![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
|
|
|
|
|
||
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
NEUROPHARMACOLOGY
1 Receptors: A Microinjection StudyThe Laboratory of Molecular Neuropharmacology, Memorial Sloan-Kettering Cancer Center, New York, New York
Received February 9, 2007; accepted May 31, 2007.
 |
Abstract |
|---|
 Top Abstract Materials and MethodsResultsDiscussionReferences |
|---|
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 µ and 
receptors, (+)-pentazocine, in contrast, has no opioid activity, but it is a potent 1 agonist. (+)-Pentazocine reduced systemic opioid analgesia for µ, 
, 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.
|
Materials and Methods |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
). (+)-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).
|
). 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 µg; and RVM and LC, 5 µg. 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 µg/µl.
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 µg in 2 µl 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 (µ). 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, 2001).
Data Analysis. Rat analgesia data were compared by both the area under the time-action curve and at peak effect using one-way analysis of variance followed by a post hoc Tukey's analysis. ED50 values were calculated by Pharm/PCS software (Springer-Verlag, New York, NY) using nonlinear regression analysis. Results are presented as means ± S.E.M. of triplicate experiments unless specified otherwise.
|
|
Results |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
1 Receptor Agonist. Microinjection of morphine into any one of a number of sites within the rat brain elicits a profound naloxone-sensitive analgesia (Pert and Yaksh, 1974; Bodnar et al., 1988). We chose three sites sensitive to morphine analgesia to explore the interaction between supraspinal opioid and 1 systems: PAG, LC, and RVM.
Microinjection of morphine into each of the three regions produced a dose-dependent analgesia, with peak effects of 30 min in the PAG and the RVM and 15 min in the LC, and durations of action were between 60 and 90 min (Fig. 1). These results are consistent with our earlier study (Rossi et al., 1993).
We next explored whether 1 systems modulated morphine action in these regions. The 1 agonist (+)-pentazocine significantly blocked morphine analgesia in the PAG, RVM, and LC (Fig. 2, A–C) in a dose-dependent manner (Fig. 2D), but the sensitivity of the regions varied markedly. The RVM was the most sensitive to (+)-pentazocine, with an ID50 of 2.6 ng. The LC also was quite sensitive, with an ID50 of 17.4 ng. In contrast, the doses of (+)-pentazocine needed to reduce morphine analgesia in the PAG were up to 1000-fold greater, with an ID50 of 4090 ng (Fig. 2D).
In both the PAG site and RVM sites, the effects of (+)-pentazocine could be overcome by increasing the morphine dose (Fig. 3; Table 1). In the PAG, (+)-pentazocine significantly shifted the morphine dose-response curve more than 4-fold, increasing the ED50 from 1.5 to 6.5 µg. We observed a similar significant shift in the morphine dose-response curve in the RVM, with the morphine ED50 significantly increasing from 2.8 to 12 µg. In both regions, morphine retained the ability to fully overcome the inhibitory actions of (+)-pentazocine.
|
|
Modulation of PAG Analgesia through RVM 1 Receptor Systems. Opioid administration into the PAG activates a circuit between the PAG and the RVM, with release of endogenous opioids (Osborne et al., 1996; Fields, 2000). We therefore examined potential interactions between the PAG and RVM. Administration of (+)-pentazocine into the RVM attenuated the analgesic activity of morphine microinjected into the PAG (Fig. 4A), consistent with the prior demonstration of the circuit. (+)-Pentazocine was equally effective in attenuating the actions of morphine given into either the PAG or RVM (Fig. 4B). The ability of RVM (+)-pentazocine to block PAG morphine analgesia is further supported by the sensitivity of PAG morphine analgesia to RVM (+)-pentazocine at only 50 ng, a dose that was inactive against morphine when both were given directly into the PAG.
|
This ability of (+)-pentazocine administered into the RVM to block PAG morphine analgesia raised the question of whether the high doses of (+)-pentazocine needed to lower morphine analgesia when both drugs were given into the PAG might be due to diffusion of (+)-pentazocine from the PAG to the RVM. However, this seems to be unlikely for several reasons. Autoradiographically, we failed to see evidence of [125I](–)-pentazocine diffusing in the RVM following its injection into either the LC or PAG after 45 min. However, the sensitivity of autoradiography is limited, and it might not be sufficient to detect (+)-pentazocine doses necessary for activity in the PAG and RVM. Therefore, we examined the ability of the 1 antagonist haloperidol to reverse (+)-pentazocine actions. If the activity of PAG (+)-pentazocine was due to its diffusion into the RVM, haloperidol administered into the RVM should reverse the actions of (+)-pentazocine and increase morphine analgesia. Haloperidol did not reverse (+)-pentazocine (Fig. 5), making it unlikely that the actions of (+)-pentazocine in the PAG result from diffusion into the RVM. However, it leaves open the question of why this region is so insensitive, particularly because it has readily demonstrable levels of binding sites autoradiographically (Gundlach et al., 1986).
