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Utilizing agonist-stimulated GTPgammaS autoradiography, we analyzed the ability of preproorphanin FQ (ppOFQ) peptides to stimulate [35S]-GTPgammaS binding in adult rat brain. Orphanin FQ (OFQ) stimulated [35S]-GTPgammaS binding in a pattern similar to that described for [125I]-OFQ at the endogenous opioid receptor-like (ORL1) receptor. The ppOFQ peptides nocistatin and orphanin FQ2 (OFQ II(1-17)) had no effect, suggesting that they do not mediate their reported analgesic effects via a G(i/o)-coupled receptor (i.e. opioid or ORL1). Unlike OFQ II(1-17), high concentrations of its C-terminal extension, OFQ II(1-28), stimulated [35S]-GTPgammaS binding in a mu (mu) opioid receptor-like distribution and the effect was blocked by naloxone. To explore these observations, we evaluated the receptor binding profile of OFQ II(1-28) at the cloned ORL1 and mu opioid receptors. OFQ II(1-28) had no specific binding at either ORL1 or mu opioid receptors at concentrations up to 50 microM. This lack of affinity was not consistent with a mu-mediated effect, as suggested by preliminary observation using functional autoradiography in rat brain sections. Although behavioral studies suggest that OFQ II(1-28) possesses analgesic activity, this effect does not appear to be mediated via direct binding at the mu opioid receptor. Taken together, these findings support the view that (1) OFQ is the only ppOFQ peptide that binds to and activates the ORL1 receptor and (2) OFQ II(1-28) does not bind or stimulate [35S]-GTPgammaS binding in cells expressing the mu opioid receptor.

The orphanin peptide system, although structurally similar to the endogenous opioid family of peptides and receptors, has been established as a distinct neurochemical entity. The distribution of the opioid receptor-like (ORL1) receptor and its endogenous ligand orphanin FQ (OFQ) in the central nervous system of the adult rat has been recently reported, and although diffusely disseminated throughout the brain, this neuropeptide system is particularly expressed within stress and pain circuitry. Little is known concerning the normal expression of the orphanin system during gestation, nor how opiate or stress exposure may influence its development. Using in situ hybridization techniques, the present study was undertaken to determine the normal pattern of expression of ORL1 mRNA in the human and rat brain at various developmental stages. Rat embryos, postnatal rat brains and postmortem human brains were collected, frozen and cut into 15 microm coronal sections. In situ hybridization was performed using riboprobes generated from cDNA containing representative human and rat ORL1 and OFQ sequences. Both ORL1 and OFQ mRNA is detected as early as E12 in the cortical plate, basal forebrain, brainstem and spinal cord. Expression for both ORL1 and OFQ is strongest during the early postnatal period, remaining strong in the spinal cord, brainstem, ventral forebrain, and neocortex into the adult. Human ORL1 and OFQ expression is observed at 16 weeks gestation, remaining relatively unchanged up to 36 weeks. The influence of early orphanin expression on maturation of stress and pain circuitry in the developing brain remains unknown.

The technique of in situ hybridization with specific ribonucleotide probes was used to determine the distribution patterns of mRNA encoding the alpha 1a-, alpha 1b- and alpha 1d-adrenoceptor (AR) subtypes in rat brain and spinal cord. The expression pattern of alpha 1a-AR mRNA has not been reported previously, and was found to be widespread throughout the rat central nervous system. High levels were found in regions of the olfactory system, several hypothalamic nuclei, and regions of the brainstem and spinal cord, particularly in areas related to motor function. Regions expressing moderate levels of mRNA for this receptor were the septum, bed nucleus of the stria terminalis, cerebral cortex, amygdala, cerebellum and pineal gland. Low expression levels were detected in the hippocampal formation. Most nuclei in the basal ganglia and thalamus expressed extremely low or undetectable levels of alpha 1a-AR mRNA. The expression patterns of the alpha 1b- and alpha 1d-AR mRNAs were similar to those described using oligonucleotide probes in earlier studies. High expression of alpha 1b-AR mRNA was noted in the pineal gland, most thalamic nuclei, lateral nucleus of the amygdala and dorsal and median raphe nuclei. Moderate expression levels were noted throughout the cerebral cortex, and in some olfactory, septal, and brainstem regions. The distribution of alpha 1d-AR mRNA was the most discrete of the three receptors examined. Expression was strong in the olfactory bulb, cerebral cortex, hippocampus, reticular thalamic nucleus, regions of the amygdala, motor nuclei of the brainstem, inferior olivary complex and spinal cord. Comparison of the distributions of the alpha 1a-, alpha 1b- and alpha 1d-AR mRNA suggests unique functional roles for each of these receptors.

Conventional in situ hybridization methods have been useful in characterizing the anatomical distribution of cells in the central nervous system that express dopamine D2 receptor mRNA. However, due to the large size of the D2 mRNA pool, this method may be insensitive to changes in D2 gene transcription. We have developed a method of hybridizing a 35S-labelled cRNA probe to an intron in the D2 receptor gene in order to measure the amount of primary transcript or heteronuclear RNA (hnRNA) in D2-expressing cells. Introns are found uniquely in hnRNA and are thought to be short-lived intermediates. Thus, monitoring introns could represent a more direct measure of D2 gene transcription. The anatomical distribution of the D2 hnRNA is similar to the distribution of D2 mRNA in the rat brain. D2 heteronuclear RNA was found in the nuclei of cells in the caudate putamen, nucleus accumbens, hippocampus, olfactory tubercle, substantia nigra, ventral tegmental area, and zona incerta. Other regions that contain D2 mRNA, but do not demonstrate intronic signal, include the globus pallidus, prefrontal, cingulate, entorhinal, and piriform cortex, septum, and amygdala. However, these areas have low amounts of D2 mRNA and may contain levels of D2 hnRNA that are below detection. Heteronuclear RNA quantitation by solution hybridization followed by RNase protection was performed on striatum, substantia nigra, cerebral cortex, hippocampus, hypothalamus, and pituitary using a D2 intron 7/exon 8 border probe. These results corroborate the distribution of hnRNA revealed with intronic in situ hybridization. In addition, protection assays were able to detect hnRNA in areas that express low levels of D2 like the cortex, hippocampus and hypothalamus. hnRNA/mRNA ratios calculated from intron/exon border probe protection assays were not equivalent for all the tissue areas studied, indicating that transcription and/or hnRNA half lives may differ between tissues that express D2 receptors. The combined use of intronic in situ hybridization and intron/exon border protection assay as an index of D2 gene transcription and RNA processing provides more information than measuring the mRNA pool alone. It may also prove to be a more useful measure of gene regulation, allowing for evaluation of gene responses to acute treatments.