Rebecca C. Schreiber

Galanin expression increases in adult rat sympathetic neurons after axotomy

Changes in neuropeptide expression occur in sensory, motor, and sympathetic neurons following axotomy. The particular pattern of peptide changes that occurs varies among the three cell types. We have studied the regulation in the rat superior cervical ganglion of the expression of galanin, a peptide previously shown to increase in axotomized sensory and motor neurons. While normally only an occasional neuron exhibiting galanin-like immunoreactivity is found in this ganglion, at two days after transection of the postganglionic internal and external carotid nerves, immunostaining can be observed in many neurons throughout the ganglion. Similar changes are found when ganglia are placed in organ culture for two days. The distribution of immunostained neurons after section of only one of the postganglionic trunks suggests that changes in galanin-like immunoreactivity occur only within neurons whose axons are transected. None the less, even when both nerve trunks are transected, only about half of the neurons in the ganglion exhibit galanin-like immunoreactivity, indicating that only a proportion of the axotomized neurons exhibit a detectable response. The few immunostained neurons seen after section of the cervical sympathetic trunk may also represent axotomized neurons. Galanin-like immunoreactivity extracted from the ganglion co-chromatographs with authentic galanin, and the level of this immunoreactivity increases dramatically after axotomy and explantation, and modestly after decentralization. These same manipulations produce parallel increases in the level of galanin messenger RNA. Together, the findings indicate that the expression of galanin increases in sympathetic neurons after axotomy. Galanin is thus the first neuropeptide whose expression has been shown to increase after transection of all three types of peripheral axons that have been studied.

Monocyte chemoattractant protein (MCP)-1 is rapidly expressed by sympathetic ganglion neurons following axonal injury.

ED1-immunoreactive macrophages, absent from the superior cervical ganglia (SCG) of normal rats, appear in these ganglia within 48 h after postganglionic axotomy. Further, resident macrophages show changes after axotomy. Since chemokines function as chemoattractants and activators of leukocytes, the effects of axotomy on chemokine expression in the SCG were examined. Within 6 h after nerve transection, increases were seen in mRNA levels for monocyte chemoattractant protein (MCP)-1. MCP-1 mRNA was concentrated in a population of neurons, while MCP-1 protein was localized to endothelial cells. This axotomy-induced neuronal MCP-1 expression may trigger the infiltration and/or activation of macrophages in SCG after injury.

Chemical sympathectomy and postganglionic nerve transection produce similar increases in galanin and VIP mRNA but differ in their effects on peptide content

Large changes in neuronal gene expression occur in adult peripheral neurons after axonal transection. In the rat superior cervical ganglion, for example, neurons that do not normally express vasoactive intestinal peptide (VIP) or galanin do so after postganglionic nerve transection. These effects of axotomy could result from a number of aspects of the surgical procedure. To test the idea that the important variable might be the disconnection of axotomized neuronal cell bodies from their target tissues, we examined the effects of producing such a disconnection by means of the compound 6-hydroxydopamine (6-OHDA), a neurotoxin that causes degeneration of sympathetic varicosities and avoids many of the complications of surgery. Two days after 6-OHDA treatment, VIP and galanin immunoreactivities had increased two- and 40-fold, respectively. Nevertheless, these increases were substantially smaller than the 30- and 300-fold changes seen after surgical axotomy. When expression of VIP and galanin was examined at the mRNA level, however, comparable increases were found after either procedure. The results indicate that chemical destruction of sympathetic varicosities produces an equivalent signal for increasing VIP and galanin mRNA as does axonal transection. The differences in the neuropeptide levels achieved suggests that peptide expression after nerve transection is regulated both at the mRNA and protein levels.

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.

POLYAMINES INCREASE IN SYMPATHETIC NEURONS AND NON- NEURONAL CELLS AFTER AXOTOMY AND ENHANCE NEURITE OUTGROWTH IN NERVE GROWTH FACTOR-PRIMED PC12 CELLS

Following axonal damage, sympathetic neurons are capable of regenerating and reinnervating their target tissues. Some years ago exogenous administration of polyamines was shown to enhance this regeneration. Recently, it was found that axonal injury leads to a dramatic up-regulation of the expression of arginase I in sympathetic neurons. This enzyme catalyzes the conversion of arginine to ornithine, which can subsequently be converted to the diamine putrescine and, ultimately, to the polyamines spermidine and spermine. In the present study, using an antiserum that reacts with both spermidine and spermine, we have found an increase in polyamine levels in both neurons and non-neuronal cells in the superior cervical ganglion 2 and 5 days following transection of the ganglions postganglionic trunks. Using PC12 cells primed with nerve growth factor and then stripped off the culture dish and replated as a model system for axotomized sympathetic neurons, we found that spermidine treatment, with or without nerve growth factor, resulted in an increased percentage of cells with a neurite whose length was at least twice the diameter of the neurons cell body. These increases could be seen within 48 h and were still evident after 8 days. Together, these data support the possibility that endogenous polyamines are involved in the normal regeneration which occurs following sympathetic axonal damage.

Changes in Gene Expression in Adult Sympathetic Neurons after Axonal Injury

Publisher Summary This chapter summarizes the advances in the knowledge of the phenotype of axotomized peripheral neurons, concentrating on the phenotypic changes that occur in axotomized sympathetic neurons and the signals that trigger these changes. The decrease in tyrosine hydroxylase (TH) activity in axotomized sympathetic neurons is accompanied by a decrease in TH mRNA. Changes can also be found in the mRNA levels of other proteins involved in synaptic transmission. Decreases in transmitter synthesis also occur in axotomized sensory and motor neurons. In addition to changes in neurotransmitter-neuromodulator levels in peripheral neurons after axotomy, changes have been found in their complement of receptors for these signaling molecules. Axotomy of peripheral neurons also produces increases in expression of certain proteins known to be involved in regeneration, such as tubulin and growth-associated protein (GAP)-43. In addition, several neuropeptides, not normally expressed by these neurons, are induced in response to axotomy. Some of these peptides might promote neuronal survival and/or fiber outgrowth under conditions in which the neurons are deprived of their target-derived trophic factors. Two events involved in triggering these phenotypic changes following axotomy have been identified: the induction and release of leukemia inhibitory factor (LIF) in nonneuronal cells within the superior cervical ganglion (SCG) and/or at the site of nerve transection and the reduction in levels of nerve growth factor (NGF) in the SCG as a consequence of the disconnection of the neurons from their target tissues. Other evidence confirms that dramatic changes occur in the nonneuronal population of the SCG after axotomy. Evidence for an endogenous LIF-inducing factor has also been reported in the chapter. In addition to LIF induction, a reduction in NGF levels in the SCG also plays an important role in triggering these changes.