Modulation of Brainstem Opiate Analgesia in the Rat by 1 Receptors: A Microinjection Study

1 Receptors have been implicated in the modulation of opioid analgesia. In the current study, we examined the role of 1 systems in the periaqueductal gray (PAG), the rostroventral medulla (RVM), and the locus coeruleus (LC) of the rat, regions previously shown to be sensitive to morphine. Morphine was a potent analgesic in all three regions. Coadministration of the 1 agonist ( )-pentazocine diminished the analgesic actions of morphine in all three regions, although the PAG was far less sensitive than the other two regions. Blockade of the 1 receptors with haloperidol in the RVM markedly enhanced the analgesic actions of coadministered morphine, implying a tonic activity of the 1 system in this region. This effect was mimicked by down-regulation of RVM 1 receptors using an antisense approach. However, no tonic 1 activity was observed in either the LC or the PAG. The RVM also was important in modulating analgesia elicited from morphine microinjected into the PAG. The analgesic actions of morphine given into the PAG could be attenuated by ( )-pentazocine placed into the RVM, whereas haloperidol in the RVM enhanced PAG morphine analgesia. These studies illustrate the pharmacological importance of 1 receptors in the brainstem modulation of opioid analgesia. Receptors are unique proteins of approximately 28 kDa that have been cloned from guinea pigs (Hanner et al., 1996), humans (Kekuda et al., 1996), mice (Pan et al., 1998), and rats (Seth et al., 1998; Mei and Pasternak, 2001) with distinct pharmacological characteristics (Martin et al., 1976; Bowen, 2000; Matsumoto, 2007). They have been implicated in a wide range of actions. They have been associated with potassium channels (Aydar et al., 2002) and aspects of cell proliferation and cancer (Bowen, 2000; Aydar et al., 2004; Casellas et al., 2004). Among their actions, receptors comprise a tonically active antiopioid system (Chien and Pasternak, 1993, 1994, 1995a,b; Pasternak, 1994; King et al., 1997; Mei and Pasternak, 2002). The 1 antagonist haloperidol greatly potentiated systemic opioid analgesia. Although ( )pentazocine is an effective opioid with activity at both and receptors, ( )-pentazocine, in contrast, has no opioid activity, but it is a potent 1 agonist. ( )-Pentazocine reduced systemic opioid analgesia for , , 1, 3, and orphanin FQ/ nociceptin ligands. This enhanced activity of opioid actions with haloperidol implied that the 1 system was tonically active. Furthermore, the differences in sensitivity to opioids among several strains of mice could be eliminated by blocking 1 actions with haloperidol, raising the possibility that these sensitivity differences might reflect varying levels of tonic activity of the 1 system. The cloning of 1 receptors facilitated their study at the molecular level. Down-regulation of 1 receptors in either the mouse or rat using antisense techniques had effects similar to those of the antagonist haloperidol. Thus, the evidence implicating the ability of 1 receptors to modulate opioid analgesia is strong. A number of brainstem structures have shown potent morphine analgesic activities (Pert and Yaksh, 1974; Bodnar et al., 1988; Rossi et al., 1993, 1994a). These studies revealed complex synergistic interactions among three morphine-sensitive sites, the periaqueductal gray (PAG), the locus coeruleus (LC), and the rostroventral medulla (RVM). Autoradiographic studies indicate that 1 receptors are present within the brainstem, including these morphine sensitive sites (Walker et al., 1992). Furthermore, the 1 receptor antagonist haloperidol and agonist ( )-pentazocine, influence supraspinal, but not spinal, morphine analgesia (J. F. Mei and G. W. Pasternak, unpublished data). This supraspinal modulation of opioid analgesia by 1 receptors raises questions regarding the regional localization of these interactions. We now report the mapping of rat 1 receptor modulation of morphine analgesia in brainstem nuclei. This work was supported from a Senior Scientist Award and National Institute on Drug Abuse Grants DA07241, DA02615, and DA00220 (to G.W.P.) and National Cancer Institute Core Grant CA08748 (to Memorial SloanKettering Cancer Center). Article, publication date, and citation information can be found at http://jpet.aspetjournals.org. doi:10.1124/jpet.107.121137. ABBREVIATIONS: PAG, periaqueductal gray; LC, locus coeruleus; RVM, rostroventral medulla; %MPE, percent maximal possible effect. 