Alfred Mansour

Opioid-receptor mRNA expression in the rat CNS: anatomical and functional implications

The cloning of the opioid receptors has profoundly affected our understanding of opioid-receptor expression, regulation and function. This review focuses on the impact that cloning has had on our understanding of opioid-receptor anatomy, and provides broad anatomical maps of the three opioid-receptor mRNAs in relation to their binding sites. In addition, three model anatomical systems, the nigrostriatal and mesolimbic dopamine systems, the hypothalamic neuroendocrine axes, and the ascending and descending pain pathways, have been highlighted to discuss issues of receptor transport, trafficking and pre- versus postsynaptic localization.

Anatomy of CNS opioid receptors

Abstract There is a wide body of evidence to suggest the existence of at least three distinct opioid receptor types in the CNS, referred to as μ, δ, and κ. This paper reviews some of the findings that have led to this conclusion and the anatomical distributions of these sites in the rat brain. Their relation to the opioid peptides and some of the proposed functions mediated by these receptor systems are also discussed.

Cloning and pharmacological characterization of a rat μ opioid receptor

We have isolated a rat cDNA clone that displays 75% amino acid homology with the mouse delta and rat kappa opioid receptors. The cDNA (designated pRMuR-12) encodes a protein of 398 amino acids comprising, in part, seven hydrophobic domains similar to those described for other G protein-linked receptors. Data from binding assays conducted with COS-1 cells transiently transfected with a CMV mammalian expression vector containing the full coding region of pRMuR-12 demonstrated mu receptor selectivity. In situ hybridization mRNA analysis revealed an mRNA distribution in rat brain that corresponds well to the distribution of binding sites labeled with mu-selective ligands. Based upon these observations, we conclude that pRMuR-12 encodes a mu opioid receptor.

Localization of orphanin FQ (nociceptin) peptide and messenger RNA in the central nervous system of the rat

Orphanin FQ (OFQ) is the endogenous agonist of the opioid receptor‐like receptor (ORL‐1). It and its precursor, prepro‐OFQ, exhibit structural features suggestive of the opioid peptides. A cDNA encoding the OFQ precursor sequence in the rat recently has been cloned, and the authors recently generated a polyclonal antibody directed against the OFQ peptide. In the present study, the authors used in situ hybridization and immunohistochemistry to examine the distribution of OFQ peptide and mRNA in the central nervous system of the adult rat. OFQ immunoreactivity and prepro‐OFQ mRNA expression correlated virtually in all brain areas studied. In the forebrain, OFQ peptide and mRNA were prominent in the neocortex endopiriform nucleus, claustrum, lateral septum, ventral forebrain, hypothalamus, mammillary bodies, central and medial nuclei of the amygdala, hippocampal formation, paratenial and reticular nuclei of the thalamus, medial habenula, and zona incerta. No OFQ was observed in the pineal or pituitary glands. In the brainstem, OFQ was prominent in the ventral tegmental area, substantia nigra, nucleus of the posterior commissure, central gray, nucleus of Darkschewitsch, peripeduncular nucleus, interpeduncular nucleus, tegmental nuclei, locus coeruleus, raphe complex, lateral parabrachial nucleus, inferior olivary complex, vestibular nuclear complex, prepositus hypoglossus, solitary nucleus, nucleus ambiguous, caudal spinal trigeminal nucleus, and reticular formation. In the spinal cord, OFQ was observed throughout the dorsal and ventral horns. The wide distribution of this peptide provides support for its role in a multitude of functions, including not only nociception but also motor and balance control, special sensory processing, and various autonomic and physiologic processes. J. Comp. Neurol. 406:503–547, 1999.

Immunohistochemical localization of the cloned μ opioid receptor in the rat CNS

Three opioid receptor types have recently been cloned that correspond to the pharmacologically defined mu, delta and kappa 1 receptors. In situ hybridization studies suggest that the opioid receptor mRNAs that encode these receptors have distinct distributions in the central nervous system that correlate well with their known functions. In the present study polyclonal antibodies were generated to the C terminal 63 amino acids of the cloned mu receptor (335-398) to examine the distribution of the mu receptor-like protein with immunohistochemical techniques. mu receptor-like immunoreactivity is widely distributed in the rat central nervous system with immunoreactive fibers and/or perikarya in such regions as the neocortex, the striatal patches and subcallosal streak, nucleus accumbens, lateral and medial septum, endopiriform nucleus, globus pallidus and ventral pallidum, amygdala, hippocampus, presubiculum, thalamic and hypothalamic nuclei, superior and inferior colliculi, central grey, substantia nigra, ventral tegmental area, interpeduncular nucleus, medial terminal nucleus of the accessory optic tract, raphe nuclei, nucleus of the solitary tract, spinal trigeminal nucleus, dorsal motor nucleus of vagus, the spinal cord and dorsal root ganglia. In addition, two major neuronal pathways, the fasciculus retroflexus and the stria terminalis, exhibit densely stained axonal fibers. While this distribution is in excellent agreement with the known mu receptor binding localization, a few regions, such as neocortex and cingulate cortex, basolateral amygdala, medial geniculate nucleus and the medial preoptic area fail to show a good correspondence. Several explanations are provided to interpret these results, and the anatomical and functional implications of these findings are discussed.

