In situ hybridization histochemistry with a probe directed against an intron sequence of the rat arginine vasopressin (AVP) gene was used to demonstrate localization and regulation of AVP heteronuclear RNA in discrete brain regions. Hybridization with an AVP intron I (AVPinI) probe revealed specific hybridization confined to cell nuclei of paraventricular nucleus, supraoptic nucleus (SON), and suprachiasmatic nucleus neurons of the rat hypothalamus. Grain counts revealed that the signal generated by the AVPinI probe represented 1.9% of that derived from an AVP exon C probe (AVPexC) in the SON. Interestingly, in the suprachiasmatic nucleus the proportion of AVPinI to AVP exon C ratio was much higher (12%), suggesting either increased transcription of the AVP gene or changes in posttranscriptional RNA processing. Regulatory experiments revealed that 2.6-fold increases in AVPinI signal could be visualized in the SON as little as 30 min after an acute salt load, a period during which no significant change in cytoplasmic AVP mRNA could be observed. In response to chronic salt loading, both AVP heteronuclear RNA and AVP mRNA were up-regulated. These data compared favorably with transcription rate values determined by nuclear run-on assay, suggesting that intronic in situ hybridization affords a relatively reliable method for assessment of rapid changes in gene transcription in individual central nervous system neurons.

In situ hybridization histochemistry is a useful method for localizing specific mRNA and studying the regulation of gene expression in an anatomical context. Previously, classical recombinant DNA and microbiological techniques have been required to identify and nick-translate the cloned DNAs necessary for in situ hybridization experiments. These requirements can be circumvented by the use of synthetic oligonucleotides complementary to the mRNA of interest. Compared to cloned cDNA probes, oligonucleotides are easy to manufacture, penetrate tissue much more easily, can be made to correspond to a sequence at any point in a known cDNA structure, and allow for the design of more precise controls for in situ studies. We describe a number of considerations in oligonucleotide probe design, including unique probe design from cDNA sequences and mixed probe design from protein primary structure data. The issues of species specificity, G-C content, probe length, tissue-specific mRNA expression, repeated sequences, non-coding region specific probes, and gene family homologies are discussed in an in situ hybridization context. Alternative strategies for mixed probe design are also considered. Information on the synthesis, purification, and sequence confirmation of oligonucleotides is then presented, followed by methods for labeling and using these probes for in situ hybridization histochemistry. The special considerations of specificity controls are addressed, including combined in situ hybridization histochemistry and immunocytochemistry, competition studies, the use of multiple hybridization probes, Tm studies, and Northern analysis of extracted RNA. The current and future directions of research with this technique are considered, with emphasis on the need to improve quantitation in order to facilitate the study of gene expression and regulation at the single cell level.

Peptides derived from pro-dynorphin and pro-vasopressin precursors coexist within neurosecretory vesicles of magnocellular neurons in the rat hypothalamus projecting to the posterior pituitary. The secretory activity of these neurons can be stimulated using physiological manipulations known to increase plasma vasopressin levels, such as dehydration and salt-loading. With chronic osmotic challenge, the mRNAs for both pro-dynorphin and pro-vasopressin increase in parallel in the supraoptic and paraventricular nuclei of the hypothalamus, but not within the nonmagnocellular suprachiasmatic nucleus cell groups projecting elsewhere than the neural lobe. The results indicate an example of coordinate regulation of mRNA expression for coexisting peptides within the brain.

Rat brain expresses two types of corticosteroid-binding proteins. The type I receptor binds corticosterone with high affinity and is structurally related to the kidney mineralocorticoid receptor (MR), while the type II or classical glucocorticoid receptor binds corticosterone with lower affinity and displays an in vivo preference for dexamethasone. Here we describe the isolation and characterization of a cDNA coding for the MR, from a rat hippocampus cDNA library, by low stringency hybridization to radiolabeled human glucocorticoid receptor cDNA. The nucleotide and deduced amino acid sequence for rat hippocampal MR displays extensive homology to a MR cDNA isolated from human kidney, suggesting that they are orthologous genes. Southern analysis suggests that there is only one gene for the MR, and in vitro expression of the receptor generates a high affinity corticosterone-binding protein. These data provide evidence to support the contention that a single gene gives rise to the MR in renal tissues and type I receptors in the brain.