I. Introduction

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The use of molecular biological approaches has led to extraordinary advances in our understanding of opioid action in the last decade. Soon after the cloning of the  opioid peptide receptor (DOP₁, originally termed DOR-1) (Evans et al., 1992; Kieffer et al., 1992), a number of laboratories identified clones corresponding to the µ opioid peptide (MOP₁, originally termed MOR-1) and  opioid peptide (KOP₁, originally termed KOR-1) receptors. A meeting held in Washington, DC, as a tribute to the memory of Dr. William Martin,² documented these advances in opioid receptor pharmacology (Uhl et al., 1994). At this meeting, several laboratories first described a fourth receptor clone closely homologous to the traditional opioid receptors (Table 1). These clones were isolated from a number of species and were typical G-protein-coupled receptors with the expected predicted seven transmembrane domains. These novel clones displayed approximately 50% identity with the traditional opioid receptors overall, with the transmembrane regions showing even higher homologies of up to 80%.

Despite their close homology to the other opioid receptors, the novel clones were difficult to characterize and were considered by many to be an orphan receptor. Few opioids labeled these novel clones, and their affinities were markedly lower than those seen with the cloned opioid receptors. Functionally, the ability of opioids to modulate adenylate cyclase with these clones also was markedly limited (Mollereau et al., 1994; Pan et al., 1995). Several early papers uncovered a close relationship between this clone and the ₃ receptor but concluded that they were not identical (Pan et al., 1994, 1995). The evidence for a relationship between them came from several lines of investigation. A monoclonal antibody capable of neutralizing ₃ opioid binding in brain tissue and ₃ analgesia in vivo recognized the expressed receptor generated through in vitro translation in Western blot analysis. Furthermore, in antisense mapping studies a number of antisense probes directed against the second and third coding exons of the murine clone (KOR-3) blocked the analgesic activity of ₃ analgesic naloxone benzoylhydrazone in mice without affecting the analgesic actions of traditional µ, , and ₁ drugs. Yet, the inability of a range of antisense probes targeting the first coding exon and the markedly different binding profile of the new clone indicated that the novel clone was not identical to the ₃ receptor. The differences between the two were further documented with the identification of the endogenous ligand for this receptor, a heptadecapeptide termed orphanin FQ or nociceptin (OFQ/N³) (Fig. 1). Despite an exceedingly high affinity for the cloned receptor, OFQ/N does not compete binding to the traditional µ opioid receptors.

View larger version (36K): [in this window] [in a new window]   Fig. 1.   OFQ/N and its related peptides. Sequences of the endogenous opioid peptides and ppOFQ/N peptides are presented. In some situations, sequences are species-dependent. The sequence of ppOFQ/N160-187 is from the mouse and nocistatin from human.

The nomenclature in this field is confusing, particularly for the receptor. In the early papers each laboratory used its own nomenclature for the receptor (Table 1), but as the years went by the term ORL1 gained favor. Recently, it has been suggested that all the opioid receptor family of receptors be renamed, with the term NOP₁ (nociceptin/orphanin FQ peptide) receptor referring to the ORL1 clone in all species. Similarly, the ligand is known as nociceptin or orphanin FQ, having been isolated by two groups independently (Meunier et al., 1995; Reinscheid et al., 1995). Nociceptin was chosen by one group to denote its presumed pronociceptive activity. The term orphanin FQ refers to its affinity for the "orphan" opioid receptor, while the F and Q refer to the first and last amino acids, phenylalanine and glutamine. Neither term predominates and most laboratories use the two together, as is done in this review: orphanin FQ/nociceptin, or OFQ/N.

This field has burgeoned enormously since the cloning of the receptor and the identification of its peptides. Aspects of this field have been reviewed previously (Henderson and McKnight, 1997; Meunier, 1997; Civelli et al., 1998; Darland et al., 1998; Taylor and Dickenson, 1998; Zaki and Evans, 1998; Yamamoto et al., 1999; also see a special issue of the journal Peptides, volume 21, number 7, 2000). The current review will focus rather comprehensively upon the behavioral pharmacology of OFQ/N, with an attempt to understand it from the molecular perspective.

	II. Molecular Biology of the Orphanin FQ/Nociceptin Receptor

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NOP₁ was cloned from various species by a number of laboratories at about the same time. NOP₁ is a typical G-protein-coupled receptor with seven predicted transmembrane domains (Fig. 2A) and is localized to murine chromosome 2 (Chen et al., 1994; Nishi et al., 1994). It was readily detected by Northern analysis, where a major band of 3.4 kb was detected in mice (Pan et al., 1995). Several laboratories found a similar band in rats at approximately 3.4 kb, as well as additional bands (Chen et al., 1994; Wang et al., 1994; Lachowicz et al., 1995). One group reported additional bands of 7.5 and 10 kb (Chen et al., 1994), another observed a single additional band of approximately 7.6 kb (Lachowicz et al., 1995), and a third group reported two additional bands of 13 and 23 kb (Wang et al., 1994). The significance of these additional larger bands is not clear, particularly with the differences noted among groups. However, the differing ratios of these larger bands to the 3.4-kb band among regions raising interesting questions regarding regional processing (Wang et al., 1994; Lachowicz et al., 1995).

View larger version (38K): [in this window] [in a new window]   Fig. 2.   Schematic of the NOP1 receptor and its gene, Oprl1. A, schematic of the NOP1 receptor is presented with the sites of the protein corresponding to the splice sites in the mRNA indicated. B, schematic of the structure of the murine Oprl1 gene.