0022-3565/07/3223-1278–1285$20.00 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 322, No. 3 Copyright © 2007 by The American Society for Pharmacology and Experimental Therapeutics 121137/3240822 JPET 322:1278–1285, 2007 Printed in U.S.A. 1278 at A PE T Jornals on Jne 4, 2017 jpet.asjournals.org D ow nladed from Materials and Methods Materials. Na[I] was purchased from PerkinElmer Life and Analytical Sciences (Boston, MA). [I]( )-Pentazocine labeling was performed using the chloramine T/sodium metabisulfate method (Letchworth et al., 2000). ( )-Pentazocine and morphine sulfate were gifts from the Research Technology Branch of National Institute on Drug Abuse (Bethesda, MD). Halothane was purchased from Halocarbon Laboratories (Hackensack, NJ). All other chemicals were purchased from Sigma-Aldrich (St. Louis, MO). All the drugs used in the in vivo studies were dissolved in saline. Glass fiber filters (number 32) were purchased from Whatman Schleicher and Schuell (Keene, NH). Formula 963 liquid scintillant was purchased from PerkinElmer Life and Analytical Sciences. Animals and Analgesic Testing. Male albino Sprague-Dawley rats (175–250 g) were purchased from Charles River Laboratories, Inc. (Wilmington, MA), and they were maintained on a 12-h light/ dark cycle with rodent chow and water available ad libitum. Rats were housed in groups of two in clear plastic cages at ambient room temperatures between 21 and 25°C until they were tested. All animal studies were approved by the Institutional Animal Care and Use Committee of the Memorial Sloan-Kettering Cancer Center (New York, NY), and they adhered to National Institutes of Health guidelines (Institute of Laboratory Animal Resources (1996). Analgesia was determined using the radiant heat tail-flick technique as reported previously (Chien and Pasternak, 1995b). The thermal stimulus was positioned 8 cm above the dorsum and 3 to 9 cm proximal to the tip of the tail of a lightly restrained animal. The mean of two latency readings was taken for each animal at each indicated time. The baseline latencies ranged between 2 and 3 s, and maximal latency of 10 s was used to minimize the tissue damage. Studies used groups of four to seven rats. Saline injections did not appreciably alter the latencies over time compared with the baseline value, as shown in Fig. 2A. The percent maximal possible effect (%MPE) was calculated as the (observed latency baseline latency)/ (maximal latency baseline latency). Cannulations. Rats were anesthetized with chlorpromazine HCl (3 mg/kg i.p.) given 20 min before ketamine HCl (100 mg/kg i.m.) injection. The animal was set on the Kopf stereotaxic instrument (David Kopf Instruments, Tujunga, CA), and a stainless steel guide cannula (26-gauge; Plastic Products, Roanoke, VA) was implanted stereotaxically in the specified location. Cannulae coordinates were the same as in prior studies (Rossi et al., 1993). Cannulae were secured to the skull with three anchor screws and dental acrylic. The cannulae were then capped with a dummy cannulae (Plastic Products). Each animal was housed in a single cage, and it was allowed 1 week to recover from surgery before behavioral testing began. All cannulated rats were tested with morphine to confirm cannula placement at least 5 days before the experiment: PAG, 2.5 g; and RVM and LC, 5 g. Animals displaying a poor response were assumed to have a faulty cannulae placement, and they were not used in further experiments (Bodnar et al., 1988, 1991). After behavioral testing, cannulae placement was confirmed anatomically. Rat brains were fixed in 10% buffered formalin overnight and transferred into 30% sucrose until section cutting. The brains were cut coronally and stained with cresyl violet, and placement was confirmed by light microscopy. Animals whose placements were inaccurate were not included in the result (Bodnar et al., 1988, 1991; Rossi et al., 1993). Antisense and in Vivo Assay. Antisense oligodeoxynucleotides were designed using the rat 1 receptor cDNA sequences with the Align and Gene Runner programs (Scientific & Educational Software, Cary, NC), and they were purchased from Midland Certified Reagent (Midland, TX). The oligodeoxynucleotides were repurified using sodium acetate precipitation, and they were dissolved in saline to make a final concentration of 5 g/ l. The antisense oligodeoxynucleotide sequence (RS357AN) corresponding to the cloned rat 1 receptor (rs2-2) was 5 -CCAGCCGCCCGCGTTCAC-3 , and the mismatch control differed by switching six bases, 5 -CCACGCGCCGCCGTTACC-3 . Groups of mice were treated with antisense oligodeoxynucleotides (10 g in 2 l of saline per injection) or vehicle (saline) on days 1, 2, and 4, and they were tested for analgesia on day 5 with morphine sulfate ( ). Previous studies in rats and mice have shown that antisense treatment can down-regulate opioid or 1 receptors at both the mRNA and protein levels (Rossi et al., 1994b; Mei and Pasternak,