Dopamine receptor mRNA and protein expression in the mouse corpus striatum and cerebral cortex during pre- and postnatal development

Abstract

The outcome of dopaminergic signaling and effectiveness of dopaminergic drugs depend on the relative preponderance of each of the five dopamine receptors in a given brain region. The separate contribution of each receptor to overall dopaminergic tone is difficult to establish at a functional level due to lack of receptor subtype specific pharmacological agents. A surrogate for receptor function is receptor protein or mRNA expression. We examined dopamine receptor mRNA expression by quantitative reverse transcription real-time PCR in the striatum, globus pallidus, frontal cortex and cingulate cortex of embryonic and postnatal mice. Samples of each region were collected by laser capture microdissection. D1- and D2-receptor mRNAs were the most abundant in all the regions of the mature brain. The D1-receptor was predominant over the D2-receptor in the frontal and cingulate cortices whereas the situation was reversed in the striatum and globus pallidus. In the proliferative domains of the embryonic forebrain, D3-, D4- and D5-receptors were predominant. In the corpus striatum and cerebral cortex, the D3- and D4-receptors were the only receptors that showed marked developmental regulation. By analyzing D1 receptor protein expression, we show that developmental changes in mRNA expression reliably translate into changes in protein levels, at least for the D1-receptor.

Introduction

Dopamine receptors are associated with a variety of cellular functions. In the mammalian central nervous system, dopamine receptor activation is critical for regulation of mood, motivation and motor function. Dopamine receptors are coupled to G-proteins. Typically, dopamine receptor activation leads to changes in intracellular cAMP levels (Monsma et al., 1990, Robinson and Caron, 1996, Schinelli et al., 1994) and triggers a signaling cascade that culminates in gene transcription (Gerfen, 2000). Based on G-protein partners and intracellular signaling mechanisms, two classes of dopamine receptors are recognized (Monsma et al., 1990, Robinson and Caron, 1996, Schinelli et al., 1994). The D1-like receptors are Gs/olf coupled and their activation results in increased intracellular cAMP. The D2-like receptors are Gi/o coupled and their activation decreases intracellular cAMP. Genes encoding five dopamine receptors have been cloned and based on genetic evidence, the D1-like receptors are further divided into D1- and D5-receptors (D1R and D5R, respectively) and the D2-like receptors into D2-, D3- and D4-receptors (D2R, D3R and D4R, respectively) (Sibley and Monsma, 1992). Although signaling via the adenylyl cyclase–cAMP system is the principal mode of action, dopamine receptors also activate phospho-lipase C via the Gq/11 system and increase intracellular calcium levels (Jin et al., 2003, Rashid et al., 2007). Dopamine receptors also interact with glutamate receptors (Cepeda and Levine, 1998, Lee et al., 2002, Schoffelmeer et al., 2000) and mobilize intracellular Ca²⁺ stores (Lezcano and Bergson, 2002, Tang and Bezprozvanny, 2004).

Activation of the D1-like and D2-like receptors typically produces opposite effects on intracellular signaling cascades and cell physiology (Stoof and Kebabian, 1984). A physiological balance between the activities of the two receptors is critical for normal neurological function. In fact, perturbation of the physiological balance appears to underlie a variety of neurological and neuro-psychiatric conditions such as Parkinson's disease, depression, schizophrenia, attention deficit hyperactivity, anxiety and drug addiction. Many of the pharmacological agents used in the management of these conditions produce their effects by selectively activating or antagonizing the D1-like or D2-like receptors. Therefore, understanding the relative abundance of each of the five dopamine receptors in a given brain region is helpful for understanding the overall impact of dopaminergic signaling during normal development as well as in disease states.

Accurate estimates of dopamine receptor levels and function in specific brain regions is hampered by low abundance of some of the receptor subtypes, lack of subtype-specific agonists or antagonists, and unreliable antibodies. Low receptor abundance and antibody non-specificity is a particular problem in the developing brain. A surrogate marker for receptor function is receptor mRNA expression. We have used real-time quantitative PCR to compare mRNA levels for each of the five dopamine receptors in different regions of the embryonic and postnatal brain. Samples of the different brain regions were collected by using laser capture microdissection (LCM). Using western blots, we estimated D1-receptor protein levels in the developing and mature striatum and cerebral cortex and compared the protein levels to mRNA levels. Our data show that all five receptor mRNAs are expressed in the proliferative and postmitotic domains as early as embryonic day 12 (E12). The mRNA levels change dynamically during development and each receptor and each brain region show independent patterns of developmental regulation. Furthermore, developmental changes in mRNA levels reliably translate into changes in protein levels, at least for the D1-receptor, validating the usefulness of mRNA measurement as a surrogate for receptor expression.