Opioid receptor‐like (ORL1) receptor distribution in the rat central nervous system: Comparison of ORL1 receptor mRNA expression with 125I‐[14Tyr]‐orphanin FQ binding

The recently discovered neuropeptide orphanin FQ (OFQ), and its opioid receptor‐like (ORL1) receptor, exhibit structural features suggestive of the μ, κ, and δ opioid systems. The anatomic distribution of OFQ immunoreactivity and mRNA expression has been reported recently. In the present analysis, we compare the distribution of orphanin receptor mRNA expression with that of orphanin FQ binding at the ORL1 receptor in the adult rat central nervous system (CNS). By using in vitro receptor autoradiography with 125I‐[14Tyr]‐OFQ as the radioligand, orphanin receptor binding was analyzed throughout the rat CNS. Orphanin binding sites were densest in several cortical regions, the anterior olfactory nucleus, lateral septum, ventral forebrain, several hypothalamic nuclei, hippocampal formation, basolateral and medial amygdala, central gray, pontine nuclei, interpeduncular nucleus, substantia nigra, raphe complex, locus coeruleus, vestibular nuclear complex, and the spinal cord. By using in situ hybridization, cells expressing ORL1 mRNA were most numerous throughout multiple cortical regions, the anterior olfactory nucleus, lateral septum, endopiriform nucleus, ventral forebrain, multiple hypothalamic nuclei, nucleus of the lateral olfactory tract, medial amygdala, hippocampal formation, substantia nigra, ventral tegmental area, central gray, raphe complex, locus coeruleus, multiple brainstem motor nuclei, inferior olive, deep cerebellar nuclei, vestibular nuclear complex, nucleus of the solitary tract, reticular formation, dorsal root ganglia, and spinal cord. The diffuse distribution of ORL1 mRNA and binding supports an extensive role for orphanin FQ in a multitude of CNS functions, including motor and balance control, reinforcement and reward, nociception, the stress response, sexual behavior, aggression, and autonomic control of physiologic processes. J. Comp. Neurol. 412:563–605, 1999.

μ-Opioid receptor mRNA expression in the rat CNS: comparison to μ-receptor binding

Abstract The distribution of cells expressing μ-receptor mRNA and μ-receptor binding sites were compared in brain and spinal cord tissue sections using a combination of in situ hybridization and receptor autoradiographic techniques. μ-Receptor mRNA was visualized with a 35S-labeled cRNA probe directed to transmembrane III–VI of the rat μ-receptor, while μ-receptor binding sites were labeled with the μ-selective ligand [3H]DAMGO. A high correspondence between the μ-receptor mRNA and receptor binding distributions was observed in the nucleus of the accessory olfactory bulb, anterior olfactory nuclei, striatal patches of the nucleus accumbens and caudate-putamen, endopiriform nucleus, claustrum, diagonal band of Broca, globus pallidus, ventral pallidum, bed nucleus of stria terminalis, most thalamic nuclei, medial and posteriocortical medial amygdala, lateral, dorsomedial, posterior and mammillary nuclei of the hypothalamus, presubiculum, subiculum, rostral interpeduncular nucleus, median raphe, inferior colliculus, parabrachial nucleus, locus coeruleus, central grey, nucleus ambiguus, nucleus of the solitary tract, nucleus gracilis, nucleus cuneatus, and the dorsal motor nucleus of vagus. Differences in μ-receptor mRNA and receptor binding distributions were observed in several regions, including the olfactory bulb, cortex, hippocampus, superior colliculus, spinal trigeminal nucleus, cochlear nucleus and spinal cord, and may be due to μ-receptor transport to presynaptic terminals.

