicine-sensitive mechanism as does the orthograde rapid axonal transport. However, the uptake of NGF may not only take place from the nerve terminals but also from the preterminal parts, as has been shown in other studies with horseradish peroxidase. Autoradiographic studies strongly supported the existence of a retrograde transport by showing a clear localization of radioactivity in a small number of neurones in the superior cervical ganglion on the injected side, whereas on the non-injected side there was only a diffuse distribution of radioactivity throughout the ganglion.     

Endogenous nerve growth factor is required for regulation of the low affinity neurotrophin receptor (p75) in sympathetic but not sensory ganglia

During development, many sympathetic and sensory neurons are dependent on nerve growth factor (NGF) for survival. The low affinity neurotrophin receptor (p75), expressed in these neurons, is regulated by exogenous NGF in vitro and in vivo. However, whether p75 expression in vivo is under the control of endogenous NGF has not been determined. The role of NGF in regulating the expression of p75 in sympathetic and sensory nerves was investigated in Sprague-Dawley rats treated with an antiserum specific for NGF. P75 was differentially regulated. P75 immunoreactivity (-ir) within sympathetic neurons in the superior cervical ganglia (SCG) was reduced after 2 days, and disappeared after 5 days, of treatment with the NGF antiserum. In contrast, a significant increase in p75-ir was detected in nerve bundles within and close to the SCG from 3 to 14 days after treatment. A similar pattern of p75 expression was observed in the stellate and coeliac ganglia. In contrast, p75 expression in nerve terminals of the mesenteric arteries and irides was reduced. However, in the same animals the expression of p75 was not significantly affected by the treatment in dorsal root, trigeminal or nodose ganglia, salivary gland or small intestine. In contrast to p75, the NGF high affinity receptor trkA was little affected in sympathetic neurons by depletion of endogenous NGF for 2 weeks. These results indicate that endogenous NGF is required in sympathetic ganglia for the expression of p75 but not trkA in neurons, but for the down-regulation of p75 in glia. In contrast, endogenous NGF is not essential for the regulation of p75 in neurons or glia within sensory ganglia. © Wiley-Liss, Inc.