Benito Anton

Immunohistochemical localization of ORL-1 in the central nervous system of the rat

A novel member of the opioid receptor family (ORL‐1) has been cloned from a variety of vertebrates. ORL‐1 does not bind any of the classical opioids, although a high affinity endogenous agonist with close homology to dynorphin has recently been identified. We have generated a monoclonal antibody to the N‐terminus of ORL‐1 to map areas of receptor expression in rat central nervous system (CNS). Intense and specific immunolabeling was observed in multiple areas in the diencephalon, mesencephalon, pons/medulla, and spinal cord. In the telencephalon, intense labeling was observed in the neuropil throughout layers II–V in the neocortex, the anterior olfactory nuclear complex, the pyriform cortex, the CA1–CA4 fields and dentate gyrus of the hippocampus, and in many of the septal and basal forebrain areas. In contrast to other members of the opioid receptor family, light labeling for ORL‐1 was observed in telencephalic areas such as caudate‐putamen. In the cerebellum, ORL‐1 immunoreactivity was only observed in the deep nuclei. Throughout the CNS the majority of labelling was localized to fiber processes and fine puncta, although labeled scattered perikarya were observed in a few brain areas such as the hilus dentate in the hippocampus and some nuclei in the brainstem and spinal cord. The present mapping study is consistent with the reported distribution of ORL‐1 mRNA and provides the first immunohistochemical report on anatomical and cellular distribution of ORL‐1 receptor in the rat CNS.

Pentylenetetrazol-induced kindling: early involvement of excitatory and inhibitory systems

Alterations in the brain of rats receiving a single non-convulsive administration pentylenetetrazol (PTZ), 30 mig/kg, i.p. (single PTZ group) were investigated and compared with those detected in fully PTZ kindled rats (chronic PTZ group). In vitro receptor autoradiography experiments showed that both single and chronic PTZ groups presented mu opioid and benzodiazepine (BDZ) receptor binding in specific brain areas. Using an antibody generated against the delta opioid receptor (DOR-1), it was found that DOR-1 like immunoreactivity was reduced in cortex and amygdala in mice following single and chronic PTZ administration. Microdialysis experiments revealed that the administration of PTZ 30 mg/kg, i.p. in freely moving rats without previous experience with the drug, induces a rise in glutamate release, detected in the first and second 10 min dialysates collected from amygdala (138% and 50%, respectively) and frontal cortex (70% and 45%, respectively) as well as aspartate in frontal cortex in the first and second PTZ-dialysates (143% and 80%, respectively). Subsequently, values returned to basal conditions. It may be speculated that decreased BDZ receptor binding results from enhanced release of GABA. On the other hand, the decrease of mu receptor binding and DOR-1 immunoreactivity observed after PTZ administration may be the result of enhanced levels of opioid peptides probably released over the kindling procedure. In conclusion, the present study indicates that PTZ-kindling is associated with an imbalance between excitatory and inhibitory systems which is apparent early in the epileptogenic process.

ORL‐1 and Mu opioid receptor antisera label different fibers in areas involved in pain processing

Mu opioid receptors (MOR) mediate the analgesic effects of opioid drugs such as morphine. The opioid receptor‐like (ORL‐1) receptor is structurally related to opioid receptors and the ORL‐1 receptor agonist, orphanin FQ/nociceptin, induces analgesia at the spinal level, but appears to recruit different circuitry than that used by mu opioids. When administered intracerebroventricularly, orphanin FQ/nociceptin produces hyperalgesia and/or reverses opioid analgesia. The functionally distinct actions elicited by MOR and ORL‐1 receptors, which activate similar intracellular signaling systems and show similar regional distributions, could be explained by their differential cellular localization. By using double label immunohistochemistry and confocal microscopy, the present study investigates the distribution of MOR and ORL‐1 receptors in regions of the rat nervous system that are involved with nociceptive processing. In general co‐localization of MOR and ORL‐1 receptor immunoreactivity was not observed in either perikarya or neuropil in the dorsal root ganglia, nor in the Lissauers tract and superficial laminae of the spinal cord. Likewise, there was no evidence for co‐localization of these receptors within the periaqueductal gray, the nucleus raphe magnus, the gigantocellular reticular nucleus, and the nucleus of the solitary tract. These observations indicate that MOR and ORL‐1 receptors are expressed predominantly on different fiber systems in these regions. This differential distribution is consistent with the distinct pharmacology of ORL‐1 and MOR receptor agonists and suggests that the antisera to MOR and ORL‐1 receptors may provide useful markers for further investigations of analgesic and counteranalgesic pathways modulating pain perception. J. Comp. Neurol. 399:373–383, 1998.

