The effects of drugs which destroy the sympathetic nervous system on the retrograde transport of nerve growth factor

It has been proposed that the drugs (6-hydroxydopamine, guanethidine, vinblastine) which are known to destroy sympathetic neurons in neonatal animals do so by preventing the accumulation of retrogradely transported nerve growth factor (NGF). It was found, consistent with the proposal, that administration of 6-hydroxydopamine (100 mg/kg s.c.) or vinblastine (0.4 mg/kg s.c.) 16 h prior to the administration of [¹²⁵I]NGF completely prevented the accumulation of retrogradely transported [¹²⁵I]NGF in superior cervical ganglia of neonatal rats. Administration of 6-hydroxydopamine or vinblastine to adult rats (where it does not cause sympathetic neuron cell death) didnot completely prevent the retrograde transport of NGF, although 6-hydroxydopamine produced an alteration of the time course of accumulation (early times unaffected, later times depressed). The administration of guanethidine to adult rats (50 mg/kg/day) produced a modest decrease in the accumulation of NGF (40–60%). It would appear, however, that this decrease cannot account for the cytotoxic effects of guanethidine since: (1) sub-cytotoxic doses of guanethidine and non-cytotoxic guanidinium blocking agents also produce modest decreases in the retrograde transport in NGF; and (2) the retrograde transport of [¹²⁵I]NGF is not affected in neonatal animals until after the neurons are clearly damaged. Hence, the data are entirely consistent with the hypothesis that NGF deprivation caused by 6-hydroxydopamine and vinblastine is the mechanism of the cytotoxic effects of these drugs on sympathetic neurons in neonatal animals. Guanethidine destroys sympathetic neurons by some other mechanism.     

Characteristics of the retrograde axonal transport system for nerve growth factor in the sympathetic nervous system

Retrograde axonal transport of¹²⁵I labeled nerve growth factor (NGF) occurs in the adrenergic neurons of the sympathetic nervous system of rats as well as mice. This transport is saturable, it can be blocked by an excess of unlabeled NGF and shows a specificity for the biologically active form of the protein. Other proteins such as serum albumin, chymotrypsin or insulin are transported in varying, but much smaller, amounts. Although the transport of albumin is blocked by an excess of NGF, the opposite is not true, again suggesting a specific mechanism. NGF antibody, injected subcutaneously, prevents retrograde transport in neonatal but not in more mature animals and the relative resistance of the latter to the action of NGF antiserum may be related to this phenomenon.     

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.     

Short- and long-term effects of nerve growth factor on the sympathetic nervous system in the adult mouse.

Nerve growth factor (NGF) induced a marked increase in the adrenergic nerve terminal networks of several peripheral organs in intact adult and young adult mice. The animals were investigated at 3 days and at 2 months after 6 daily subcutaneous injections of the 7S species of NGF (1 mug/g body weight each dose). At 3 days after the end of the treatment an increased fluorescence intensity, as well as an increased density of the adrenergic terminal plexuses was observed by fluorescence histochemical techniques in iris, salivary glands, heart, intestine, spleen and pancreas. These changes were paralleled by significant increases (up to 55%) in the endogenous NA levels. Increased NA levels were also detected in the brain of the NGF-treated animals. At 2 months after treatment the effects had almost totally disappeared, demonstrating that the NGF-induced overgrowth of the sympathetic axons was only temporary. The increased adrenergic innervation probably resulted from a stimulatory effect of NGF on collateral sprouting from the intact adrenergic axons. In line with the idea that the terminals of peripheral axons are continuously renewed, it is suggested that endogenous NGF could play a regulatory role in such a continuous growth process of the fully developed, adult sympathetic nervous system, directed at the maintenance of an adequate adrenergic terminal network.     

