Tyrosine hydroxylase is expressed in a subpopulation of small dorsal root ganglion neurons in the adult mouse

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Abstract

The expression of tyrosine hydroxylase (TH) was studied in adult mouse dorsal root ganglia (DRGs) and spinal cord by means of immunohistochemistry and in situ hybridization. TH immunoreactivity and TH mRNA were present in 10–15% of lumbar DRG neurons, in most cases being small/medium-sized. Only very few of these neurons coexpressed calcitonin gene-related peptide (CGRP), and only around 6% bound isolectin B4 (IB4). Dopamine β-hydroxylase-positive(+) or aromatic amino acid decarboxylase (AADC)+ DRG neurons were rare and did not colocalize TH. No evidence for dopamine transporter expression was obtained. Axotomy of the sciatic nerve only showed a tendency towards reduction in the number of TH+ neurons. In the dorsal horn of the spinal cord, moderately dense and widespread TH+ nerve terminals were observed, mainly in the gray matter and they did not show a typical primary afferent pattern. Also, dorsal rhizotomy or peripheral axotomy had no apparent effect on TH-LI in the dorsal horn. In the skin, along with an abundant TH+ innervation of blood vessels and sweat gland acini, a number of fibers was observed in close relation to the skin surface, some even penetrating into the epithelium. These results demonstrate presence, in normal adult mouse DRGs, of a subpopulation of TH+, essentially CGRP- and IB4-negative small/medium-sized neurons. No evidence for transport of TH into central afferents was obtained, but the enzyme may be present in some sensory fibers in the skin. The fact that neither AADC nor the dopamine transporter could be visualized suggests of non-dopaminergic transmitter phenotype, but the levels of these two dopaminergic markers may be too low to be detected with the present methodology. A further alternative is that l-DOPA after release is extracellularly converted to dopamine.

Introduction

Tyrosine hydroxylase (TH) is the enzyme catalyzing the production of levo-DOPA (l-DOPA) from the amino acid tyrosine, and is the rate-limiting step in the catecholamine biosynthesis (Levitt et al., 1965, Nagatsu et al., 1964). TH is abundantly expressed in sympathetic neurons of the autonomic nervous system. Two other enzymes, aromatic amino acid decarboxylase (AADC) and dopamine β-hydroxylase (DBH), are also produced by sympathetic neurons, and they will act in sequence to synthesize dopamine (DA) and then noradrenaline (NA), the principal neurotransmitter of the sympathetic nervous system (see von Euler, 1971). In the mammalian adrenal gland and brain, and especially in the nervous system of certain other species such as frogs, a fourth enzyme, phenylethanolamine N-methyl transferase (PNMT), converts NA to adrenaline (Connett and Kirshner, 1970).

Early studies in sensory neurons demonstrated presence of a subpopulation of TH-positive (+) neurons in rat dorsal root ganglia (DRGs) and cranial sensory ganglia. Thus, a small number of small TH+ neuron profiles (NPs) was observed in DRGs (Miller Jonakait et al., 1984, Price, 1985, Price and Mudge, 1983, Vega et al., 1991), whereas larger numbers were detected in nodose and petrosal ganglia (Ichikawa et al., 1993, Katz and Black, 1986, Katz et al., 1983, Matsumoto et al., 2003). Since sensory neurons seem to lack DBH and PNMT (Katz et al., 1983, Katz et al., 1986, Price, 1985, Price and Mudge, 1983, Vega et al., 1991), and neurons in the petrosal ganglia have been shown to express AADC (Finley et al., 1992), the DA-synthesizing enzyme, it has been suggested that the TH+ cells in sensory ganglia have a dopaminergic phenotype.

In the mouse, only few studies have analyzed the expression and activity of catecholamines and their related enzymes in sensory neurons. Thus, enzymatic activity of the monoamino oxidase has been detected in DRGs (Dubovy et al., 1988), and some recent studies show expression of TH in embryonic mouse DRG sensory neurons (Forgie et al., 2000, Ichikawa et al., 2005).

In the present study, we have analyzed expression of TH in lumbar DRGs and some other tissues of the adult untreated mouse, as well as the effect of sciatic nerve transection and dorsal rhizotomy, using immunohistochemistry and in situ hybridization. In addition, we ‘phenotyped’ the TH+ DRG neurons using double-staining with antisera raised against calcitonin gene-related peptide (CGRP), galanin, the neuropeptide tyrosine (NPY), the NPY Y1 and -2 receptors (Y1R and Y2R, respectively), and also studying isolectin B4 (IB4) binding. Furthermore, we probed for a possible expression of DBH, AADC and the DA transporter (DAT). We also included galanin-overexpressing (GalOE) mice under the control of the DBH promoter and their littermates (wild type, WT), as a tool to further describe the identity of TH+ neurons in DRGs.

Section snippets

Animals

The experiments were performed on twenty-four wild type (WT) male NMRI mice (B and K, Stockholm, Sweden), as well as four male GalOE (Steiner et al., 2001) and four male WT littermate mice (all with a b.wt. of 20–30 g), the latter from Jackson Laboratories (Bar Harbour, MA, USA). The animals were maintained under standard conditions on a 12-h day/night cycle (light on 07:00 a.m.), with water and food ad libitum.

Axotomy

In eleven deeply anaesthetized mice (Isoflurane; Baxter Medical AB, Sweden; 2% for

DRGs, trigeminal and superior cervical ganglia

Approximately 15% of all mouse L5 DRG NPs were TH+ (14.5 ± 3.2, naive, n = 2) (Figs. 1a, c). They were mostly small, ranging between 100 to 600 μm2 and with a peak between 250 and 400 μm2 (Fig. 2). TH+ fibers were observed between cell bodies or in fiber bundles within the DRGs, and occasionally such fibers were observed to originate from TH+ NPs (Fig. 1c). Frequently, TH+ fibers were surrounding blood vessels (Fig. 1a), but only in few cases, pericellular baskets of TH+ fibers, of unknown

Discussion

In the present study, we have explored the presence and phenotype of a subpopulation of adult male mouse lumbar DRG neurons expressing TH, the rate limiting enzyme in the catecholamine synthesis. We have found that these neurons comprise 10–15% of all DRG neurons, are small in size and exhibit neither a ‘peptidergic’ nor a ‘non-peptidergic’ phenotype, as shown by a low degree of coexistence with CGRP and IB4 binding, respectively. Thus, these TH+ neurons seem to represent a novel and select

Acknowledgments

This study was supported by the Swedish Research Council (04X-2887), the Wallenberg Foundation, the Knut and Alice Wallenberg Foundation, the Wallenberg Consortium North, a Carrillo Oñativia Grant and the Austral University.

We are grateful to Drs. Davor Stanic, Jan Mulder, Brun Ulfhake, Britta Robertson and Esbjörn Bergman for valuable technical advice. We thank the late Professor Menek Goldstein, New York University Medical School, New York, NY, USA, for generous donation of rabbit TH

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