Quantitative immunolocalization of mu opioid receptors: regulation by naltrexone

The present study utilized a newly developed quantitative immunohistochemical assay to measure changes in mu opioid receptor abundance following chronic administration of the opioid receptor antagonist naltrexone. These data were compared with those obtained from mu receptor radioligand binding on adjacent tissue sections, in order to determine whether the characteristic antagonist-induced increase in radioligand binding is due to an increase in the total number of mu receptors and/or to an increase in the proportion of receptors that are in an active binding conformation in the absence of a change in the total number of receptors. Adult male Sprague-Dawley rats were administered naltrexone, 7-8 mg/kg per day, or saline continuously for seven days by osmotic minipumps, after which time their brains were processed for immunohistochemistry and receptor autoradiography on adjacent fresh frozen tissue sections. Semiquantitative immunohistochemistry was performed using a radiolabelled secondary antibody for autoradiographic determination and a set of radioactive standards. Results demonstrate an overall concordance between the distribution of mu opioid receptors as measured by the two different methods with a few exceptions. Following naltrexone administration, mu receptor immunoreactivity was significantly higher in the amygdala, thalamus, hippocampus, and interpeduncular nucleus as compared with the saline-treated control animals. [3H]D-Ala2,N-Me-Phe4,Gly-ol5-enkephalin binding to mu opioid receptors was significantly higher in the globus pallidus, amygdala, thalamus, hypothalamus, hippocampus, substantia nigra, ventral tegmental area, central gray, and interpeduncular nucleus of the naltrexone-treated rats. These findings indicate that in some brain regions chronic naltrexone exposure increases the total number of mu opioid receptors, while in other regions there is an increase in the percent of active receptors without an observable change in the total number of receptors. Quantitative receptor immunodetection together with ligand autoradiography provides a new approach for investigating the regulation of mu opioid receptors on tissue sections.

Expression of the lacZ Reporter Gene in the Rat Basal Forebrain, Hippocampus, and Nigrostriatal Pathway Using a Nonreplicating Herpes Simplex Vector

We recently demonstrated the efficacy of a nonreplicating herpes simplex type 1 virus construct, employing the Moloney murine leukemia virus long terminal repeat promoter, in providing long-term expression of the lacZ gene in rat hippocampal neurons. We now report the utility of this construct in expressing the reporter gene in neurons of the basal forebrain and substantia nigra and examine the spread of the virus to other brain regions. Dorsal and ventrolateral hippocampal formation injection of the virus resulted in numerous beta-gal-expressing cells in the stratum pyramidale, stratum oriens, stratum lacunosum-moleculare, and stratum granulosum. Scattered cells of the medial septum/diagonal band were positively stained following direct injection into this region. More intense staining of the basal forebrain was observed following hippocampal injection as a result of retrograde transport of the virus as shown by PCR analysis of viral DNA. Hippocampal injection also resulted in positive cell staining in several other afferent projection nuclei, namely, the supramammillary bodies, dorsal and caudal linear raphe, and perirhinal/entorhinal cortex. Very few cells were labeled around injection sites in the striatum or substantia nigra. However, substantia nigra zona compacta cells were blue following striatal injection, as were pallidal neurons following nigral injection. These data demonstrate the feasibility of using this virus construct to express foreign genes such as neurotrophic factors in basal forebrain and substantia nigra neurons, taking advantage of retrograde transport of the virus to preserve local anatomy.

Studies on a cDNA encoding a delta opioid receptor

Abstract From an NG108-15 cDNA eucaryotic expression library, we recently identified and characterized a ∂-opioid receptor clone (DOR-1) with the anticipated affinity and selectivity for ligands binding ∂-opioid receptors(1). Using a similar strategy an essentially identical clone was isolated by Keiffer et al. (2) from the same cell line. Based upon Southern analysis DOR-1 is a murine clone and the NG108-15 cells (a rat/mouse hybrid line) have lost the genetic material encoding the rat DOR-1 sequence. Here we present some insights into the gene structure and regulation of DOR-1 in NG108-15 cells. Finally, we report on the generation of antisera to DOR-1 and our findings concerning the anatomical distribution of this delta opioid receptor in rodent brain.