|
1 Receptor Antagonist. Haloperidol enhances opioid analgesia, presumably by blocking a tonically active 1 system (Chien and Pasternak, 1993, 1994, 1995b; King et al., 1997; Mei and Pasternak, 2002). To assess the possibility of tonic 1 activity within individual brainstem regions, we examined the effects of the 1 antagonist haloperidol on morphine analgesia in each region. At the maximal doses, we could test due to solubility constraints (5 µg), haloperidol had little effect on morphine analgesia when both were microinjected into either the PAG or the LC (Fig. 6). However, haloperidol coadministered with morphine into the RVM markedly enhanced the analgesic response. Full dose-response curves revealed that haloperidol shifted the ED50 of morphine in the RVM by approximately 2-fold (Fig. 7; Table 1). These findings confirm tonic 1 activity in the RVM but not in the PAG and LC sites. Haloperidol can interact with both 1 and dopamine D2 receptors. To ensure that the actions of haloperidol were mediated through 1 sites, we also tested (–)-sulpiride, a D2 receptor-selective antagonist. Administering (–)-sulpiride along with morphine into the RVM failed to enhance the analgesic actions of morphine in the RVM, supporting the suggestion that haloperidol was acting through blockade of 1 receptors (J. F. Mei and G. W. Pasternak, unpublished observations), results similar to those in mice (Chien and Pasternak, 1994).
|
|
1 Receptors on Morphine Analgesia in the RVM. Antisense approaches have proven valuable in selectively down-regulating opioid receptors and receptors within the central nervous system, with a selectivity greater than that of most antagonists (Standifer et al., 1994; Pasternak and Pan, 2000). To confirm the role of 1 receptors in the potentiation of morphine analgesia, we used an antisense approach similar to that used to down-regulate µ opioid receptors (Rossi et al., 1994b). In earlier work, our group demonstrated the ability of antisense treatment to down-regulate 1 receptors and to potentiate opioid analgesia in both mice and rats (Pan et al., 1998; Mei and Pasternak, 2001, 2002). In this study, we administered the antisense directly into RVM, and we tested morphine given into the same region. Antisense treatment enhanced morphine analgesia in the RVM [Fig. 8), as shown both by the peak effect in the time-action curve (p < 0.001) and by the area under the curves (p < 0.001)]. To ensure the specificity of the effect, we used a mismatch antisense probe in which the order of three sets of adjacent bases was switched, keeping the total base composition the same. The mismatch was without effect, confirming the selectivity of the antisense effect.
|
1 Actions in the RVM can modulate the analgesic actions of morphine in the PAG. We therefore examined of the effects of antisense treatment in the RVM on the analgesic activity of morphine administered into the PAG (Fig. 8). Morphine in the PAG at a dose that failed to significantly elevate tail-flick latencies above baseline levels elicited a profound analgesic response following antisense treatment in the RVM. Again, the inactivity of the mismatch antisense oligodeoxynucleotide confirmed the selectivity of the response. These antisense studies confirmed the role of 1 receptors in the modulation of morphine analgesia and the ability of 1 receptors within the RVM to modulate the analgesic actions of PAG morphine.
|
Discussion |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
; Pert and Yaksh, 1974; Bodnar et al., 1988, 1991; Rossi et al., 1993). Although each can induce analgesia alone, they also interact with each other, as shown by analgesic synergy between morphine administered in the PAG and the RVM (Rossi et al., 1994a). This pharmacological interaction is consistent with detailed studies demonstrating the importance of the RVM in mediating the actions of opioids in the PAG (Osborne et al., 1996; Christie et al., 2000; Fields, 2000).
The receptor system reportedly plays an important role in antiopioid, antipsychotic, neuroprotective, and antidepressant activities (Bowen, 2000; Hayashi and Su, 2005). (+)-Pentazocine, a selective 1 receptor agonist, blocks opioid analgesia, whereas the 1 antagonist haloperidol potentiates opioid analgesia (Chien and Pasternak, 1993, 1994, 1995a,b), results supported by antisense approaches in mice (Mei and Pasternak, 2001, 2002). The object of the current study was to explore the role of 1 receptor systems within a series of brainstem nuclei of the rat known to be important in opioid analgesia.