Southern analysis from a number of groups implies a single copy of the gene for the NOP₁ receptor, which is termed Oprl1. The murine NOP₁ gene structure was elucidated soon after the initial reports of the receptor (Pan et al., 1996b). The receptor has three coding exons, similar to the other opioid receptors. The first coding exon yields the amino terminus and the first transmembrane domain (Fig. 2A). The second coding exon is responsible for the next three transmembrane domains. The splice site between the second and third coding exons is located in the second extracellular loop, and the third coding exon is responsible for the remainder of the protein, including the last three transmembrane domains and the intracellular carboxyl tail (Fig. 2A). The binding pocket has been proposed to comprise several of the transmembrane domains (Topham et al., 1998; Mouledous et al., 2000). The initial gene structure identified five exons, with a noncoding exon preceding and following the three coding exons (Fig. 2B) (Pan et al., 1996b). More recent work has identified two mini-exons between the first and second coding exons that are alternatively spliced for a total of seven. The numbering of the exons has changed, as new ones have been uncovered. In the current review, exon 2 corresponds to the first coding exon of the original clone, exon 5 to the second coding exon and exon 6 to the last one.

Like other members of the opioid receptor family, NOP₁ undergoes alternative splicing. The first variant, NOP_1d, was identified in lymphocytes and contains a 15-bp deletion from the 3' end of the first coding exon (exon 2) (Halford et al., 1995; Wick et al., 1995). Similar variants were obtained from mouse (Pan et al., 1998b), rat (Wick et al., 1994; Xie et al., 1999), and human (Peluso et al., 1998) brains. An intron retention variant, NOP_1e, containing the intron between the second and third coding exons (exons 5 and 6) was reported in mice (Pan et al., 1998b), rats (Chen et al., 1994; Xie et al., 1999, 2000), and human brain (Xie et al., 1999). The initial report in rats found an 84-bp insertion that could be translated through to generate a full-length receptor. The mouse version, however, had only 81 bp, and the presence of a stop codon prevented translation of the last three transmembrane domains (Pan et al., 1998a). A more recent report in rats found a similar 81-bp insertion with a stop codon (Xie et al., 1999). It is not clear which of the two rat variants predominates, but this is an important issue since one has the potential of being a functional variant whereas the other does not.

An additional three NOP₁ variants have been described that contain mini-exons located between the first and second coding exons (exons 2 and 5) (Fig. 2B) (Xie et al., 1999). NOP_1a contains a 34-bp insertion (exon 3) between the first two coding exons. Due to a frameshift, it gives a predicted stop codon in exon 5, yielding a truncated protein lacking the seven transmembrane domains typically associated with G-protein-coupled receptors. NOP_1c contains a different mini-exon insertion of 139 bp (exon 4) and predicts a truncated protein due to a frameshift and a predicted stop-codon. The other variant, NOP_1b, shows a different splice site within exon 4, the same mini-exon as NOP_1c, and contains only the 3' portion of the mini-exon (98 bp). Like the others, it also gives a predicted truncated protein. The significance of these truncated proteins is still not fully understood. Clearly, they do not function like traditional G-protein-coupled receptors. Yet, this does not necessarily imply that they have no functional significance. It is interesting that similar truncated variants have been reported for all the other opioid receptor genes.

Although NOP₁ itself has little affinity for traditional opioids, it can be converted into an opioid-like receptor either by simple mutations or by generating chimeras. In the rat NOP₁, a series of mutations within the transmembrane regions that had little effect upon the affinity of OFQ/N itself markedly transformed the affinity of the receptor up to 50-fold for dynorphin A and several of its analogs, although the mutants still did not show appreciable affinity for -endorphin (Meng et al., 1996). These mutations were dispersed throughout the protein, including an A213K mutation in TM5, a triple mutation VQV276-278IHI in TM6, and a T302I mutation near the top of TM7. When the T302I and VQV276-278IHI mutations were combined, the affinity of dynorphin A increased even further, with a K_i under 1 nM.

Chimeras also illustrate the close relationship between the NOP₁ receptor and the traditional opioid receptors. Chimeras combining the NOP₁ and KOP₁ (originally termed KOR-1) receptors were able to maintain high affinity for OFQ/N and dynorphin A (Lapalu et al., 1998; Mollereau et al., 1999). The first coding exon of the NOP₁ receptor encodes the first transmembrane domain, just as in the traditional opioid receptors. Exchanging the first coding exon of the NOP₁ receptor with the first exon of the µ opioid receptor MOP₁ (originally termed MOR-1) or the  opioid receptor DOP₁ (originally termed DOR-1) did not appreciably affect the affinity of the receptor for OFQ/N, but it did enhance the affinity of the ₃ ligand naloxone benzoylhydrazone (NalBzoH) approximately 5-fold and diminished the affinity of the truncated OFQ/N derivative OFQ/N(1-11) (Pan et al., 1996c). More interesting, however, the exchange with the first exon of DOP₁ converted NalBzoH from an agonist into an antagonist without changing its affinity. Opiates such as morphine still did not show appreciable affinity for any of the chimeras.

	III. Receptor Binding

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Structurally, the NOP₁ receptor has the anticipated seven transmembrane domains expected from members of the G-protein-coupled class of receptors. NOP₁ binding is sensitive to sodium and divalent cations (Ardati et al., 1997), as first described with opioid receptors (Pert and Snyder, 1973; Pasternak et al., 1975a,b; Wilson et al., 1975), and OFQ/N and its analogs modulate guanosine 5'-3-O-(thio)triphosphate binding in a pertussis toxin-sensitive manner (Reinscheid et al., 1995, 1996; Knoflach et al., 1996; Shimohira et al., 1997; Sim and Childers, 1997; Meis and Pape, 1998; Narita et al., 1999). The first description of OFQ/N binding to NOP₁ utilized an iodinated analog, ¹²⁵I-[Tyr¹⁴]OFQ/N (Reinscheid et al., 1995). Although many laboratories have used this ligand, others have used ³H-OFQ/N (Dooley and Houghten, 1996), which yields results virtually identical to those of the iodinated ligand (Ardati et al., 1997). More recently, a novel radioligand for the NOP₁ receptor, ³H-ac-RYYRWK-NH₂, was reported (Thomsen et al., 2000).

