**Abstract**

The post-ganglionic sympathetic neurons play an important role in modulating visceral functions and maintaining homeostasis through complex and reproducible axonal and dendritic connections between individual neurons and with their target tissues. Disruptions in these connections and in sympathetic nervous system function are observed in several neurological, cardiac and immune-related disorders, which underscores the need for understanding the mechanisms underlying neuronal polarity, axonal growth and dendritic growth in these neurons. The goals of this chapter are to explore our current understanding of the various growth factors, their signaling pathways, downstream effectors and interplay between these pathways to regulate different stages of axonal and dendritic growth in sympathetic neurons.

**Keywords:** sympathetic neurons, growth factors, neurotrophins, cytokines, BMPs, axons, dendrites

## **1. Introduction**

The sympathetic nervous system is an important component of the peripheral autonomic nervous system responsible for controlling the visceral functions of the body to maintain homeostasis and the "flight or fight response" [1]. The sympathetic pathway is composed of two neurons – a preganglionic neurons located in the intermediolateral horn of the spinal cord, originating from the thoracolumbar region of the spinal cord and the postganglionic neuron that is, in most cases, located in the paravertebral sympathetic ganglia chain on either side of the spinal cord. Some of the preganglionic axons synapse with pre-vertebral sympathetic ganglia such as the celiac, mesenteric and pelvic ganglia, which innervate the gastrointestinal and urinary tracts and are not part of the sympathetic chain [2]. The superior cervical ganglia (SCG) is the first and the largest ganglia in the sympathetic chain and innervates most of the tissues in the head and neck region including the pineal gland, cerebral blood vessels, carotid body, vestibular system, muscles in the iris, lacrimal glands and piloerector muscles. Of the sympathetic neurons, SCG neurons are one of the most studied to understand various aspects of neuronal development in the peripheral nervous system. In recent years, the observation of autonomic dysfunction in many diseases such

as Parkinson's disease, cardiac disorders, multiple system atrophy, multiple sclerosis, diabetes and immune-related disorders, has renewed an interest in understanding neuronal development and maintenance of sympathetic neurons [3–11].

During early development, the precursors of the post-ganglionic sympathetic neurons are derived from the trunk neural crest cells, which then migrate ventrally along the neural tube, through the anterior portion of the sclerotome and coalesce near the dorsal aorta to form the sympathetic ganglia [12]. In rodents, the neural crest migration occurs between E8 and E11, with cells forming coalesced sympathetic ganglia around E12–E14 with the more rostral ganglia forming before the caudal ones. Studies on the early sympathetic neuron specification and neural crest migration show that growth factors such as neurotrophins, semaphorins and ephrins are important for migration of these neural crest cells, with bone morphogenetic proteins (BMPs) being important for their differentiation into sympathetic neuronal lineage. The exposure to BMPs leads to the induction o of transcription factors such as Phox2b, Mash1, Hand2, Gata3, Insm1, Sox4 and Sox 11, which lead to the survival of these neurons and their differentiation into noradrenergic neurons [12]. Following the specification of these neurons, the next crucial step to create a functional sympathetic network is the extension and maturation of axons and dendrites. In this chapter, we will explore the pathways that are important for establishing and refining axonal and dendritic arbors in sympathetic neurons.

## **2. Growth factors and signaling pathways involved in axonal growth**

Following the specification of sympathetic neurons, the first sign of neuronal polarity is the extension of a single axon from the cell body [13]. In rodents, although the initiation of axonal growth from sympathetic ganglia starts as early as E12, most of the axonal growth occurs around E14–E15, with target innervation continuing into first few weeks of postnatal life [13–15]. Axonal growth has three stages – initiation of axons from the post-ganglionic neurons, elongation of the axons towards the final targets and finally target innervation which involves branching as well as restriction of axonal growth. Research using cultured sympathetic neurons *in vitro* and *in vivo* studies have identified multiple growth factors, extracellular matrix molecules and downstream signaling targets, involved in different stages of axonal growth and axonal guidance, functioning either as activators or inhibitors of axonal growth. In this section, we will examine the various molecules and their roles in these three stages of axonal growth.

### **2.1 Hepatocyte growth factor**

Hepatocyte growth factor (HGF) or scatter factor is one of the few growth factors that appears to be involved in initiation of axonal growth in sympathetic neurons. Both HGF and its receptor Met tyrosine kinase are co-expressed in the sympathetic neurons throughout embryonic, starting as early as E12.5, with HGF being secreted by the sympathetic neurons and functioning as an autocrine regulator of axonal growth [16–18]. Treatment of cultured sympathetic neurons with HGF induces axonal growth and enhances the axonal growth promoted by nerve growth factor [16]. Also, inhibition of HGF activity through treatment with anti-HGF antibodies and *Met* signaling mutants show decreased axonal growth and branching compared to wildtype embryos [17–19]. Although HGF promotes survival of sympathetic neuroblasts, it is not necessary for the survival of post-mitotic sympathetic neurons [17]. Furthermore, *in vitro* studies and studies on docking site mutants for Met receptors suggest that HGF exerts its effects on axonal growth in mice through activation of the mitogen-activated protein kinase (MAPK) pathway and PI-3 K pathway [20]. Although lack of HGF signaling *in vivo* results in decreased axonal growth, it does not lead complete lack of axons in sympathetic neurons, suggesting the involvement of other factors in the first step of axonal growth.
