After axotomy, substance P and vasoactive intestinal peptide expression occurs in pilomotor neurons in the rat superior cervical ganglion

doi:10.1016/S0306-4522(99)00576-X

Neuroscience

Volume 96, Issue 3, March 2000, Pages 611-618

https://doi.org/10.1016/S0306-4522(99)00576-X Get rights and content

Abstract

Autonomic sympathetic postganglionic neurons normally express distinct combinations of neuropeptides which are often highly correlated with the projection of the neurons. When sympathetic postganglionic neurons are axotomized, they can express quite different neuropeptides, notably substance P, vasoactive intestinal peptide or galanin. In this study, we have examined rat sympathetic postganglionic neurons in the superior cervical ganglion that project to the skin, the vasculature of the skeletal muscle or to the submandibular salivary gland, and assessed whether the neuropeptides that they express after axotomy depend on which target tissue they previously innervated. In all three populations, around half of the postganglionic neurons expressed galanin after axotomy. In contrast, only skin-projecting neurons showed a significant increase in the number of neurons that expressed substance P (22%) and vasoactive intestinal peptide (17%) following axotomy. Within the skin-projecting neurons, as judged on the basis of cell body size, substance P and vasoactive intestinal peptide were expressed predominantly in pilomotor neurons, but only rarely were the two neuropeptides present in the same nerve cell body.

In conclusion, we have demonstrated that three different neuropeptides, which can be induced by axotomy in postganglionic neurons, follow quite different patterns of expression when they are viewed in relation to the function of the postganglionic neurons in the superior cervical ganglion.

Section snippets

Retrograde tracer injections

Adult male Sprague–Dawley rats (36–52 days old) were used for the following experiments. All experiments were approved by and conformed to the guidelines of the University of Melbourne Animal Experimentation Ethics Committee. Every effort was made to minimize the numbers and any suffering of animals used in the following experiments. The exact numbers used in each experiment are given in Table 1, Table 3. Animals were anaesthetized by intramuscular (i.m.) injection with a mixture of ketamine and

Neuropeptide expression in control ganglia

Immunohistochemical analysis revealed that peptide expression was very rare in control SCGs. No VIP or substance P immunoreactivity was seen in neurons retrogradely labelled from any of the three targets and galanin immunoreactivity was only present in 2.2% of the neurons projecting to forehead skin (Table 1).

Neuropeptide expression following nerve injury

Retrograde tracing studies using either Fast Blue or cholera toxin produced identical results. The results from experiments using either of these methods have hence been combined in the

Discussion

The most significant finding of this study was that the pattern of expression of neuropeptides by rat sympathetic postganglionic neurons following axotomy is related to the targets, and hence functions, of the neurons. Neurons projecting to the skin showed an increased expression of either substance P or VIP immunoreactivity following axotomy, while neurons projecting to the vasculature of skeletal muscle and to the salivary gland showed no significant increase in substance P or VIP expression.

Acknowledgements

This work was supported by a National Health and Medical Research Council project grant. We would like to thank Prof. Elspeth McLachlan for helpful discussions and Dr Heather Young for critical comments on the manuscript.

