Corticotropin-releasing factor (CRF) is the primary factor involved in controlling the release of ACTH from the anterior pituitary and also acts as a neurotransmitter in a variety of brain systems. The actions of CRF are mediated by G-protein coupled membrane bound receptors and a high affinity CRF receptor, CRF1, has been previously cloned and functionally characterized. We have recently isolated a cDNA encoding a second member of the CRF receptor family, designated CRF2, which displays approximately 70% homology at the nucleotide level to the CRF1 receptor and exhibits a distinctive pharmacological profile. The present study utilized in situ hybridization histochemistry to localize the distribution of CRF2 receptor mRNA in rat brain and pituitary gland and compared this with the distribution of CRF1, receptor expression. While CRF1 receptor expression was very high in neocortical, cerebellar, and sensory relay structures, CRF2 receptor expression was generally confined to subcortical structures. The highest levels of CRF2 receptor mRNA in brain were evident within the lateral septal nucleus, the ventromedial hypothalamic nucleus and the choroid plexus. Moderate levels of CRF2 receptor expression were evident in the olfactory bulb, amygdaloid nuclei, the paraventricular and suraoptic nuclei of the hypothalamus, the inferior colliculus and 5-HT-associated raphe nuclei of the midbrain. CRF2-expressing cells were also evident in the bed nucleus of the stria terminalis, the hippocampal formation and anterior and lateral hypothalmic areas. In addition, CRF2 receptor mRNA was also found in cerebral arterioles throughout the brain. Within the pituitary gland, CRF2 receptor mRNA was detectable only at very low levels in scattered cells while CRF1 receptor mRNA was readily detectable in anterior and intermediate lobes. This heterogeneous distribution of CRF1 and CRF2 receptor mRNA suggests distinctive functional roles for each receptor in CRF-related systems. The CRF1 receptor may be regarded as the primary neuroendocrine pituitary CRF receptor and important in cortical, cerebellar and sensory roles of CRF. The anatomical distribution of CRF2 receptor mRNA indicates a role for this novel receptor in hypothalamic neuroendocrine, autonomic and general behavioral actions of central CRF.

The present study reports the isolation of a cDNA clone that encodes a second member of the corticotropin-releasing factor (CRF) receptor family, designated as the CRF2 receptor. The cDNA was identified using oligonucleotides of degenerate sequence in a PCR paradigm. A PCR fragment obtained from rat brain was utilized to isolate a full-length cDNA from a rat hypothalamus cDNA library that encoded a 411-amino acid protein with "70% identity to the known CRF1 receptor over the entire coding region. When expressed in mouse Ltk-cells, this receptor stimulates cAMP production in response to CRF and known CRF-like agonists. CRF and the nonmammalian CRF-related peptides sauvagine and urotensin I stimulate adenylate cyclase activity in a dose-dependent manner with a rank order of potency different from that of the CRF1 receptor: sauvagine > urotensinrat/human CRF > ovine CRF. Tissue distribution analysis of the mRNAs by reverse transcriptase-PCR shows CRF2 receptor mRNA is present in rat brain and detectable in lung and heart. In situ hybridization studies indicate specific expression within the brain in the ventromedial nuclei of the hypothalamus, the lateral septum, the amygdala, and entorhinal cortex, but there is unremarkable expression in the pituitary. An additional splice variant of the CRF2 receptor with a different N-terminal domain has been identified by PCR, encoding a putative protein of 431 amino acids. Thus, the data demonstrate the presence of another functional CRF receptor, with significant differences in the pharmacological profile and tissue distribution from the CRF1 receptor, which would predict important functional differences between the two receptors.Corticotropin-releasing factor (CRF), a 41-amino acid peptide, regulates the secretion of adrenocorticotropin and other proopiomelanocortin products from the anterior pituitary. CRF also coordinates the endocrine, behavioral, and autonomic responses to stress. Within the past few years, substantial evidence has accumulated from both laboratory and clinical studies implicating CRF as a physiological mediator of stress responses and stress-induced disorders (1-6). Immunocytochemical studies have shown that CRF is found within the paraventricular nucleus of the hypothalamus as well as limbic areas such as the central and medial nuclei of the amygdala, the bed nucleus of the stria terminalis, substantia inominata, septum, preoptic area, the lateral hypothalamus, and brain stem nuclei involved in stress responses and regulation of autonomic function, such as the locus coeruleus, the parabrachial nucleus, and the dorsal vagal complex (see ref. 7). CRF, when administered intracerebroventricularly, results in behavioral, physiological, and autonomic responses that are similar to those observed when animals are exposed to a stressful environment (4-6).

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The present study examined the comparative distribution of 5-HT1A receptor mRNA and 5-HT1A receptors in rat brain using a combination of in situ hybridisation histochemistry and in vitro receptor autoradiography. 5-HT1A mRNA was visualized using a 910 bp cRNA probe synthesised from a BalI-PvuII fragment of the rat 5-HT1A reetor gene, while 5-HT1A receptors were labelled with the 5-HT1A-selective ligand 8-OH-DPAT. In general terms, there was a complementary distribution of cells expressing 5-HT1A receptor mRNA and 5-HT1A receptor sites. High levels of both 5-HT1A mRNA and 5-HT1A receptors were evident in the hippocampal formation (CA1, CA3, dentate gyrus), entorhinal cortex, and raphe nuclei and lower levels in neocortex and thalamus. Although 5-HT1A mRNA was not expressed in any regions which did not also exhibit 5-HT1A receptors, within both the diagonal band and the medial septal nucleus mRNA levels were proportionately higher than 5-HT1A receptor levels, possibly reflecting receptor transport or a heterogeneity in 5-HT1A receptor turnover mechanisms. 5-HT1A receptor mRNA and 5-HT1A binding sites were undetectable in caudate/putamen and cerebellar regions. The present data indicate the synthesis of 5-HT1A receptors both in raphe serotonergic cells and anatomically specific serotonergic projection areas, further supporting both a presynaptic autoregulatory and postsynaptic modulatory role for this receptor in serotonergic transmission.