Expression and distribution of all dopamine receptor subtypes (D1–D5) in the mouse lumbar spinal cord: A real-time polymerase chain reaction and non-autoradiographic in situ hybridization study

doi:10.1016/j.neuroscience.2007.07.052

Neuroscience

Volume 149, Issue 4, 23 November 2007, Pages 885-897

https://doi.org/10.1016/j.neuroscience.2007.07.052 Get rights and content

Abstract

Dopamine is a catecholaminergic neuromodulatory transmitter that acts through five molecularly-distinct G protein–coupled receptor subtypes (D₁–D₅). In the mammalian spinal cord, dopaminergic axon collaterals arise predominantly from the A11 region of the dorsoposterior hypothalamus and project diffusely throughout the spinal neuraxis. Dopaminergic modulatory actions are implicated in sensory, motor and autonomic functions in the spinal cord but the expression properties of the different dopamine receptors in the spinal cord remain incomplete. Here we determined the presence and the regional distribution of all dopamine receptor subtypes in mouse spinal cord cells by means of quantitative real time polymerase chain reaction (PCR) and digoxigenin-label in situ hybridization. Real-time PCR demonstrated that all dopamine receptors are expressed in the spinal cord with strongly dominant D₂ receptor expression, including in motoneurons and in the sensory encoding superficial dorsal horn (SDH). Laser capture microdissection (LCM) corroborated the predominance of D₂ receptor expression in SDH and motoneurons. In situ hybridization of lumbar cord revealed that expression for all dopamine receptors was largely in the gray matter, including motoneurons, and distributed diffusely in labeled cell subpopulations in most or all laminae. The highest incidence of cellular labeling was observed for D₂ and D₅ receptors, while the incidence of D₁ and D₃ receptor expression was least. We conclude that the expression and extensive postsynaptic distribution of all known dopamine receptors in spinal cord correspond well with the broad descending dopaminergic projection territory supporting a widespread dopaminergic control over spinal neuronal systems. The dominant expression of D₂ receptors suggests a leading role for these receptors in dopaminergic actions on postsynaptic spinal neurons.

Section snippets

DA actions in the spinal cord

Descending dopaminergic projections are implicated in exerting antinociceptive actions (Fleetwood-Walker et al., 1988). These actions may occur at least in part by modulating activity on primary afferent terminals since DA has excitatory, inhibitory or mixed effects on dorsal root ganglia neurons (Gallagher et al 1980, Abramets and Samoilovich 1991, Molokanova and Tamarova 1995). Actions on spinal neurons themselves are also implicated since DA directly inhibits neurons in the pain-encoding

Animals and slide preparations

All experimental procedures complied with the NIH guidelines for animal care and the Emory Institutional Animal Care and Use Committee. Care was taken to minimize the number of animals used and their suffering. A total of 13 male C57BL/6 mice (postnatal day 14) were used for this study. For the ISH experiments, animals (n=10) were anesthetized with i.p. injection of urethane (2 mg/kg body weight) before being perfused with 1/3 volume:weight cold heparinized saline followed by equal

Results

Using the primers listed in Table 1, we first performed real-time PCR to assess the relative expression levels of DA receptors in the cord. Fig. 1A shows the relative expression values reported normalized to D₂ receptor expression in the whole spinal cord. Note that D₂ receptors are expressed at much higher values than the other DA receptors. The expression of D₁, D₃, D₄, and D₅ was significantly lower than the D₂ receptor (P<0.001). The low levels of D₃ and D₄ receptors may relate to a

Discussion

This study used real-time PCR and ISH to examine the expression properties of DA receptors in the lumbar spinal cord of young (day 14) mice. Specifically, we attempted to provide a map of their presence in the lumbar cord of juvenile animals, by describing and comparing the spatial distribution of the five known DA receptor subtypes. The principal findings are that; (i) all five receptors are expressed in the lumbar spinal cord with dominant D₂ receptor expression, (ii) the receptor

Acknowledgments

We thank Dr. Dapeng Cui and the laboratories of Drs. Gary Miller (Minzhen Wang) and Doug Eaton (Otor Al-Khalili) for assistance with real-time PCR. This project was supported by National Institute of Neurological Disorders and Stroke grant NS045248.

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¹: Present address: Emory University School of Medicine and Georgia Institute of Technology, Department of Biomedical Engineering, 313 Ferst Drive, Atlanta, GA 30332-0535, USA.

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NeuropharmacologyExpression and distribution of all dopamine receptor subtypes (D1–D5) in the mouse lumbar spinal cord: A real-time polymerase chain reaction and non-autoradiographic in situ hybridization study

Abstract

Section snippets

DA actions in the spinal cord

Animals and slide preparations

Results

Discussion

Acknowledgments

Eur J Pharmacol

Life Sci

Neuroscience

Brain Res

Neurosci Lett

Neuroscience

Neuroscience

Neuropsychopharmacology

Eur J Pharmacol

Brain Res

J Chem Neuroanat

Brain Res

Neuropharmacology

Brain Res

Brain Res

Dev Biol

Neurosci Lett

Methods

Biol Psychiatry

Neuroscience

Neuroscience

Neuroscience

Brain Res Bull

Neuroscience

Eur J Pharmacol

J Biol Chem

Brain Res

Neuroscience

Analysis of two types of dopaminergic responses of neurons of the spinal ganglia of rats

Neurosci Behav Physiol

Neuromodulation of the locomotor network by dopamine in the isolated spinal cord of newborn rat

Eur J Neurosci

Regional, cellular, and subcellular variations in the distribution of D1 and D5 dopamine receptors in primate brain

J Neurosci

Neuropharmacology
Expression and distribution of all dopamine receptor subtypes (D₁–D₅) in the mouse lumbar spinal cord: A real-time polymerase chain reaction and non-autoradiographic in situ hybridization study