The ability of the 1 agonist (+)-pentazocine to diminish opioid analgesia in all three brainstem regions clearly demonstrates the presence and potential importance of the 1 system in the modulation of opioid mechanisms. However, there were significant differences among the sites. (+)-Pentazocine was quite potent in both the LC and the RVM but not the PAG, despite the high levels of 1 binding as assessed autoradiographically (Gundlach et al., 1986). Indeed, the doses necessary to influence morphine analgesia in the PAG were several orders of magnitude greater than the other two regions. Initially, we assumed that this might reflect the need for the drug to diffuse from the PAG to the RVM, particularly because (+)-pentazocine at far lower doses administered directly into the RVM attenuated morphine analgesia following morphine injection into the PAG. However, several lines of evidence suggest that this is not likely. First, directly looking at the diffusion of [125I]pentazocine failed to show any appreciable levels in the RVM after administering the compound into the PAG. In addition, the 1 antagonist haloperidol given into the RVM did not reverse the actions of (+)-pentazocine administered into the PAG, which would have been expected if the (+)-pentazocine were acting in the RVM. Yet, it is still not clear why (+)-pentazocine is so weak in the PAG. It may simply reflect the limitations of the 1 system in this region in modulating analgesia, but at these high doses, (+)-pentazocine may be acting through alternative, non- mechanisms.
Although morphine analgesia in both the LC and RVM was lowered by low doses of (+)-pentazocine, implying a highly sensitive 1 system, only the RVM seemed to have tonic 1 activity based upon the ability of the 1 antagonist haloperidol to enhance morphine actions. Earlier work in mice (Chien and Pasternak, 1993, 1994, 1995a; King et al., 1997; Mei and Pasternak, 2002) and in rats (Chien and Pasternak, 1995b; Mei and Pasternak, 2001) showed that haloperidol enhanced opioid analgesia. Interestingly, varying levels of tonic 1 receptor activity seemed to be responsible for many of the differences in sensitivities of some strains to opioids.
Although haloperidol is a potent 1 antagonist, it also blocks dopamine D2 receptors quite effectively, making the interpretation of some of the studies difficult. Unlike haloperidol, the selective dopamine D2 receptor antagonist (–)-sulpiride does not interact with 1 receptors. In our prior studies in CD-1 mice, (–)-sulpiride did not reproduce the actions of haloperidol. However, in other mouse strains and models, dopamine D2 receptors can influence opioid action (Calcutt et al., 1971; Kunihara et al., 1993; Kamei and Saitoh, 1996), a finding that was supported in a more recent study looking at opioid actions in a dopamine D2 receptor knockout mouse (King et al., 2001). Although there may be situations in which D2 receptors modulate opioid actions, 1 systems also are important. This was clearly shown by the ability of haloperidol to potentiate and (+)pentazocine to block opioid analgesia in the D2 knockout mice. Because these mice have no D2 receptors, the actions must be mediated through 1 sites.
To further verify the role of 1 receptors in the RVM, we also examined the effects of antisense. Antisense approaches can effectively down-regulate receptors and influence opioid behavior (Standifer et al., 1994). Down-regulation of 1 receptors in the RVM by antisense, but not by mismatch controls, potentiated morphine analgesia in much the same way as haloperidol. This confirms the role of 1 receptors in the RVM in morphine analgesia.
The interactions between the PAG and RVM were particularly interesting. A large literature has established the importance of the PAG-to-RVM pathway in opioid analgesia (Osborne et al., 1996; Fields, 2000). This is further supported by the synergistic opioid interactions in microinjection studies (Rossi et al., 1993). The current studies support these PAG/RVM interactions. (+)-Pentazocine administered into the RVM attenuated morphine analgesia from the PAG, whereas haloperidol in the RVM enhanced it. Finally, antisense treatment targeting 1 receptors in the RVM potentiated morphine analgesia from the PAG, much like the actions of haloperidol. Thus, the 1 system in the RVM also modulates opioid actions from the PAG.
In conclusion the current study supports a role for 1 receptor actions in the brainstem. Its activity within the three regions differed widely. Whereas both the LC and RVM were sensitive to (+)-pentazocine, the PAG was not, despite the demonstration of 1 receptors in this site autoradiographically (Walker et al., 1992). The RVM was particularly interesting in that it was the only regions with evidence for tonic 1 activity, and it could modulate the analgesia from morphine given into the PAG.
|
Acknowledgements |
|---|
|
Footnotes |
|---|
Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
ABBREVIATIONS: PAG, periaqueductal gray; LC, locus coeruleus; RVM, rostroventral medulla; %MPE, percent maximal possible effect.