Most groups found a high affinity of OFQ/N for the transfected NOP₁ receptor, typically around 50 pM, although there is a moderately wide range of values that likely reflect differences in assay techniques, buffers, and cell lines (Dooley and Houghten, 2000) and ligand stability (Quigley et al., 2000). Yet, there is good agreement regarding the specificity of the labeling, which clearly distinguished the NOP₁ receptor from traditional opioid receptors. Of a wide variety of opiates, only NalBzoH showed a significant affinity for the site (K_i = 310 nM; Table 2), and even this was far less than its potency at opioid sites (K_i < 10 nM). Morphine and other alkaloids have K_i values well above 1000 nM.

The initial characterization of NOP₁ binding studies examined the interactions of OFQ/N and its analogs with the cloned receptor. Subsequent studies on brain tissue have yielded more diverse results. Although several laboratories found K_D values in brain similar to those in transfected cell lines (Albrecht et al., 1998; Nicholson et al., 1998; Thomsen et al., 2000), a number of other groups report far lower affinities (Dooley and Houghten, 1996, 2000; Wu et al., 1997; Mathis et al., 1998, 1999). Wide variations of binding levels also were reported. For example, reports of binding in rat cortex range from 22 fmol/mg of protein (Thomsen et al., 2000) to 291 fmol/mg of protein (Albrecht et al., 1998). The reasons underlying these differences are not clear. However, there are a number of variables that may play a role that include the choice of radioligand and binding conditions (Dooley and Houghten, 2000).

	IV. NOP1 Receptor Heterogeneity

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A major question regarding the NOP₁ receptor involves binding site heterogeneity. Evidence raising the possibility of multiple OFQ/N receptors comes from both pharmacological and binding sources. As noted earlier, the Oprl1 gene that encodes the NOP₁ receptor undergoes alternative splicing, but none of the additional variants identified to date encode a full-length receptor. Although the combined evidence for multiple classes of OFQ/N binding sites is suggestive, none of the studies provides conclusive evidence for NOP₁ receptor heterogeneity. Yet, it is worthwhile reviewing the evidence.

The behavioral pharmacology of OFQ/N is reviewed in detail in subsequent sections. However, several issues played a major role in exploring the possibility of multiple OFQ/N receptors. As discussed below, OFQ/N reportedly has both hyperalgesic and analgesic activities. The hyperalgesic activity was insensitive to opioid antagonists, as expected based upon their poor affinity for the OFQ/N binding sites. However, several laboratories have observed that opioid antagonists reverse the analgesic responses of OFQ/N (Rossi et al., 1996b, 1997, 1998b; Jhamandas et al., 1998; Kolesnikov and Pasternak, 1999). Many assumed that OFQ/N activated a neural circuit with a downstream opioid link, which could be blocked by the antagonists, and this is still a consideration. However, the opioid antagonist Win44,441 blocks the inhibition of cAMP accumulation produced by OFQ/N in mouse brain (Mathis et al., 1997). Since this assay directly measures the effects of the receptor coupled to cyclase in membrane fragments, it is difficult to envision how the antagonist could act other than directly blocking the receptor activated by OFQ/N. Thus, this OFQ/N action in brain membranes argues for an opioid antagonist-sensitive OFQ/N receptor.

Antisense mapping studies also differentiated between OFQ/N analgesia and hyperalgesia. Whereas probes targeting the second and third coding exons of the NOP₁ receptor down-regulated OFQ/N and NalBzoH analgesia (King et al., 1997; Rossi et al., 1997), a probe targeting the first coding exon was ineffective. Yet, the same antisense probe based upon the first coding exon blocked the hyperalgesia and anti-opioid activity of OFQ/N, whereas the antisense probes targeting exons 2 and/or 3 that were active against OFQ/N analgesia had no effect against hyperalgesia (King et al., 1997; Rossi et al., 1997, 1998). Although these pharmacological assays are suggestive, behavioral approaches have many potential subtleties and alternative explanations. Thus, they do not provide conclusive evidence for multiple OFQ/N receptors.

Binding studies also suggest binding site heterogeneity. Early studies with ¹²⁵I-[Tyr¹⁴]OFQ/N in mouse brain revealed curvilinear Scatchard plots, suggestive of sites of differing affinity (Mathis et al., 1997). However, this does not necessarily imply different receptors. In NOP₁-transfected HEK293 cells with high levels of expression, ³H-OFQ/N binding also yielded biphasic Scatchard plots, presumably reflecting two conformations of the receptor (Ardati et al., 1997). Alternatively, curvilinear Scatchard plots can result from radioligand degradation. Saturation studies alone cannot distinguish among these possibilities and must be interpreted in conjunction with other types of studies. However, other approaches also implied NOP₁ receptor binding heterogeneity.

