Activating transcription factor 3 induction in sympathetic neurons after axotomy: response to decreased neurotrophin availability.

Activating transcription factor 3 (ATF3) is induced in a high proportion of axotomized sensory and motor neurons after sciatic nerve transection. In the present study, we looked at the expression of this factor in the superior cervical ganglion (SCG) after axotomy and after other manipulations that induce certain aspects of the cell body response to axotomy. Sympathetic ganglia from intact rats and mice exhibit only a very occasional neuronal nucleus with activating transcription factor 3-like immunoreactivity (ATF3-IR); however, as early as 6 h and as late as 3 weeks postaxotomy, many of the neurons showed intense ATF3-IR. A second population of cells had smaller and generally less intensely stained nuclei, and at least some of these cells were satellite cells. Lesions distal to the SCG induced by administration of 6-hydroxydopamine or unilateral removal of the salivary glands produced increases in ATF3-IR similar to those seen after proximal axotomy, indicating that this response is not strictly dependent on the distance of the lesion from the cell body. Two proposed signals for triggering ATF3 expression were examined: reduction in nerve growth factor (NGF) availability and induction of the cytokine leukemia inhibitory factor (LIF). While administration of an antiserum raised against NGF to intact animals induced ATF3-IR, induction of ATF3-IR after axotomy was not reduced in LIF null mutant mice. Since axotomy, 6-hydroxydopamine, and sialectomy are known to decrease the concentration of NGF in the SCG, our data suggest that these decreases in NGF lead to increases in ATF3-IR. Furthermore, since the number of neurons in the SCG expressing ATF3-IR was greater after axotomy than after antiserum against NGF treatment, this raises the possibility that decreased NGF is not the only process regulating ATF3 expression after axotomy.

Reduction in nerve growth factor availability leads to a conditioning lesion-like effect in sympathetic neurons.

Axotomized peripheral neurons are capable of regeneration, and the rate of regeneration can be enhanced by a conditioning lesion (i.e., a lesion prior to the lesion after which neurite outgrowth is measured). A possible signal that could trigger the conditioning lesion effect is the reduction in availability of a target-derived factor resulting from the disconnection of a neuron from its target tissue. We tested this hypothesis with respect to nerve growth factor (NGF) and sympathetic neurons by administering an antiserum to NGF to adult mice for 7 days prior to explantation or dissociation of the superior cervical ganglion (SCG) and subsequently measuring neurite outgrowth. The antiserum treatment dramatically lowered the concentration of NGF in the SCG and increased the rate of neurite outgrowth in both explants and cell cultures. The increase in neurite outgrowth was similar in magnitude to that seen after a conditioning lesion. To determine if exogenous NGF could block the effect of a conditioning lesion, mice were injected with NGF or cytochrome C immediately prior to unilateral axotomy of the SCG, and for 7 days thereafter. A conditioning lesion effect of similar magnitude was seen in NGF-treated and control animals. While NGF treatment increased NGF levels in the contralateral control ganglion, it did not significantly elevate levels in the axotomized ganglion. The results suggest that the decreased availability of NGF after axotomy is a sufficient stimulus to induce the conditioning lesion effect in sympathetic neurons. While NGF administration did not prevent the conditioning lesion effect, this may be due to the markedly decreased ability of sympathetic neurons to accumulate the growth factor after axotomy.

A conditioning lesion enhances sympathetic neurite outgrowth.

Axonal regeneration can be influenced by a conditioning lesion (an axonal injury made prior to a second test lesion). Previously, sympathetic neurons in vivo were shown to respond to a conditioning lesion with decreased neurite outgrowth, in contrast to the enhanced outgrowth observed in all other peripheral neurons examined. The present experiments tested the effects of a conditioning lesion on neurite outgrowth in vitro from the superior cervical ganglion (SCG) and the impact of several factors on that response. Ganglia axotomized 1 week earlier and then explanted in Matrigel or collagen gel responded with a significant increase in neurite extension compared to sham-operated ganglia. A distal axotomy produced by unilateral removal of the salivary glands (sialectomy) caused an increase in neurite outgrowth similar to that of a proximal axotomy. These conditioning lesions induced both an increase in the rate of elongation, and, in the case of the proximally axotomized SCG, a shorter initial delay of outgrowth. The enhanced outgrowth following sialectomy was specific to the nerve containing the majority of axons projecting to the salivary glands, suggesting that the conditioning lesion effect is restricted to previously injured neurons. Deletion of the gene for leukemia inhibitory factor (LIF), a gene induced by axotomy, did not abolish the conditioning lesion effect in SCG explants or dissociated cell cultures. In conclusion, sympathetic neurons are capable of responding to a conditioning lesion with increased neurite outgrowth. The hypothesis that the neuronal cell body response to axotomy plays an important role in the conditioning lesion response is discussed.

The effects of deafferentation and exogenous NGF on neurotrophins and neurotrophin receptor mRNA expression in the adult superior cervical ganglion.