Address correspondence to: Dr. Gavril W. Pasternak, Department of Neurology, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, NY 1002. E-mail: pasterng{at}mskmail.mskcc.org
|
References |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
Aydar E, Palmer CP, and Djamgoz MB (2004) Sigma receptors and cancer: possible involvement of ion channels. Cancer Res 64: 5029–5035.
Aydar E, Palmer CP, Klyachko VA, and Jackson MB (2002) The sigma receptor as a ligand-regulated auxiliary potassium channel subunit. Neuron 34: 399–410.[CrossRef][Medline]
Bodnar RJ, Paul D, and Pasternak GW (1991) Synergistic interactions between the periaqueductal gray and the locus coeruleus. Brain Res 558: 224–230.[CrossRef][Medline]
Bodnar RJ, Williams CW, and Pasternak GW (1988) Role of mu1 opiate receptors in supraspinal opiate analgesia: a microinjection study. Brain Res 447: 45–52.
Bowen WD (2000) Sigma receptors: recent advances and new clinical potentials. Pharm Acta Helv 74: 211–218.[CrossRef][Medline]
Calcutt CR, Doggett NS, and Spencer PSJ (1971) Modification of the anti-nociceptive activity of morphine by centrally administered ouabain and dopamine. Psychopharmacologia 21: 111–117.[CrossRef][Medline]
Casellas P, Galiegue S, Bourrie B, Ferrini JB, Jbilo O, and Vidal H (2004) SR31747A: a peripheral sigma ligand with potent antitumor activities. Anticancer Drugs 15: 113–118.[CrossRef][Medline]
Chien C-C and Pasternak GW (1993) Functional antagonism of morphine analgesia by (+)-pentazocine: evidence for an anti-opioid 1 system. Eur J Pharmacol 250: R7–R8.[CrossRef][Medline]
Chien C-C and Pasternak GW (1994) Selective antagonism of opioid analgesia by a system. J Pharmacol Exp Ther 271: 1583–1590.
Chien C-C and Pasternak GW (1995a) (–)-Pentazocine analgesia in mice: interactions with a receptor system. Eur J Pharmacol 294: 303–308.[CrossRef][Medline]
Chien C-C and Pasternak GW (1995b) Sigma antagonists potentiate opioid analgesia in rats. Neurosci Lett 190: 137–139.[CrossRef][Medline]
Christie MJ, Connor M, Vaughan CW, Ingram SL, and Bagley EE (2000) Cellular actions of opioids and other analgesics: implications for synergism in pain relief. Clin Exp Pharmacol Physiol 27: 520–523.[CrossRef][Medline]
Fields HL (2000) Pain modulation: expectation, opioid analgesia and virtual pain. Prog Brain Res 122: 245–253.[Medline]
Gundlach AL, Largent BL, and Snyder SH (1986) Phencyclidine (PCP) receptors: autoradiographic localization in brain with the selective ligand, [3H]TCP. Brain Res 386: 266–279.[CrossRef][Medline]
Hanner M, Moebius FF, Flandorfer A, Knaus HG, Striessnig J, Kempner E, and Glossmann H (1996) Purification, molecular cloning, and expression of the mammalian sigma1-binding site. Proc Natl Acad Sci U S A 93: 8072–8077.
Hayashi T and Su TP (2005) The potential role of sigma-1 receptors in lipid transport and lipid raft reconstitution in the brain: implication for drug abuse. Life Sci 77: 1612–1624.[CrossRef][Medline]
Institute of Laboratory Animal Resources (1996) Guide for the Care and Use of Laboratory Animals 7th ed. Institute of Laboratory Animal Resources, Commission on Life Sciences, National Research Council, Washington DC.
Jacquet YF and Lajtha A (1973) Morphine action at central nervous system sites in rat: analgesia or hyperalgesia depending on site and dose. Science 182: 490–492.
Kamei J and Saitoh A (1996) Involvement of dopamine D2 receptor-mediated functions in the modulation of morphine-induced antinociception in diabetic mouse. Neuropharmacology 35: 273–278.[CrossRef][Medline]
Kekuda R, Prasad PD, Fei Y-J, Leibach FH, Ganapathy V, and Fei YJ (1996) Cloning and functional expression of the human type 1 sigma receptor (hSigmaR1). Biochem Biophys Res Commun 229: 553–558.[CrossRef][Medline]
King MA, Bradshaw S, Chang AH, Pintar JE, and Pasternak GW (2001) Potentiation of opioid analgesia in dopamine2 receptor knockout mice: evidence for a tonically active anti-opioid system. J Neurosci 21: 7788–7792.