OFQ/N(1-11) is a truncated peptide derived from OFQ/N. In vivo, it is functionally active, eliciting analgesia (Rossi et al., 1997) and inhibiting cAMP accumulation in brain membranes (Mathis et al., 1997). These actions were not anticipated based upon its very poor affinity for the cloned NOP₁ receptor in binding assays. To more accurately assess the possibility of a novel OFQ/N(1-11) binding site, tyrosine-containing analogs were generated that could be iodinated and used to examine binding sites directly (Mathis et al., 1998), as done earlier with OFQ/N itself (Reinscheid et al., 1995). Of the analogs, [Tyr¹⁰]OFQ/N(1-11) proved most valuable. Like OFQ/N(1-11), [Tyr¹⁰]OFQ/N(1-11) is analgesic when given supraspinally in mice and it competes ¹²⁵I-[Tyr¹⁴]OFQ/N binding in mouse brain more potently (K_i = 79 nM) than OFQ/N(1-11) itself (K_i = 262 nM). Iodinating the analog to iodo[Tyr¹⁰]OFQ/N(1-11) further enhanced its potency (K_i = 39 nM).

In mouse brain, ¹²⁵I-[Tyr¹⁰]OFQ/N(1-11) labeling strongly suggested a novel binding site distinct from the binding of ¹²⁵I-[Tyr¹⁴]OFQ/N. Binding parameters of ¹²⁵I-[Tyr¹⁰]OFQ/N(1-11) revealed an affinity (K_D) of 0.24 nM, which is over 100-fold lower than its K_i against ¹²⁵I-[Tyr¹⁴]OFQ/N binding in mouse brain and more than 10-fold lower than its K_D determined in CHO cells transfected with the NOP₁ receptor. Furthermore, in brain it displayed a B_max of only 43 fmol/mg of protein, which is far fewer sites than observed in companion assays with ¹²⁵I-[Tyr¹⁴]OFQ/N (Table 3) or from the literature (Dooley and Houghten, 1996; Albrecht et al., 1998; Nicholson et al., 1998). It also is interesting that the capacity of the ¹²⁵I-[Tyr¹⁰]OFQ/N(1-11) site is similar to the higher affinity (K_D = 4 pM) site observed in mouse brain for ¹²⁵I-[Tyr¹⁴]OFQ/N. A possible association of the two is further suggested by saturation studies with ¹²⁵I-[Tyr¹⁴]OFQ/N in which the inclusion of OFQ/N(1-11) appeared to selectively reduce the higher affinity binding component of ¹²⁵I-[Tyr¹⁴]OFQ/N.

The difference in selectivity between OFQ/N(1-11) and the standard OFQ/N radioligands was quite revealing (Mathis et al., 1999). OFQ/N and its analogs labeled the ¹²⁵I-[Tyr¹⁰]OFQ/N(1-11) site with very high affinity, confirming its classification as an OFQ/N site. The affinity of OFQ/N(1-11) increases about 30-fold, with its K_i dropping to only 8.7 nM. It is interesting, however, that the affinity of OFQ/N(1-7) is unchanged and remains quite poor.

As previously noted, ¹²⁵I-[Tyr¹⁴]OFQ/N binding is insensitive to opioids. In contrast, ¹²⁵I-[Tyr¹⁰]OFQ/N(1-11) binding is competed by a wide variety of opioid ligands. Although the affinities of most of the opioids examined remain lower than against traditional opioid receptors, a number of compounds showed high affinity for this site (Table 4). Among the opiates, NalBzoH was the most impressive. In brain membranes, its affinity against ¹²⁵I-[Tyr¹⁰]OFQ/N(1-11) binding (K_i = 3.9 nM) is similar to that seen with traditional opioid binding sites and almost 100-fold greater than ¹²⁵I-[Tyr¹⁴]OFQ/N binding. The affinity of fentanyl is increased over 100-fold against the OFQ/N(1-11) site. Some opioid peptides also show high affinity for the ¹²⁵I-[Tyr¹⁰]OFQ/N(1-11) site, particularly dynorphin A and -neoendorphin. Indeed, dynorphin A labels the ¹²⁵I-[Tyr¹⁰]OFQ/N(1-11) site as potently as µ and  opioid receptors.

Together, along with the dramatic anatomical differences between ¹²⁵I-[Tyr¹⁴]OFQ/N and ¹²⁵I-[Tyr¹⁰]OFQ/N(1-11) binding (see below), these studies suggest the possibility of OFQ/N receptor heterogeneity. If multiple OFQ/N sites exist, they may correspond to splice variants of NOP₁ receptor. Alternatively, they might correspond to post-translational modifications of the receptors, result from modulation of the receptor by additional proteins, or be expressed by a totally different gene. However, without more definitive biochemical evidence, OFQ/N binding site heterogeneity remains tentative.

	V. Orphanin FQ/Nociceptin

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The unusual properties of the "orphan opioid receptor" soon led to the identification of an endogenous peptide, termed orphanin FQ (Reinscheid et al., 1995) or nociceptin (Meunier et al., 1995) (OFQ/N), that labeled the cloned receptor with very high affinity (Fig. 1). OFQ/N is a heptadecapeptide with some interesting structural homologies to the classical opioid peptide dynorphin A (Fig. 1). Both peptides are comprised of 17 amino acids bounded by pairs of basic amino acids important in their production from their precursors. Furthermore, both have internal pairs of basic amino acids, raising the possibility of further processing. The opioid peptides share a YGGF motif, where the fifth amino acid is either leucine or methionine. The amino terminus of OFQ/N is a phenylalanine instead of a tyrosine, followed by GGF. Finally, both peptides contain the same last two amino acids at the carboxyl terminus. Despite these similarities, the peptides are functionally quite distinct. OFQ/N has no appreciable affinity for any of the opioid receptors. Alanine scanning reveals the importance of the amino acids in positions 1, 2, 4, and 8 (Dooley and Houghten, 1996). Of these, the phenylalanine in position 1 is particularly important in establishing the selectivity of binding since replacing it with a tyrosine yields analogs with far greater affinity at traditional opioid receptors, although the [Tyr¹] analog still can induce naloxone-insensitive actions presumably mediated through NOP₁ receptors (Champion and Kadowitz, 1997a,b). The basic structure can be modified and even truncated at its carboxyl terminus without major loss of activity, but the initial FGGF motif is required for activity (Guerrini et al., 1997).