Demonstration of the retrograde transport of nerve growth factor receptor in the peripheral and central nervous system

NGF acts on responsive neurons by binding to specific NGF receptors on axonal termini, after which a critical biochemical signal is retrogradely transported to the cell body. The identity of the signal(s) is unknown; candidates include NGF itself or some other "second messenger." A possible second messenger is the NGF receptor. As a first step in assessing the possible role of NGF receptor in the generation of the NGF-dependent signal, and in understanding the economy of NGF receptor synthesis and utilization, we determined whether the NGF receptor is retrogradely transported. Using immunohistochemical staining with a monoclonal antibody (192-IgG) against rat NGF receptor, we looked for accumulation of NGF receptor molecules distal (retrograde transport), as well as proximal (anterograde transport), to sites of axonal ligation or transection. By 10-12 hr in both the ligated sciatic nerve and the lesioned fimbria-fornix, accumulated NGF receptor was detected proximal and distal to the ligation/lesion site. The transported receptor presumably was located in sympathetic and sensory neurons in the sciatic nerve and in forebrain cholinergic neurons projecting from the medial septum to the hippocampus. In both anatomical sites, accumulation of NGF receptor on the proximal (anterograde) side occurred in streams of fine axonal processes, whereas staining on the distal (retrograde) side occurred in varicose or granular configurations. These results raise the possibility that the NGF receptor has a role in the mechanism of NGF beyond the initial binding event at the plasma membrane of the axonal terminus.     

Specificity of the retrograde axonal transport of nerve growth factor

The specificity of the retrograde axonal transport of nerve growth factor (NGF) has been investigated by injecting¹²⁵I-labelled NGF and similar proteins (both with respect to molecular weight and electrical charge at physiological pH) into the anterior eye chamber of adult mice. Previous studies have shown that the difference between the accumulation of radioactivity in the superior cervical ganglion of the injected and non-injected sides is a measure for retrograde axonal transport. Of all the proteins studied (NGF, cytochrome c, insulin, horseradish peroxidase, ovalbumin, bovine albumin, ferritin), NGF was the only which exhibited a statistically significant (2–3 fold) difference between injected and non-injected sides. The specificity of the retrograde transport of NGF was further supported by the finding that relatively small chemical changes, such as oxidation of the tryptophan moieties of the NGF molecule, resulted in a marked reduction of retrograde transport.It is concluded that the system for providing information to the cell body from the adrenergic nerve terminals in the form of NGF is highly specific, and that this specificity does not depend on general physico-chemical properties of the molecule such as size of the molecule and electrical charge at physiological pH. However, the information available so far does not allow one to decide as to whether the selectivity resides in the transport system itself or in the specificity of the uptake mechanism at the nerve terminals.     

The effect of the retrograde axonal transport of nerve growth factor on the morphology of adrenergic neurones.

An injection of nerve growth factor (NGF) into one eye of neonatal rats results in an increase in the tyrosine hydroxylase activity of the ipsilateral superior cervical ganglion. This effect was seen maximally after the intraocular injection of a depot preparation of NGF linked to cellulose. The sympathetic neurones that innervate the eye can be identified by autoradiography after the retrograde axonal transport of either NGF or tetanus toxin labelled with [125I]iodine. It was only those cells having their terminals in the vicinity of the depot preparation. This demonstrates that NGF transported from the periphery to the cell bodies is effectively retained within the transporting cell and is not released to act on extracellular receptors within the ganglion. It is suggested that this specificity of action for NGF reaching the ganglion in this fashion is important during normal development in determining the survival of adrenergic neurones.     

Binding and retrograde transport of leukemia inhibitory factor by the sensory nervous system.

Leukemia inhibitory factor (LIF), a peptide growth factor with multiple activities, has recently been shown to support the generation and survival of sensory neurons in cultures of mouse neural crest and dorsal root ganglia (DRG). We have conducted binding experiments with 125I-LIF on cultures of DRG to determine the receptor distribution for LIF on these cells and found that at least 60% of the sensory neurons in the cultures bound 125I-LIF, all of which could be eliminated by the addition of unlabeled LIF. The other cells in the culture, which morphologically appeared to be Schwann cells, did not bind appreciable quantities of 125I-LIF. In order to investigate whether LIF is retrogradely transported to sensory neurons in vivo, 125I-LIF was injected into the footpads and gastrocnemius muscles of newborn and adult mice, following sciatic nerve ligation. Radioactivity accumulated in the distal portion of the sciatic nerve, indicating retrograde transport of LIF. Subsequent experiments on mice with unligated sciatic nerves showed that 125I-LIF is specifically transported into the sensory neurons of the DRG. There was no apparent transport of 125I-LIF into motor neurons in the spinal cord. These experiments demonstrate that LIF can specifically bind to and be transported by sensory neurons and further support the idea that LIF acts as a target-derived neurotrophic factor, analogous to NGF.     

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.     

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.     

