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Quantitative in situ hybridization allows measurement of mRNA level modifications in a variety of experimental conditions. This analysis may be performed both at the regional anatomical and cellular levels by densitometry, neuronal counting and silver grain measurements.

Dopamine transporter knock-out mice display locomotor hyperactivity due to increased extracellular striatal levels of dopamine. Hyperdopaminergic activity within this mesolimbic pathway is involved in the rewarding properties of morphine which are also increased in these mice. Due to the hyperdopaminergia, profound alterations in gene expression for dopamine receptors and neuropeptides are observed in the caudate putamen and nucleus accumbens. Here we investigated (1) the levels of mu-, delta- and kappa-opioid receptors mRNAs in normal mice from gestational day 13 (G13) to adult, and (2) the adaptive changes in the expression of these receptors in mice lacking the dopamine transporter. Our results show that, in wild-type mice, mu-opioid receptor mRNA expression appears early during development (G13) with a homogeneous distribution that evolves towards a patchy distribution in adult. Delta-opioid receptor mRNA appears only at G17 and kappa-opioid receptor mRNA is not observed before adulthood. The levels of delta-opioid receptor mRNA are not modified during development in knock-out mice compared to wild-type, but are increased in adult caudate putamen (+39%, P<0.05) and nucleus accumbens (+66%, P<0.05) at a time when these receptors are believed to be functional. The mu- and kappa-opioid receptors mRNA levels are not modified in the adult knock-out mice. In addition, we observed no differences in any opioid receptor mRNA level in dopamine transporter knock-out mice during prenatal ontogeny compared to wild-type. Our results constitute a detailed neuroanatomical description of opioid receptor mRNA expression from the time of their appearance during prenatal development until adulthood. Furthermore, we show here that chronic constitutive hyperdopaminergia only affects delta-opioid receptor mRNA levels in adult. Even if the propensity of knock-out mice to show increased rewarding properties to morphine seems to be mainly due to the substantial and further increase in hyperdopaminergic activity following drug treatment, the involvement of increased delta-opioid receptor mRNA levels in this behavior remains to be elucidated.

In situ hybridization experiments were performed in rat brain sections from normal and 6-hydroxydopamine-treated rats in order to map and identify the neurons expressing the D1 receptor gene in the striatum and the substantia nigra. Procedures of combined in situ hybridization, allowing the simultaneous detection of two mRNAs in the same section or in adjacent sections, were used to characterize the phenotypes of the neurons expressing the D1 receptor gene. D1 receptor mRNA was found in neurons all over the caudate-putamen, the accumbens nucleus, and the olfactory tubercle but not in the substantia nigra. In the caudate-putamen and accumbens nucleus, most of the neurons containing D1 receptor mRNA were characterized as medium-sized substance P neurons and distinct from those containing D2 receptor mRNA. Nevertheless, 15-20% of the substance P neurons did not contain D1 receptor mRNA. The neurons containing preproenkephalin A mRNA did not contain D1 receptor mRNA but contained D2 receptor mRNA. A small number of cholinergic and somatostatinergic neurons exhibited a weak reaction for D1 receptor mRNA. These results demonstrate that dopamine acts on efferent striatal neurons through expression of distinct receptors--namely, D1 and D2 in separate cell populations (substance P and preproenkephalin A neurons, respectively)--and can also act on nonprojecting neurons through D1 receptor expression.

In situ hybridization with D2 receptor probe and immunohistochemistry with choline acetyltransferase (ChAT) antibody performed on adjacent sections demonstrate dopamine D2 receptor gene expression in cholinergic neurons of the rat caudate-putamen and nucleus accumbens. Eighty per cent of cholinergic neurons in the striatum contain detectable D2 receptor mRNA. The other neurons without detectable D2 mRNA do not display specific localization or aspect in the caudate-putamen and nucleus accumbens as compared to the other cholinergic neurons. The absence of detectable D2 mRNA in certain cholinergic neurons can be due to the limited sensitivity of the procedure that would not detect low mRNA levels, or alternatively can reflect the existence of two cholinergic cell populations in the striatum, one of which would not express the D2 receptor gene. The other forebrain cholinergic neurons do not contain D2 mRNA.

In situ hybridization experiments were performed with brain sections from normal, control and haloperidol-treated rats to identify and map the cells expressing the D2 dopamine receptor gene. D2 receptor mRNA was detected with radioactive or biotinylated oligonucleotide probes. D2 receptor mRNA was present in glandular cells of the pituitary intermediate lobe and in neurons of the substantia nigra, ventral tegmental area, and forebrain, especially in caudate putamen, nucleus accumbens, olfactory tubercle, and piriform cortex. Hybridization with D2 and preproenkephalin A probes in adjacent sections, as well as combined hybridization with the two probes in the same sections, demonstrated that all detectable enkephalin neurons in the striatum contained the D2 receptor mRNA. Large neurons in caudate putamen, which were unlabeled with the preproenkephalin A probe and which may have been cholinergic, also expressed the D2 receptor gene. Haloperidol treatment (14 or 21 days) provoked an increase in mRNA content for D2 receptor and preproenkephalin A in the striatum. This suggests that the increase in D2 receptor number observed after haloperidol treatment is due to increased activity of the D2 gene. These results indicate that in the striatum, the enkephalin neurons are direct targets for dopamine liberated from mesostriatal neurons.