Full descriptions of all the structure-activity relationships of OFQ/N is beyond the scope of this review. However, several analogs are important. The first analog, [Tyr¹⁴]OFQ/N, was developed to enable the detection of receptor binding and has been particularly important (Reinscheid et al., 1995). Replacing the leucine at position 14 yielded a peptide that could be iodinated and still maintain affinity for the receptor similar to the parent compound (Reinscheid et al., 1995). This analog has proven extremely valuable in the characterization of the receptor in both transfected cell lines and in the brain.

OFQ/N has two pairs of basic amino acids within its structure, raising the possibility of further processing to yield OFQ/N(1-11) and OFQ/N(1-7). Although there are studies showing the activity of these peptides and suggesting that their pharmacology may differ from that of OFQ/N itself (Rossi et al., 1997), as described below, the physiological significance of these truncated peptides has not been fully established. The possibility that the truncated peptides also might be relevant led to the development of a tyrosine-containing analog of OFQ/N(1-11) suitable for iodination (Mathis et al., 1998). Analogs were synthesized with tyrosine at positions 1, 10, or 11. The placement of tyrosine at position 1 lowered its affinity against NOP₁ binding in transfected cells, but enhanced its potency against the traditional opioid receptors. [Tyr¹¹]OFQ(1-11) and its iodinated version, iodo[Tyr¹¹]OFQ/N(1-11), on the other hand, were devoid of activity against traditional opioid receptors and more potent against NOP₁ binding in transfected cells than OFQ/N(1-11) itself. Both [Tyr¹¹]OFQ(1-11) and iodo[Tyr¹¹]OFQ/N(1-11) were pharmacologically active, eliciting analgesia in mice. As discussed earlier, these agents have proven valuable in binding studies.

The evaluation of the pharmacology of OFQ/N was hindered for a number of years by the lack of an effective antagonist. The first proposed antagonist, [Phe¹(CH₂-NH)Gly²]-nociceptin(1-13)-NH₂, was subsequently found to be a partial agonist, with many groups observing OFQ/N-like actions in a variety of models. A recently described small molecule antagonist, J-113397 (1-[3R,4R)-1-cyclooctylmethyl-3-hydroxymethyl-4-piperidyl]-3-ethyl-1,3-dihydro-2H-benzimidazol-2-one) (Kawamoto et al., 1999; Ozaki et al., 2000a,b), has proven valuable in a number of models (see Section VIII.G.2.)_.

The OFQ/N sequence contains pairs of basic amino acids that might imply additional processing of the peptide to OFQ/N(1-11) and/or OFQ/N(1-7). Both of these truncated peptides are functionally active when administered in vivo (Rossi et al., 1997), producing analgesia that is reversed by opioid antagonists. Neither peptide shows appreciable affinity for any of the traditional opioid receptors, but OFQ/N(1-11) does label cloned NOP₁ receptors moderately well (K_i = 55 nM), although its affinity still is far lower than OFQ/N itself. OFQ/N(1-7) does not compete with binding to the NOP₁ receptor at doses as high as 1 µM. The true significance of these peptides remains to be demonstrated.

Like most neuropeptides, OFQ/N is generated from a larger precursor peptide, prepro-OFQ/N (ppOFQ/N) that has been cloned from mouse, rat, and human (Fig. 3) (Meunier et al., 1995; Pan et al., 1996a; Reinscheid et al., 2000) and that has been localized in man to chromosome 8 (8p21) (Mollereau et al., 1996). Overall, there is high interspecies homology, with 80% identity among the three organisms. Within the precursor, there are several additional peptides suggested by the presence of pairs of basic amino acids. Nocistatin has been examined most extensively (see Section IX.A.). Nocistatin possesses analgesic actions and presumably acts through a distinct receptor since it has no appreciable affinity for any of the traditional opioid receptors or NOP₁. It is interesting that the nocistatin sequence shows the most variability of the putative peptides within ppOFQ/N among species. The mouse version is the longest, containing 41 amino acids, whereas the rat peptide has 35 and the human form only 30. The mouse sequence has an interesting DAEPGA motif that is repeated three times. The rat form has a similar double repeat, but the human form does not. The differences between the species rests primarily over the length of this repeat, with the human form lacking 10 of the amino acids of the mouse version at this location.

View larger version (31K): [in this window] [in a new window]   Fig. 3.   Schematic of the prepro-OFQ/N gene.

Another peptide was predicted from the sequence of ppOFQ/N based upon the presence of pairs of basic amino acids suggesting sites of peptide processing. Orphanin FQ2 is a heptadecapeptide, like OFQ/N and dynorphin A, with a phenylalanine (F) and glutamine (Q) at the first and last position, leading to its name, OFQ2 (also called NocII; and hereinafter called OFQ2/NocII). The placement of OFQ2/NocII within ppOFQ/N is interesting in that OFQ2/NocII is immediately downstream from OFQ, much like dynorphin B is immediately downstream of dynorphin A in preprodynorphin. When administered centrally, OFQ2/NocII is pharmacologically active, raising the possibility that it is physiologically relevant (Rossi et al., 1998a; Florin et al., 1999) (see Section IX.B.). A longer peptide containing the OFQ2/NocII sequence at its amino terminus, ppOFQ/N(180-187), has been described and it also is functionally active in mice (Mathis et al., 2001). It is still an open question as to whether ppOFQ/N(180-187) is active itself or whether it is further processed to OFQ2/NocII.