King MA, Pan Y-X, Mei J, Chang A, Xu J, and Pasternak GW (1997) Enhanced kappa opioid analgesia by antisense targeting the 1 receptor. Eur J Pharmacol 331: R5–R6.[CrossRef][Medline]
Kunihara M, Ohyama M, and Nakano M (1993) Effects of spiradoline mesylate, a selective kappa-opioid-receptor agonist, on the central dopamine system with relation to mouse locomotor activity and analgesia. Jpn J Pharmacol 62: 223–230.[Medline]
Letchworth SR, Mathis JP, Rossi GC, Bodnar RJ, and Pasternak GW (2000) Autoradiographic localization of 125I[Tyr(14)]orphanin FQ/nociceptin and 125I[Tyr(10)]orphanin FQ/nociceptin(1-11) binding sites in rat brain. J Comp Neurol 423: 319–329.[CrossRef][Medline]
Martin WR, Eades CG, Thompson JA, Huppler RE, and Gilbert PE (1976) The effects of morphine and nalorphine-like drugs in the nondependent and morphine-dependent chronic spinal dog. J Pharmacol Exp Ther 197: 517–532.
Matsumoto RR (2007) Sigma receptors: historical perspective and background, in Sigma Receptors: Chemistry, Cell Biology and Clinical Implications (Matsumoto RR, Bowen WD, and Su T-P eds) pp 1–24, Springer, New York.
Mei J and Pasternak GW (2001) Molecular cloning and pharmacological characterization of the rat sigma1 receptor. Biochem Pharmacol 62: 349–355.[CrossRef][Medline]
Mei JF and Pasternak GW (2002) 1 Receptor modulation of opioid analgesia in the mouse. J Pharmacol Exp Ther 300: 1070–1074.
Osborne PB, Vaughan CW, Wilson HI, and Christie MJ (1996) Opioid inhibition of rat periaqueductal grey neurones with identified projections to rostral ventromedial medulla in vitro. J Physiol 490: 383–389.
Pan YX, Mei JF, Xu J, Wan BL, Zuckerman A, and Pasternak GW (1998) Cloning and characterization of a mouse 1 receptor. J Neurochem 70: 2279–2285.[Medline]
Pasternak GW (1994) Anti-opioid activity of sigma1 systems. Regul Pept 54: 219–220.[CrossRef]
Pasternak GW and Pan Y-X (2000) Antisense mapping: assessing functional significance of genes and splice variants. Methods Enzymol 314: 51–60.[CrossRef][Medline]
Pert A and Yaksh TL (1974) Sites of morphine induced analgesia in primate brain: relation to pain pathways. Brain Res 80: 135–140.[CrossRef][Medline]
Rossi GC, Pasternak GW, and Bodnar RJ (1993) Synergistic brainstem interactions for morphine analgesia. Brain Res 624: 171–180.[CrossRef][Medline]
Rossi GC, Pasternak GW, and Bodnar RJ (1994a) µ and opioid synergy between the periaqueductal gray and the rostro-ventral medulla. Brain Res 665: 85–93.[CrossRef][Medline]
Rossi G, Pan YX, Cheng J, and Pasternak GW (1994b) Blockade of morphine analgesia by an antisense oligodeoxynucleotide against the mu receptor. Life Sci 54: L375–L379.[CrossRef]
Seth P, Fei YJ, Li HW, Huang W, Leibach FH, and Ganapathy V (1998) Cloning and functional characterization of a receptor from rat brain. J Neurochem 70: 922–931.[Medline]
Standifer KM, Chien C-C, Wahlestedt C, Brown GP, and Pasternak GW (1994) Selective loss of opioid analgesia and binding by antisense oligodeoxynucleotides to a opioid receptor. Neuron 12: 805–810.[CrossRef][Medline]
Walker JM, Bowen WD, Goldstein SR, Roberts AH, Patrick SL, Hohmann AG, and DeCosta B (1992) Autoradiographic distribution of [3H](+)-pentazocine and [3H]1,3-di-o-tolylguanidine (DTG) binding sites in guinea pig brain: a comparative study. Brain Res 581: 33–38.[CrossRef][Medline]
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
 |
 |
 |
|
 |
 |
 |
4) were pretreated with either vehicle, haloperidol (5 µg), or (+)-pentazocine (6.5 µg) 10 min before injection of at least three different doses of morphine in either the PAG or the RVM site, and analgesia was assessed in the tail-flick assay. ED50 with 95% confidence limits were calculated and presented here. The ED50 value of morphine PAG analgesia with haloperidol pretreatment was not determined because no significant change compared with naïve animals was seen in single-dose studies.
