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.Biochemical and pharmacological investigations have demonstrated the existence of two dopamine receptor subtypes, D1 and D2, which differentiate the signal transduction mediated by dopamine as either an activation or an inhibition of adenylate cyclase (1-4). Rat D2 receptor cDNA has been cloned and sequenced, and it appears that the D2 receptor belongs to a family of receptors that are coupled to guanine nucleotide-binding proteins (5). Its mRNA is abundantly represented in rat brain, especially in the mesencephalon and the basal ganglia (5). These areas contain, respectively, the cell bodies and terminals of the dopaminergic mesostriatal system (6). Striatal neuron activities are under the influence of the dopamine neurons of the substantia nigra and ventral tegmental area (7,8). Binding experiments with radiolabeled ligands have demonstrated the presence of striatal dopamine receptors (9-15). Drugs that interact with dopamine at receptor sites significantly affect neuronal activities, including neuropeptide gene expression (16)(17)(18)(19), in the striatum.While the existence of dopamine receptors on striatal neurons and on dopamine terminals in the striatum is commonly accepted (9-15), there is no anatomical information about the characteristics of the cells expressing the dopamine receptor gene in the striatum. To better understand how D2 receptor gene expression contributes to nigrostriatal interactions, we have used in situ hybridization to study the characteristics of cells containing D2 receptor mRNA in adult rat forebrain, under normal conditions and after blockade of dopamine transmission with a dopamine receptor antagonist, haloperidol. We report here that most cells conta...

We achieved histological detection of the messenger RNAs coding for vasopressin, calcitonin, or calcitonin gene-related peptide by using biotinylated synthetic oligonucleotides, and defined the technical parameters enabling optimal detection of these mRNAs. Oligonucleotides labeled by fixation of one biotin at their 5' end or by addition of a biotin-11-dUTP tail at their 3' end can be used to detect mRNAs, although the latter are more sensitive. Streptavidin-alkaline phosphatase revealed with nitroblue tetrazolium-bromo-chloro-indolyl phosphate as substrate makes possible detection of the biotinylated oligonucleotides. Increasing formaldehyde concentration in the fixative decreases the signal intensity; 1% formaldehyde fixation provides the most intense signal. Several controls, including those with addition of unlabeled oligonucleotides to the hybridization buffer, confirm the specificity of mRNA detection. The sensitivity of the biotinylated probes is identical or lower as compared to the corresponding radiolabeled oligonucleotides. Histological and subcellular resolution is greatly enhanced with biotinylated probes. The rat vasopressin probes stain magnocellular neurons in the supraoptic and paraventricular nuclei and, under optimal conditions, parvocellular neurons in the suprachiasmatic nucleus. Vasopressin mRNA is present in the cytoplasm of the cell bodies and in the roots of certain processes. Calcitonin and calcitonin gene-related peptide mRNA are found co-localized in the cytoplasm of the same tumor cells in human medullary thyroid carcinoma.

We report our experience in development of the in situ hybridization (ISH) procedure to detect messenger RNAs (mRNAs) coding for various molecules involved in endocrine glands and central nervous system activity, including mRNAs coding for endorphin precursors [preproenkephalin A (PPA), pro-opiocortin (POMC)], vasopressin, and transferrin. Various conditions of fixation and handling of the tissues were tested to establish optimal parameters for mRNA detection. Double-stranded DNA probes labeled by nick translation, synthetic oligonucleotides labeled at their 5' end, as well as single-stranded RNA probes were used, after incorporation of 32P- or 35S-labeled nucleotides. Specific requirements for efficient and reproducible ISH investigations are discussed. Cells expressing the PPA gene in the adrenal medulla and in the brain were detected by ISH. The results show that ISH is as sensitive as immunohistochemistry in detecting peptide-producing cells in the adrenal and that it allows detection of PPA cell bodies in brain in conditions in which they are inconstantly detected by immunohistochemistry. Unilateral destruction of substantia nigra provokes a dramatic decrease in the number of neurons expressing the PPA gene in the contralateral striatum. Cells expressing the POMC gene were detected in the pituitary of various species including man and in the rat arcuate nucleus. Neurons containing vasopressin mRNA were visualized in the supraoptic paraventricular and suprachiasmatic nucleus of the adult rat by using a synthetic oligonucleotide probe. Transferrin gene expression was shown in the central nervous system of the rat brain in two cell populations, the oligodendrocytes and the epithelial cells of the choroid plexus, by demonstration of simultaneous presence in them of transferrin immunoreactivity together with transferrin mRNA. These results show that the ISH procedure is a technique that can be routinely used to investigate gene transcription anatomically in complex heterocellular tissues such as the endocrine glands and the nervous system.

The messenger RNA coding for vasopressin has been detected at the ultrastructural level in normal and Brattleboro rat neurons, by using an oligonucleotide rat AVP prove labelled with 35SdATP. Vibratome sections of rat hypothalamus fixed with a mixture of 4% formaldehyde and 0.1% glutaraldehyde were hybridized with the probe, osmicated, included in Araldite and cut in semi-thin and thin sections that were coated with emulsion. The results demonstrate that vasopressin mRNA can be visualized in the cytoplasm of normal and Brattleboro rat neurons with an acceptable preservation of the ultrastructural aspect of the tissue. In Brattleboro rat neurons, the vasopressin mRNA is preferentially located at the periphery of the cytoplasm and is less abundant than in normal rat, this suggesting that the single base gene deletion observed in the Brattleboro rat provokes an altered transcription and compartmentation of the corresponding mRNA.

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