	VI. Anatomy of Orphanin FQ/Nociceptin and Its Receptor

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The regional distribution of OFQ/N and the NOP₁ receptor have been well described (Bunzow et al., 1994; Fukuda et al., 1994; Mollereau et al., 1994; Wick et al., 1994; Lachowicz et al., 1995; Nothacker et al., 1996; Riedl et al., 1996; Houtani et al., 2000; Neal et al., 1999a,b; Letchworth et al., 2000; O'Donnell et al., 2001). These series of publications provide detailed descriptions of the distribution of the NOP₁ receptor mRNA and binding in the brain which are beyond the scope of this review. Overall, they report a good correlation between receptor binding distributions and those seen with in situ hybridization. Regions with NOP₁ receptor binding typically express NOP₁ mRNA as well, although the levels of mRNA and binding do not always match very closely. Regions with high levels of NOP₁ binding/mRNA include the cortex, anterior olfactory nucleus, lateral septum, hypothalamus, hippocampus, amygdala, central gray, pontine nuclei, interpeduncular nucleus, substantia nigra, raphe complex, locus coeruleus, and spinal cord. The distribution patterns have suggested the involvement of the NOP₁ receptor system in "motor and balance control, reinforcement and reward, nociception, the stress response, sexual behavior, aggression and autonomic control of physiological processes" (Neal et al., 1999a).

The distribution of OFQ/N also has been well described in the literature (Dickenson, 1996; Riedl et al., 1996; Kummer and Fischer, 1997; Mitsuma et al., 1998; Neal et al., 1999b; Houtani et al., 2000; O'Donnell et al., 2001). In brief, the localization of OFQ/N corresponds reasonably well with the NOP₁ receptor. As with the receptor, OFQ/N immunoreactivity and mRNA levels detected using in situ hybridization are closely correlated. OFQ/N is found in lateral septum, hypothalamus, ventral forebrain, claustrum, mammillary bodies, amygdala, hippocampus, thalamus, medial habenula, ventral tegmentum, substantia nigra, central gray, interpeduncular nucleus, locus coeruleus, raphe complex, solitary nucleus, nucleus ambiguous, caudal spinal trigeminal nucleus, and reticular formation, as well the ventral and dorsal horns of the spinal cord (Neal et al., 1999b).

The distribution of ¹²⁵I-[Tyr¹⁰]OFQ/N(1-11) in the brain also is distinct autoradiographically (Fig. 4) (Letchworth et al., 2000). The distribution of ¹²⁵I-[Tyr¹⁴]OFQ/N binding was described earlier. ¹²⁵I-[Tyr¹⁰]OFQ/N(1-11) binding also shows intense labeling of the cortex, but far lower levels of labeling in deeper structures. Compared with ¹²⁵I-[Tyr¹⁴]OFQ/N, ¹²⁵I-[Tyr¹⁰]OFQ/N(1-11) labeling is far less intense in the olfactory tubercle, nucleus accumbens, striatum, lateral and medial septum, hypothalamus, as well as a number of brain stem structures such as the periaqueductal gray, medial raphe, and locus coeruleus.

View larger version (119K): [in this window] [in a new window]   Fig. 4.   Autoradiographic studies of 125I-[Tyr14]OFQ and 125I-[Tyr10]OFQ(1-11) in rat brain. Sections of rat brain were incubated with either 125I-[Tyr14]OFQ or 125I-[Tyr10]OFQ(1-11) and opposed to film. The exposure time was longer for 125I-[Tyr10]OFQ(1-11) than for 125I-[Tyr14]OFQ due to the lower binding levels. Reprinted from Letchworth et al., 2000.

	VII. Range of Effects of Orphanin FQ/Nociceptin

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Befitting its particularly wide distribution in the nervous system (see above), there are a myriad of proposed functional roles for OFQ/N. Receiving by far the most attention is the involvement of this peptide in the mediation and modulation of pain in the supraspinal, spinal, and peripheral compartments of the nervous system. Related proposed functions for OFQ/N include roles in opiate tolerance, dependence/withdrawal, and adaptive responses to anxiety and stress. However, studies based on direct injection of the peptide, measurement of peptide levels, administration of antagonist/antisense compounds and/or the evaluation of the phenotype of transgenic "knockout" mutants have implicated OFQ/N in the mediation of biological phenomena ranging from learning and memory to hearing to water balance to reproductive physiology. A list of OFQ/N-associated systems-level phenomena is presented in Table 5. Some of the more well studied and noteworthy phenomena will be discussed presently, starting with pain processing.

	VIII. Effects of Orphanin FQ/Nociceptin on Pain

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The first in vivo action of OFQ/N reported by both its discoverers was a reduction in latency to respond to noxious thermal stimuli on the tail-flick (Reinscheid et al., 1995) and hot-plate tests (Meunier et al., 1995) after supraspinal (intracerebroventricular) injection in the mouse. Both groups interpreted these data as reflective of a hyperalgesic action; i.e., a decrease in nociceptive threshold (increase in nociceptive sensitivity) produced by the peptide. This was very much a surprise, since classical opioids, with the possible exception of dynorphin (see Caudle and Mannes, 2000), produce analgesic and/or antihyperalgesic effects (see Pasternak, 1993). The apparent hyperalgesia produced by supraspinal OFQ/N inspired Meunier and colleagues (1995) to dub the peptide nociceptin.