Distribution of D5 dopamine receptor mRNA in rat brain

The distribution of the messenger RNA encoding the dopamine D5 receptor was determined in the rat brain by in situ hybridization. Using [35S]-labelled riboprobes to either the rat or human D5 receptor, this mRNA was localized to the hippocampus and the parafascicular nucleus of the thalamus. This mRNA could not be visualized in the more traditional brain regions associated with dopaminergic cell bodies or projection fields. This unusual distribution suggests a novel function in the brain for this subtype of the dopamine receptor.

A comparison of D1 receptor binding and mRNA in rat brain using receptor autoradiographic and in situ hybridization techniques

D1, a subtype of the dopamine receptors, is widely distributed in the nervous system and has been shown to be positively coupled to adenylate cyclase. Using a combination of in vitro receptor autoradiographic and in situ hybridization techniques, the present study examines the co-distribution of D1 receptor binding sites and D1 receptor mRNA in adjacent rat brain sections. D1 receptor binding sites were labeled using the selective antagonist [3H](R)-(+)-8-chloro-2,3,4,5-tetrahydro-3-methyl-5-phenyl-1H-3-benzaz epin- 7-ol (SCH23390) (4.6 nM), in the presence of 1 microM ketanserin, while the D1 receptor mRNA was visualized with a 35S-labeled riboprobe corresponding to a region between transmembrane domains III and VI of the rat D1 receptor (base pairs 383-843). Analysis of serial sections suggested a good agreement between D1 receptor binding and mRNA in several brain regions, including the paleocortex, caudate-putamen, nucleus accumbens, amygdala, and suprachiasmatic nucleus. Marked discrepancies between D1 receptor binding and mRNA were observed in other brain regions including the entopeduncular and subthalamic nuclei, substantia nigra (pars reticulata), hippocampus, and cerebellum. While technical considerations may contribute to these results, much of the discordance between the distributions is probably due to the differential localization of D1 receptor mRNA in cell bodies and receptor binding sites on fibers and may provide insights into receptor synthesis, transport, and membrane insertion. In the basal ganglia, for instance, D1 receptors are synthesized in the striatum and are either transported to efferent projections in areas such as the substantia nigra, or remain localized in striatal cells bodies. Ibotenic acid lesions in the striatum are consistent with these conclusions and demonstrate a coordinate loss of D1 receptor binding and mRNA in the caudate-putamen that is accompanied by a degeneration of fibers projecting to substantia nigra and a loss of D1 binding in the pars reticulata. Neurons in the dentate gyrus and in the granular layer of the cerebellum, on the other hand, synthesize D1 receptors and transport them entirely to either their dendritic or axonal fields, respectively, in the molecular layer. This analysis provides a better understanding of dopaminergic receptor systems in the CNS and their anatomical organization.

Corticosteroids regulate brain hippocampal 5-HT1A receptor mRNA expression

Using in situ hybridization techniques, the expression of 5-HT1A receptor mRNA was measured within the hippocampal formation after bilateral adrenalectomy (ADX). After 24 hr ADX, 5-HT1A receptor mRNA expression was significantly increased in all hippocampal subfields in ADX animals relative to sham-operated controls (SHAM). The magnitude of the increase was most pronounced within CA2 (127%) and CA3/4 (94%)- subfields of dorsal hippocampus, intermediate in the dentate gyrus (73%), and least within CA1 (60%). Administration of exogenous corticosterone (CORT) at the time of ADX maintained the level of 5-HT1A receptor mRNA expression within the range of SHAM animals. In vitro receptor autoradiographic analysis of 5-HT1A receptors in adjacent sections from the same animals indicated a simultaneous increase in 5- HT1A binding throughout the hippocampus in response to ADX. 5-HT1A binding increased to a similar extent (approximately 30%) in CA subfields and dentate gyrus but remained within SHAM levels in CORT- replaced animals. 5-HT1A receptor mRNA levels were also increased in hippocampal subregions of 1 week ADX animals relative to SHAM animals. Within both CA1 and CA2 subfields, the increments were approximately double those observed after 1 d ADX. 5-HT1A receptor binding was increased in every hippocampal subfield to a similar extent as that observed after 1 d ADX. Increases in both 5-HT1A receptor mRNA expression and 5-HT1A receptor binding were preventable by administration of exogenous CORT at the time of ADX. Hippocampal 5-HT1C receptor mRNA and D1 receptor mRNA expression were not significantly altered by either acute or chronic ADX treatment. These data indicate that adrenal steroids may selectively regulate hippocampal 5-HT1A receptors at the level of 5-HT1A receptor mRNA expression.