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Widespread expression of functional D1-dopamine receptors in fetal rat brain

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Abstract

Maternal treatment with cocaine or the D1-dopamine receptor agonist, SKF 38393, induces expression of the immediate-early gene, c-fos, in fetal rodent brain. Our previous studies have focused on the suprachiasmatic nucleus late in gestation. In the present report, we examined the anatomical distribution of functional D1-dopamine receptors throughout fetal rat brain. Functional D1 receptors were defined using three complementary methods: in situ hybridization to detect D1 receptor mRNA, autoradiographic detection of 125I-SCH 23982 binding, and in situ hybridization to detect c-fos gene expression induced by maternal treatment with SKF 38393. D1-dopamine receptor binding, receptor mRNA, and SKF 38393-induced c-fos gene expression are widespread in fetal brain by late gestation. These data indicate that the fetal brain is sensitive to dopamine receptor activation, and suggest that gestational exposure to drugs of abuse acting via dopaminergic mechanisms may influence fetal brain function.

Introduction

The suprachiasmatic nucleus (SCN) contains a biological clock which regulates circadian rhythmicity in mammals [15]. The biological clock within the SCN is functioning as an oscillator prior to birth in rodents, and its timing is set (e.g., the clock is “entrained”) prior to birth [22]. It appears that redundant signals from the mother are normally involved in entraining the fetal SCN [22]. Two manipulations have been identified which can set the phase of the fetal biological clock; these are periodic injections of either melatonin or the D1-dopamine receptor agonist, SKF 38393 5, 29. Both D1-dopamine receptors and melatonin receptors are expressed in the developing SCN 3, 7, 27, 29, 30, 32. The prenatal entrainment of the SCN by D1 receptor activation directly demonstrates that functionally relevant D1-dopamine receptors are present within the fetal brain.

The extent to which functional D1-dopamine receptors are present in other regions of fetal brain has received relatively little attention. Demonstration of ligand binding, by itself, is of limited value in assessing “functional receptors” in fetal brain, as ligand binding does not necessarily indicate the presence of coupling to appropriate transduction pathways. Our results with the dopamine system in the SCN suggest another way to define functional receptors, involving stimulus-induced expression of c-fos mRNA. Fos, the protein product of the c-fos gene, can function as a transcription factor by hetero-dimerization with the products of other immediate-early genes, most notably members of the Jun family. Immediate-early gene expression links transient events at the cell surface (e.g., receptor activation) to long-lasting cellular responses 20, 28. Induction of immediate-early genes, including c-fos, has been used as a marker of “stimulation” of specific neuronal populations. This molecular approach to “metabolic mapping at the cellular level” is well-suited to studies of developing brain, and can be used to define the presence of functional receptors in fetal brain 4, 14, 30.

In the course of our studies of the SCN, we noted that the anatomical pattern of c-fos gene expression following treatment of the mother with SKF 38393 was not restricted to the SCN, but also included the striatum and dorsal endopiriform nucleus in rats, hamsters, and mice 29, 30, 31. The objective of the present studies was to examine the distribution of functional D1-dopamine receptors in fetal rat brain, without limiting our focus to the SCN. Functional D1 receptors were defined using three complementary methods: in situ hybridization to detect D1 receptor mRNA, autoradiographic detection of 125I-SCH 23982 binding to D1 receptors, and in situ hybridization to detect c-fos gene expression induced by maternal treatment with SKF 38393. The results indicate that functional D1-dopamine receptors are present in a variety of sites in the fetal brain.

Section snippets

Animals

Timed-pregnant Sprague-Dawley rats (MBM:VAF, Zivic Miller Laboratories, Zelienople, PA) were housed inside environmental compartments (22°C) in which the light-dark cycle consisted of 12 h light:12 h dark, with lights on at 06.00 h EST (LD). During all phases of the lighting cycle, animals were exposed to dim red (620 nm) light from special fluorescent tubes (Litho-light #2, Chemical Products, N. Warren, PA). All experiments were conducted in the afternoon, during the light phase of the

c-fos gene expression in fetuses of vehicle-treated dams

Expression of c-fos mRNA was restricted to a few sites in brains of fetuses whose mothers received vehicle 40 min before tissue collection on GD 20 (Fig. 1, Table 1). The suprachiasmatic nuclei (SCN), piriform and entorhinal cortices, arcuate nucleus, superficial layers of superior colliculus, locus coeruleus, pontine nucleus, facial nucleus, trigeminal nuclear group, and prepositus hypoglossal nucleus, and pineal gland contained low levels of specific hybridization. Neuroepithelia of the

Definition of functional dopamine receptors in the fetal brain

Previous reports of D1 receptor binding in developing brain using 3H-SCH 23390 have not reported an extensive distribution in the fetus. Indeed, Schambra et al. [27]concluded that there appears to be a delay between transcription and translation of the D1 receptor mRNA. We feel that the apparent discrepancy between D1 receptor mRNA and D1 receptor binding reported previously is due to the use of a tritiated ligand with relatively low specific activity (70–90 Ci/mmol). The use of a

Acknowledgements

We thank Michael E. Greenberg and J. Stephen Fink for supplying cDNAs, Paul Batista and Sam Riley of the MGH Photography Lab for assistance in preparation of the figures, Scott A. Rivkees for performing preliminary autoradiographic studies, and Steven M. Reppert for his support and helpful discussions. This work was supported by NIH Grants HD14427 to S.M. Reppert and HD29470 to D.R.W.

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Present address: Circadian, Neuroendocrine, and Sleep Disorder Section, Brigham and Women's Hospital, Boston, MA 02115, USA.

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