Exogenous administration of an endogenous compound is not an ideal method for gleaning its true physiological role. When injected intracerebroventricularly, OFQ/N will be widely dispersed throughout the ventricular system, possibly affecting populations of ORL1 receptors that would not normally be activated by endogenously released peptide. Tissue levels are dependent upon diffusion of the agent from the cerebrospinal fluid into the brain, which results in a decreasing gradient of drug concentrations in deeper structures. The drug even can diffuse to spinal sites, particularly with high injection volumes. This makes it difficult to judge the concentration of peptide in relevant brain loci and thereby assess whether its concentration is appropriate or grossly supraphysiological. Finally, this approach entirely ignores contextual elements accompanying OFQ/N release under usual circumstances. Nonetheless, in the absence of an ORL1 antagonist and with the vast majority of the studies reviewed herein conducted before any such antagonist was available, direct injection of OFQ/N was one of only a handful of feasible experimental approaches.

In contrast to the conclusions from the initial descriptions of OFQ/N, we now recognize that there is no widely accepted "role" of OFQ/N in supraspinal pain-modulatory circuits. In fact, even the effects of supraspinal projection of OFQ/N on nociceptive sensitivity remain highly contentious. As detailed in Table 6, reports in the literature have suggested six different "effects" of supraspinal OFQ/N on nociception: 1) hyperalgesia, 2) analgesia, 3) hyperalgesia followed by analgesia, 4) neither hyperalgesia nor analgesia, 5) anti-analgesia but not hyperalgesia, and 6) anti-analgesia plus hyperalgesia. The only uncontested observation is the anti-analgesic activity of OFQ/N, first documented in 1996 (Mogil et al., 1996a)

OFQ/N blocks analgesia from a wide variety of exogenous and endogenous opioid compounds. Since OFQ/N has negligible affinity for any of the traditional opioid receptors, it must act through neural circuits as a "functional antagonist", rather than through direct molecular interactions with opioid receptors. Given intracerebroventricularly, OFQ/N can reverse and/or prevent analgesia from drugs acting at supraspinal µ-opioid receptors, including morphine (Grisel et al., 1996; Mogil et al., 1996a; Tian et al., 1997b; Zhu et al., 1997; Calo' et al., 1998; King et al., 1998; Lutfy et al., 1999; Citterio et al., 2000), DAMGO (Mogil et al., 1996b), fentanyl (Zhu et al., 1998), acetorphan (Suaudeau et al., 1998), endomorphin-1 (Wang et al., 1999a,c), and morphine-6-glucuronide (King et al., 1998). It has similar effects against supraspinal -opioid agonists, like DPDPE (Mogil et al., 1996b; King et al., 1998) and DSLET (Zhu et al., 1998; Wang et al., 1999a), ₁-opioid agonists like U50,488 (Mogil et al., 1996b; Zhu et al., 1998; Wang et al., 1999a), and dynorphin A (Citterio et al., 2000), and the ₃-opioid agonist, naloxone benzoylhydrazone (King et al., 1998).

Direct injections of OFQ/N into specific brain loci also induce anti-analgesic actions. OFQ/N placed into the periaqueductal gray (PAG) blocks morphine analgesia (Morgan et al., 1997) and its administration into the rostral ventromedial medulla (RVM) reverses DAMGO analgesia (Heinricher et al., 1997; Pan et al., 2000) (see Section VIII.H.). Importantly, OFQ/N also blocks analgesia from endogenous opioid-mediated manipulations, including electroacupuncture (Zhu et al., 1996; Tian et al., 1997a; Zhang et al., 1997) and mild stressors (Mogil et al., 1996a; Suaudeau et al., 1998; Rizzi et al., 2001). The latter phenomenon may be responsible for much of the confusion surrounding the actions of OFQ/N (see Section VII.D.3.).

The anti-opioid effect of OFQ/N against morphine analgesia is long-lasting, persisting for up to 4 to 6 h (Candeletti and Ferri, 2000). Repeated OFQ/N dosing induces tolerance, with a decreasing response over time (Lutfy et al., 1999). Although it is tempting to only assume a functional interaction between OFQ/N and other members of the opioid gene family, the anti-analgesic actions of this peptide are by no means restricted to opioid analgesia. OFQ/N equally efficaciously blocks analgesia from the ₂-adrenergic receptor agonist, clonidine (King et al., 1998), the GABA_B receptor agonist, baclofen (Citterio et al., 2000), and naloxone-insensitive forms of swim stress (Rizzi et al., 2001).

This ability to block non-opioid analgesia sets OFQ/N apart from other known functional anti-opioid peptides, including adrenocorticotrophic hormone (ACTH), cholecystokinin (CCK), dynorphin, FMRFamide (and its analogs), -melanocyte-stimulating hormone (-MSH), MIF-1/Tyr-MIF-1, neurotensin- and tyrosine-releasing hormone (Rothman, 1992), and ₁ receptor systems (e.g., Chien and Pasternak, 1993). Anti-opioid systems are thought to play important roles in a number of pain-relevant phenomena, including the mediation of individual differences in analgesic sensitivity (Chien and Pasternak, 1993; Tang et al., 1997), the induction of tolerance and dependence (Rothman, 1992) (see Section X.D.), and in plastic changes underlying neuropathic pain (Wiesenfeld-Hallin et al., 1997). Elucidation of the precise actions of OFQ/N vis-à-vis these other anti-opioid peptides will be a major research challenge for the future.