Levels of nerve growth factor (NGF) and neurotrophin-3 (NT-3) protein and neurotrophin receptor mRNA in adult sympathetic neurons were investigated following surgical removal of preganglionic input and/or in vivo administration of NGF. Expression of trkC and p75, but not trkA, was significantly decreased following a 3-week deafferentation of the superior cervical ganglion (SCG). Protein levels of NGF and NT-3 in the SCG were unchanged by deafferentation. A 2-week intracerebroventricular infusion of NGF without deafferentation resulted in enhanced mRNA levels of trkA, trkC, and p75 as well as significantly increased NGF and NT-3 protein in the SCG. When NGF infusion followed deafferentation, both trkA and p75 showed significant increases while trkC levels were similar to control values. NGF protein was not increased in the SCG when deafferentation preceded exogenous NGF, yet NT-3 was elevated and levels were similar to cases receiving NGF infusion only. These results support a role for preganglionic input in trkC and p75 expression in adult sympathetic neurons. The increased levels of NT-3 protein and trkC gene expression observed following NGF infusion suggest that NGF influences NT-3 regulation in adult sympathetic neurons. In addition, the present findings provide evidence that, when preganglionic input is removed prior to the NGF infusion, NT-3 effectively competes with NGF for trkA binding. Taken together, we propose that NT-3 may play a role in the robust sprouting of sympathetic cerebrovascular axons previously observed following NGF administration, particularly when deafferentation precedes the NGF infusion period.

Sympathetic ingrowth to the trigeminal ganglion following intracerebroventricular infusion of nerve growth factor.

The objective of the present study was to examine the remodeling of uninjured sympathetic axons in the adult rat trigeminal ganglion following a 2-week in vivo intracerebroventricular infusion of NGF. The accumulation of infused NGF in the trigeminal was assessed using ELISA and sympathetic fibers were localized immunohistochemically with an antibody to tyrosine hydroxylase (TH). In addition, high performance liquid chromatography coupled with electrochemical detection (HPLC-ECD) allowed for biochemical measurements of the catecholamines norepinephrine (NE) and dopamine (DA). Increased NGF protein in the trigeminal ganglion was paralleled by a significant increase in sympathetic fibers and pericellular plexuses (i.e. baskets) in the cell body regions. Some ganglia showed elevated NE following NGF infusion, yet the 88% increase in mean NE did not reach significance. Following bilateral removal of the sympathetic superior cervical ganglia (SCG), a significant reduction was observed in overall NE levels and in TH-immunoreactive (-ir) fibers in the cell body regions and peripheral branches, suggesting the SCG as the origin of the sympathetic ingrowth. However, mean DA levels as well as TH-ir fibers within the trigeminal central branch were unaffected by NGF infusion or removal of the SCG and likely resulted from intrinsic dopaminergic cell bodies. In conclusion, our data provide evidence that the increased availability of NGF in the young adult rat trigeminal ganglion observed following in vivo NGF infusion enhanced sympathetic associations with the sensory neurons in the trigeminal, supporting a role for NGF in the regulation of sympathosensory interactions.

Evidence that nerve growth factor mediates the formation of sensory pericellular baskets in the rat trigeminal ganglion.

A role for nerve growth factor (NGF) in the remodeling of sensory neurons in the trigeminal ganglion was examined. Intracerebroventricular NGF infusion and/or bilateral removal of the sympathetic superior cervical ganglia, both of which are believed to increase the availability of NGF to primary sensory neurons, resulted in a significant increase in the frequency of calcitonin gene-related peptide immunoreactive pericellular baskets. The results of this study suggest that increased NGF is sufficient to enhance the formation of sensory baskets in this ganglion, and provide evidence that NGF may mediate the formation of sensory baskets in the sensory ganglia following injury.

Evidence for reduced accumulation of exogenous neurotrophin by aged sympathetic neurons.

The present study investigated the potential for neurotrophin uptake by cerebrovascular axons and subsequent accumulation in the aged superior cervical ganglion (SCG) following a two week intracerebroventricular infusion of nerve growth factor (NGF). In the SCG from aged rats, NGF protein levels declined significantly compared with the SCG from young adult rats. Following NGF infusion, perivascular axons from both young adult and aged rats showed intense NGF immunostaining. In addition, significant increases in NGF protein were shown using enzyme-linked immunosorbent assay (ELISA) and in counts of NGF immunopositive cell bodies in the SCG when compared with age-matched controls. NGF accumulation in ganglia from aged rats, however, was significantly less when compared with ganglia from young adult rats. The results of the present study suggest that NGF protein is significantly reduced in aged ganglia with the neurons retaining some capacity to take up and transport exogenous neurotrophin. Even so, the potential for NGF accumulation is dramatically reduced in aged rats when compared with that of young adult rats. While previous results have shown robust NGF-induced neurotransmitter responses by sympathetic neurons from the aged animal, the present finding of reduced accumulation of NGF in aged sympathetic neurons suggests an age-related difference in the utilization or transport of NGF.