Dorsal root ganglion neurons innervating pelvic organs in the mouse express tyrosine hydroxylase

Abstract

Previous studies in rat and mouse documented that a subpopulation of dorsal root ganglion (DRG) neurons innervating non-visceral tissues express tyrosine hydroxylase (TH). Here we studied whether or not mouse DRG neurons retrogradely traced with Fast Blue (FB) from colorectum or urinary bladder also express immunohistochemically detectable TH. The lumbar sympathetic chain (LSC) and major pelvic ganglion (MPG) were included in the analysis. Previously characterized antibodies against TH, norepinephrine transporter type 1 (NET-1) and calcitonin gene-related peptide (CGRP) were used. On average, ∼14% of colorectal and ∼17% of urinary bladder DRG neurons expressed TH and spanned virtually all neuronal sizes, although more often in the medium-sized to small ranges. Also, they were more abundant in lumbosacral than thoracolumbar DRGs, and often coexpressed CGRP. We also detected several TH-immunoreactive (IR) colorectal and urinary bladder neurons in the LSC and the MPG, more frequently in the former. No NET-1-IR neurons were detected in DRGs, whereas the majority of FB-labeled, TH-IR neurons in the LSC and MPG coexpressed this marker (as did most other TH-IR neurons not labeled from the target organs). TH-IR nerve fibers were detected in all layers of the colorectum and the urinary bladder, with some also reaching the basal mucosal cells. Most TH-IR fibers in these organs lacked CGRP. Taken together, we show: (1) that a previously undescribed population of colorectal and urinary bladder DRG neurons expresses TH, often CGRP but not NET-1, suggesting the absence of a noradrenergic phenotype; and (2) that TH-IR axons/terminals in the colon or urinary bladder, naturally expected to derive from autonomic sources, could also originate from sensory neurons.

Introduction

Visceral organs such as the colorectum and the urinary bladder are innervated both by sensory and autonomic neurons (see Robinson and Gebhart, 2008), classically grouped as either “intrinsic” or “extrinsic”. The former are found along the full extent of the gut, including the colorectum, and comprise enteric sensory and motor neurons residing within the ganglionic layers of the gut wall, creating an “intrinsic” neuronal network (Furness et al., 2004). “Extrinsic” neurons in rodents (as well as in humans) belong to a variety of neuronal systems: (1) peripheral projections of thoracolumbar (TL) (from the 8th thoracic to the 1st lumbar) and lumbosacral (LS) (from the 6th lumbar to the 2nd sacral) dorsal root ganglia (DRG) neurons (see Robinson and Gebhart, 2008); (2) postganglionic projections of sympathetic neurons in the lumbar sympathetic chain (LSC), or (3) sympathetic and parasympathetic neurons present in the ‘mixed’ major pelvic ganglion (MPG) (Furness, 2006, Keast, 2006). Fibers from the afferent sensory and efferent autonomic nervous systems travel together in the pelvic (LS) and lumbar splanchnic/hypogastric (TL) nerves. In recent studies, afferent fibers in these two nerves have been characterized in mouse colorectum (Brierley et al., 2004, Brierley et al., 2005) and urinary bladder (Xu and Gebhart, 2008) with respect to mechanosensitivity, and differentiated into mucosal, muscular/mucosal, muscular, mesenteric and serosal classes.

As shown both in rat (De Groat, 1987, Keast and De Groat, 1992, Callsen-Cencic and Mense, 1997, Wang et al., 1998, Keast and Stephensen, 2000, Christianson et al., 2006, Olsson et al., 2006) and mouse (Robinson et al., 2004, Christianson et al., 2006, Spencer et al., 2008, Brumovsky et al., 2011), colorectal and urinary bladder sensory neurons synthesize a variety of neurotransmitters and associated molecules. These include excitatory neurotransmitters such as glutamate and aspartate (Keast and Stephensen, 2000), the related vesicular glutamate transporters (VGLUTs) (Olsson et al., 2006, Brumovsky et al., 2011), neuropeptides such as the calcitonin gene-related peptide (CGRP) (De Groat, 1987, Keast and De Groat, 1992, Callsen-Cencic and Mense, 1997, Wang et al., 1998, Robinson et al., 2004, Hwang et al., 2005), pituitary adenylate cyclase-activating peptide (Wang et al., 1998), substance P and somatostatin (Wang et al., 1998) or galanin (Callsen-Cencic and Mense, 1997, Wang et al., 1998). Among several receptors involved in pain mechanisms, many colorectal and urinary bladder DRG neurons also express the transient receptor potential cation channel, subfamily V, member 1 (TRPV1) (Christianson et al., 2006, Spencer et al., 2008, La et al., 2011), a nonselective cation channel activated by pH, heat and capsaicin (Caterina et al., 1997).

Tyrosine hydroxylase (TH), the rate-limiting enzyme for the catecholamine (CA) synthesis (Nagatsu et al., 1964, Levitt et al., 1965), has been traditionally utilized to detect catecholaminergic neurons, both in the central and the peripheral nervous systems. In addition to TH, the majority of sympathetic neurons in the autonomic nervous system contain aromatic aminoacid decarboxylase (AADC) and dopamine (DA) β-hydroxylase (DβH) which are sequential in the synthesis of DA to norepinephrine (NE), the principal neurotransmitter of the sympathetic nervous system (see von Euler, 1971). Some sensory neurons also express TH, as demonstrated in rat nodose and petrosal ganglia (Katz and Black, 1986, Ichikawa et al., 1991, Kummer et al., 1993, Matsumoto et al., 2003) and non-visceral DRG neurons (Price and Mudge, 1983, Jonakait et al., 1984, Price, 1985, Vega et al., 1991, Herradon et al., 2008, Kobayashi et al., 2010). The presence of TH has also been confirmed in mouse embryonic (Forgie et al., 2000, Ichikawa et al., 2005) and adult lumbar DRG neurons innervating non-visceral structures such as the glabrous (Brumovsky et al., 2006) and hairy hindpaw skin (Brumovsky et al., 2006, Li et al., 2011).

In the present study we investigated whether or not mouse visceral sensory neurons, identified by retrograde tracing with Fast Blue (FB) from the colon or the urinary bladder, also express TH. Thus, by means of immunohistochemistry, we explored the presence of this enzyme in visceral DRG, LSC and MPG neurons. We also examined colocalization of TH with CGRP in these neurons, as well as with the NE transporter type 1 (NET-1), the latter involved in the re-uptake of NE into presynaptic nerve terminals (Pacholczyk et al., 1991). Finally, we studied the distribution of axons/terminals containing TH in all layers of the colorectum and urinary bladder, including the mucosal layer.