Different effects of nerve growth factor on cultured sympathetic and sensory neurons

Long-term cultures of dissociated nodose ganglion (NG) and superior cervical ganglion (SCG) neurons from newborn rabbits were used to compare their response to nerve growth factor (7S NGF). SCG neurons required added NGF for their survival and a concentration of 1 μg/ml was found to be optimal. NG neurons, on the other hand, survived well for a long term without addition of NGF, but its application (1 μg/ml) was found to be effective in accelerating the growth of fibers (neurites) and neuronal somata. It is concluded that unlike SCG, NG neurons do not depend on exogenous NGF but may require an intrinsic trophic-like factor which may be contained in the serum of the medium, emanating from glial cells or by metabolic cooperation between neurons.     

Biological actions of nerve growth factor in the peripheral nervous system

Since the discovery of nerve growth factor (NGF), its role in the physiology/pathophysiology of nerve function has been under intense investigation. More recently, the potential of recombinant human NGF (rhNGF) as a putative treatment for peripheral neuropathies, in particular diabetic polyneuropathy and HIV-associated sensory neuropathy, is being explored. In animal models of diabetes, depletion of endogenous NGF levels has been demonstrated in foot skin and skeletal muscle; these levels reduce further with increasing disease duration. Preclinical studies in animal models of diabetes have shown that administration of NGF can reverse or alleviate impairment in nerve function.     

Intraaxonal transport of horseradish peroxidase in the sympathetic nervous system

Following a single injection of horseradish peroxidase (HRP) into the superior cervical ganglion (SCG) of the rabbit, the uptake and anterograde transport of this label was confirmed in the ganglion cell bodies, postganglionic axons, and preterminal and terminal ending axons in the ciliary processes of the eye. From the same injection site the intraaxonal HRP reaction product was demonstrated in myelinated axons, presumably by retrograde transport.

Intracytoplasmic HRP was identified in large, single membrane-bound, dense vesicles predominantly in perinuclear orientation. Intraaxonal HRP appeared throughout, either within single membrane-bound round or oblong vesicles of variable sizes and densities. Frequently, the HRP vesicles in the axons revealed elaborate membranous subunits. A limited number of whole axons or axon fascicles were diffusely stained with HRP reaction product at or near the injection site. This phenomenon may be the result of membrane injury to neurons. The HRP label was found in small amounts in axons and terminals in the ciliary processes of the eye as early as 4 h following injection into the SCG, indicating a rapid anterograde transport of HRP from a single extracellular source. Likewise the HRP label disappeared from the ganglion cell bodies and processes by the 6th day following injection. The presence of numerous HRP-labeled myelinated and non-myelinated axons in the SCG confirms the bidirectional transport of HRP in the sympathetic nervous system.     

The effect of nerve growth factor (NGF) upon axoplasmic transport in sympathetic neurons of the mouse

To study a possible agent which controls axoplasmic transport, the rates of transport were examined in postganglionic sympathetic neurons in adult mice which had been treated with nerve growth factor (NGF). Rates were measured by observing the movement along the nerve of a front of radioactive material after a prior intraganglionic injection ofL-[4,5-³H]leucine. The faster of two rates observed in female mice increased from a control value of1.22 ± 0.48mm/h (N= 15) to an experimental value of 1.83 ± 0.53mm/h (N= 6; P < 0.005)following the intramuscular injection of a total of 34 μg NGF/g body weight. In contrast, the same treatment caused the slower of the two rates to decrease from a control value of2.32 ± 0.81mm/day (N = 12) to an experimental value of 1.63 ± 0.70mm/day (N= 12; P < 0.05). The rate of slow transport was also examined in male animals. Untreated male mice exhibit a lower rate of slow transport (1.08 ± 0.38mm/day (N= 9) than do untreated females of equal age (P < 0.001). The result that nerve growth factor increases the rate of the faster phase of transport, and decreases the rate of the slower phase, indicates that this protein exerts a selective effect on transport.To obtain an independent measure of the influence of NGF, the extent of incorporation ofL-[4,5-³H]leucine into tissue proteins of excised stellate ganglia was assessed after 1 h in organ culture. Animals treated with NGF showed increased incorporation of tritiated leucine. Incorporation of radioactive leucine was correlated with the rateof transport in both control animals (r = 0.61) animals treaetd with NGF (r = 0.67). Within the framework of normal biological function, NGF may exert a controlling influence on both phases of axoplasmic transport in sympathetic postganglionic neurons.