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For example, vasoactive intestinal peptide (VIP)(Roudenok et al., 2001) and Galanin (GAL) (Ewert et al., 2008; Habecker et al., 2005) increase after myocardial infarction in cardiac sympathetic neurons. Axotomy stimulates expression of VIP, substance P (SubP), pituitary adenylate cyclase activating peptides (PACAP), and GAL in the superior cervical ganglion (Bergner et al., 2000; Boeshore et al., 2004; Hyatt-Sachs et al., 1993; Klimaschewski et al., 1994; Rao et al., 1993; Schreiber et al., 1994) by activation of the gp130 cytokine receptor (Habecker et al., 2009; Heinrich et al., 1998). A number of gp130 cytokines are elevated in the heart following myocardial infarction, including Interleukin-6 (IL-6), Leukemia Inhibitory Factor (LIF), and Cardiotrophin-1 (CT-1)(Aoyama et al., 2000; Frangogiannis et al., 2002; Gwechenberger et al., 1999), where they might stimulate neuropeptide expression in cardiac sympathetic nerves.
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Peripheral projections of NESP55 containing neurons in the rat sympathetic ganglia
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The peripheral projections of neurons expressing neuroendocrine secretory protein 55 (NESP55), a novel member of the chromogranin family, were studied by retrograde tracing technique. It was found that NESP55 positive neurons in the rat superior cervical ganglion projected to a number of targets including the submandibular gland, the cervical lymph nodes, the forehead skin, the iris, but not to the thyroid. Among these NESP55 positive target-projecting neurons, a subpopulation contained neuropeptide Y (NPY), a vasoconstrictor. Forepaw pad projecting neurons were found exclusively in the stellate ganglion, almost all of which (approximately 90%) were immunoreactive to NESP55. Colocalization of NESP55 and calcitonin gene-related peptide (CGRP), a peptide involved in sudomotor effects, was observed in a subpopulation of these paw pad projecting neurons, as was colocalization of NESP55 and NPY. The data suggest that NESP55 may have a functional role in some populations of sympathetic neurons.
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Neuroendocrine secretory protein 55 (NESP55) is a soluble, acidic and heat-stable protein, belonging to the class of chromogranins. It is expressed specifically in endocrine cells and the nervous system, and is probably involved in both constitutive and regulated secretion. In the present study, we investigated the distribution of NESP55 in various rat sympathetic ganglia by immunohistochemistry. The expression of NESP55-IR was detected in a subpopulation of principal neurons in the rat SCG, which was also TH positive, and, thus, adrenergic. In the rat stellate ganglion, more than two thirds of NESP55 positive neurons were adrenergic. Colocalization of NESP55 and calcitonin gene-related peptide (CGRP) in cholinergic neurons was also observed. In the rat thoracic chain, however, the majority of NESP55 positive neurons appeared to lack TH. No detectable NESP55-IR was found in the mouse SCG. Furthermore, in the sexually dimorphic SCG, it was demonstrated that, 80% of the NESP55 positive principal neurons were also NPY positive in the male rat, while a slightly higher, but statistically significant proportion, 87%, was found in the female. Whether or not this small difference is physiologically significant is unknown. The present data provide basic knowledge about the expression of NESP55 in the sympathetic autonomic nervous system of rat, which may further our understanding of the functional significance of NESP55.
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SP immunoreactivity has been demonstrated in the rhinencephalon, telencephalon, basal ganglia, hippocampus, amygdala, septal areas, diencephalon, hypothalamus, mesencephalon, metencephalon, pons, myelencephalon and spinal cord (Shults et al., 1984). Nerve fibres containing SP-like immunoreactivity are common in most autonomic ganglia (Helke et al., 1982; Helke and Phillips, 1988; Bergner et al., 2000), and SP immunoreactivity has also been described in trigeminal and dorsal root ganglia (Lee et al., 1985; Gibbins et al., 1987) and intrinsic neurons of the gut (Sternini et al., 1995). In the autonomic ganglia SP is thought to play a modulatory role, the best characterised response being observed in guinea pig inferior mesenteric ganglion.
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BB rats lose >50% of their islet sympathetic nerve terminals soon after diabetes onset, markedly impairing the glucagon response to activation of these nerves. In this study, we sought evidence that this degree of disease-induced nerve terminal damage affected their neuronal cell bodies. Increased galanin expression was used as a marker of the change of phenotype that occurs in neuronal cell bodies when their axons are severely damaged. The celiac ganglion (CG) was analyzed because it is a major source of the sympathetic nerves that project to the pancreatic islets. But we first needed to determine if damaging nerve terminals could increase galanin expression in this ganglion and, if so, when that expression was maximal. Severe, global nerve terminal damage produced a dramatic increase of CG galanin expression which was maximal 5 days later. We next determined if a global, but partial, nerve terminal loss would also increase galanin expression and found a significant increase of galanin mRNA and its peptide in the CG. Finally, we determined if the disease-induced, partial and islet-selective loss of nerve terminals seen in BB diabetic rats was sufficient to increase galanin: we, again, found a significant increase of galanin mRNA and its peptide in their CG. These increases did not occur in their superior cervical ganglia. We conclude that the selective damage to islet sympathetic nerve terminals seen in BB diabetic rats, rather than the systemic factors of diabetic hyperglycemia or insulin deficiency, causes the increased galanin expression observed in the CG of this animal model of type 1 diabetes.

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View full text

After axotomy, substance P and vasoactive intestinal peptide expression occurs in pilomotor neurons in the rat superior cervical ganglion

Abstract

Section snippets

Retrograde tracer injections

Neuropeptide expression in control ganglia

Neuropeptide expression following nerve injury

Discussion

Acknowledgements

J. auton. nerv. Syst.

Devl Biol.

Neuroscience

Brain Res.

Prog. Neurobiol.

Neuron

Neuroscience

Neuropeptides

Molec. cell. Neurosci.

Brain Res.

Expl Neurol.

Neuropeptides in the sympathetic system: presence, plasticity, modulation, and implications

Ann. Neurol.

Distribution of tyrosine hydroxylase and neuropeptide Y-like immunoreactive neurons in rabbit medulla oblongata, with attention to colocalisation studies, presumptive adrenaline-synthesizing perikarya, and vagal preganglionic cells

J. comp. Neurol.