Transneuronal mapping of the CNS network controlling sympathetic outflow to the rat thymus

Abstract

The thymus is a primary immune organ that is essential for the development of functional T cells. The thymus receives sympathetic innervation, and thymocytes and thymic epithelial cells express functional adrenergic receptors. In this study, we employed retrograde, transneuronal virus tracing to identify the CNS cell groups that regulate sympathetic outflow to the thymus. Pseudorabies virus (PRV) was injected into the thymus, and the pattern of PRV infection in sympathetic regulatory centers of the CNS was determined at 72 and 120 h post-inoculation. PRV infection within the CNS first appeared within the spinal cord at 72 h post-inoculation and was confined to neurons within the intermediolateral cell column at levels T₁–T₇. At 120 h post-inoculation infection had spread within the spinal cord to include the central autonomic nucleus, intercalated cell nucleus and light infection within the cells of the lateral funiculus. Within the brain, PRV positive cells were found within nuclei of the medulla oblongata, pons and hypothalamus. Infection in the hypothalamus was observed within the arcuate nucleus, dorsal, lateral, and posterior hypothalamus and in all parvicellular subdivisions of the paraventricular hypothalamic nucleus. None of the infected animals exhibited labeling of the dorsal motor nucleus of the vagus. In summary, this study provides the first anatomic map of CNS neurons involved in control of sympathetic outflow to the thymus.

Introduction

One pathway by which the brain can regulate immune function is via the sympathetic nervous system (SNS). SNS nerve fibers innervate lymphoid organs and release the neurotransmitter norepinephrine in the vicinity of immune cells. Norepinephrine released from the varicosities of sympathetic axon terminals can influence immune responses such as cytokine production via activation of adrenergic receptors expressed on target cells such as T cells (Elenkov et al., 2000). The thymus is the major site for generation of naïve T cells, and is innervated by postganglionic nerve fibers of the SNS that release norepinephrine (Vizi et al., 1995). Several studies have demonstrated that thymocytes and thymic epithelial cells express functional adrenergic receptors (Colic et al., 2000, Cupic et al., 2003, Radojcic et al., 1991). For example, incubation of thymocytes with norepinephrine or adrenergic receptor agonists in vitro alters their apoptotic and proliferative responses, and the stability of Thy-1 mRNA (Colic et al., 2000, Gu et al., 2000, Wajeman-Chao et al., 1998). Additionally, norepinephrine affects the production of certain cytokines such as IL-6 by thymic epithelial cells that influence thymocyte development (Von Patay et al., 1999). Furthermore, variation in the density of noradrenergic nerves and in norepinephrine concentration in the thymus during pregnancy, post-partum (Kendall et al., 1994) and during aging in rodents (Cavallotti et al., 1999, Madden et al., 1997) is associated with altered thymic function.

The pattern of SNS innervation within the thymus has been extensively characterized. SNS nerve fibers of the thymus originate from postganglionic cell bodies that lie within the upper paravertebral ganglia of the sympathetic chain, principally the superior cervical and stellate ganglia (Bulloch and Pomerantz, 1984, Nance et al., 1987, Tollefson and Bulloch, 1990). The primary neurotransmitter of thymic sympathetic nerves is norepinephrine, as revealed by dopamine-β-hydroxylase staining of fibers, detection of NE in thymic tissue (Felten et al., 1985, Vizi et al., 1995) and vesicle-dependent release of NE by sympathetic nerve terminals in the thymus in response to electrical stimulation (Vizi et al., 1995). Postganglionic SNS nerve fibers enter the thymus in close association with surface arteries or through the capsule, and terminate in varicose plexuses in both the cortico-medullary junction and the thymic cortex (Cavallotti et al., 1999, Felten et al., 1985, Williams and Felten, 1981). Thymocyte progenitors enter the thymus through the vasculature endothelium at the cortico-medullary junction (Schwarz and Bhandoola, 2006) and SNS nerve fibers are primarily associated with the thymic vasculature in this region. In addition to the cortico-medullary junction, SNS fibers also extend to the subcapsular and cortical parenchyma, separate from the blood vessels and in proximity to thymocytes (Vizi et al., 1995). The densest plexuses are found in the outer cortex where the most immature thymocytes reside. Although the functional significance of thymic innervation is unknown, the vascularized and parenchymal regions that receive sympathetic innervation play critical roles in thymopoiesis.

Anatomical evidence for CNS control of sympathetic outflow to the thymus is lacking. In the present study we used the retrograde migration of the pseudorabies virus (PRV) to identify the CNS neurons that regulate sympathetic tone to the thymus. PRV virus, a neurotrophic alpha-herpes virus, has proved to be a powerful neuroanatomical tool in elucidating the hierarchical order of multi-synaptic CNS circuits (Aston-Jones and Card, 2000, Loewy, 1998). Retrograde spread of the virus occurs only in synaptically-linked chains of neurons, with higher order structures becoming labeled at later time-points (Jansen et al., 1993, Strack and Loewy, 1990). Additionally, viral replication within neuron cell bodies allows the virus to be a self-amplifying marker, and second, third and even fourth order neurons can be labeled within the brain of infected animals. Many groups have used this method to determine the CNS cell groups that regulate the SNS outflow to many visceral organs, and have established a common pattern of CNS circuitry involved in the regulation of sympathetic outflows to organs such as the adrenal gland, heart, kidneys, pancreas, and spleen (Cano et al., 2001, Huang and Weiss, 1999, Jansen et al., 1995, Jansen et al., 1997, Schramm et al., 1993, Standish et al., 1994, Strack et al., 1989b, Denes et al., 2005). The general regions labeled are the paraventricular hypothalamic nucleus (PVH), A5 noradrenaline cells, ventromedial medulla (VMM), ventrolateral medulla (VML), and caudal raphe nucleus (Strack et al., 1989a). It is unknown whether these general regions, and possibly other specific CNS cell groups, play a role in regulating sympathetic outflow to the thymus. The goal of the present study was to use the transneuronal viral tracer PRV to identify the neuronal populations in the CNS that are anatomically connected to the sympathetic innervation of the thymus.