These anti-analgesic actions of OFQ/N are the most robust activities observed following supraspinal administration, having been seen by all groups examining this question. The two contentious issues, regarding OFQ/N actions, that remain involve direct analgesia and hyperalgesia. In one study, for example, higher OFQ/N doses induced analgesia in mice, although this action is not easily detected in all strains (Rossi et al., 1997). In this study, an initial hyperalgesic response was followed by analgesia. The analgesic response was reversed by opioid antagonists, but the hyperalgesic actions were not. Indeed, the biphasic hyperalgesic/analgesic activity seen with OFQ/N alone reverted to only a monophasic hyperalgesia in the presence of the opioid antagonist. Others, of course, see neither hyperalgesia or analgesia. Factors relevant to interpreting the conflicting data presented in Table 6 are discussed below.

A final comment concerns not the effect of OFQ/N on pain, but the effect of pain on OFQ/N. A recent study by Rosen and colleagues (2000) examined OFQ/N-like immunoreactivity in various nociception-related brain areas 2 weeks after the induction of a neuropathic state using Bennett and Xie's (1988) surgical model or a carrageenan inflammatory model. Both injuries increased OFQ/N levels in the cingulate cortex, and carrageenan increased levels also in the hypothalamus and the dorsal horn of the spinal cord. OFQ/N levels did not change in the PAG or RVM (in contrast to levels of dynorphin B and met-enkephalin-Arg-Phe), prompting the authors to conclude that OFQ/N is involved in the modulation of ascending nociceptive transmission pathways rather than descending nociceptive modulation pathways (but see Section VIII.H.). OFQ/N has also been identified in human cerebrospinal fluid but not at higher levels in women with ongoing labor pain compared with those presenting for elective Caesarean section (Brooks et al., 1998). Thus, any clinical relevance of supraspinal OFQ/N remains to be demonstrated.

Although the seminal investigations of OFQ/N featured supraspinal administration of the peptide, opioids play an equally crucial role in pain modulation in the spinal level (see Yaksh, 1999). Although OFQ/N injected intrathecally (10 nmol, i.t.) was initially reported to have no effect on thermal nociception (Reinscheid et al., 1995), a subsequent study reported a trend (p = 0.053) toward enhanced morphine analgesia by intrathecal OFQ/N (Grisel et al., 1996) followed by additional support for spinal OFQ/N analgesia (Xu et al., 1996; King et al., 1997). The situation has become more complicated since then, as shown in Table 7. Strikingly low OFQ/N doses spinally produce spontaneous pain, as evidenced by caudally directed scratching, biting, and licking (SBL) behaviors, and hypersensitivity to thermal and mechanical stimuli. These SBL behaviors are reminiscent of those elicited by substance P and N-methyl-D-aspartate (NMDA) and are eliminated by pretreatment with morphine and neurokinin-1 (NK₁) receptor antagonists, but not neurokinin-2 (NK₂) or NMDA receptor antagonists (Sakurada et al., 1999b). At higher OFQ/N doses, a number of laboratories have observed analgesic and anti-hyperalgesic/anti-allodynic effects. However, some have been unable to demonstrate OFQ/N analgesia at presumably effective doses. Still others have demonstrated anti-analgesic effects reminiscent of supraspinal peptide, alone or in combination with hyperalgesia (see Table 7).

Despite the many contradictions in the established literature to date, most reviewers have concluded that the dominant spinal action of high doses of OFQ/N is inhibitorycongruent with the findings of all electrophysiological studiesproducing behavioral analgesia and/or anti-hyperalgesia/anti-allodynia (Henderson and McKnight, 1997; Meunier, 1997; Harrison and Grandy, 2000; Xu et al., 2000). Wang and colleagues (1996) have arrived at the same conclusion for the trigeminal system. Assuming that spinal OFQ/N is analgesic, the potential role of classical opioid receptors remains a further unresolved issue. Of the eight studies looking at the effects of opioid antagonists on OFQ/N analgesia, only two reported a reversal (King et al., 1997; Hao and Ogawa, 1998). In another study, repeated administration of spinal OFQ/N resulted in the development of tolerance to the peptide's analgesic effects and cross-tolerance to morphine (Jhamandas et al., 1998). This finding, however, is directly contradicted by yet another study that found no cross-tolerance (Hao et al., 1997).

Nociception-relevant elements in the spinal cord undergo anatomical and functional alterations after peripheral nerve injury or inflammation and this plasticity is thought to be important in producing and maintaining chronic pain states (Woolf, 1983). OFQ/N appears to be no exception. OFQ/N levels and binding increase in the dorsal horn of the spinal cord after inflammation (Jia et al., 1998; Rosen et al., 2000). In one study, this increase was bilateral, but restricted to the superficial laminae (I and II) of the cord (Jia et al., 1998). Inflammation also induces expression of the prepro-OFQ/N gene in the dorsal root ganglion, although the increased synthesis of OFQ/N was quite short-lived (<6 h) (Andoh et al., 1997). In contrast to dynorphin, which was increased in the dorsal horn after a Bennett model nerve injury, OFQ/N levels in this study trended lower, although the decrease did not achieve statistical significance. These findings are hard to reconcile with data demonstrating that high-dose OFQ/N's depressive effect on the flexor reflex is decreased in inflamed rats and increased somewhat in nerve injured rats (Abdulla and Smith, 1998; also see Hao et al., 1998; Xu et al., 1999a). This pattern of functional changes is exactly opposite to that of µ opioids, which exhibit increased efficacy in inflammatory states (Stanfa and Dickenson, 1995) and greatly reduced efficacy against neuropathic pain (Arner and Meyerson, 1988). As pointed out by Xu and colleagues (1999a), however, the effectiveness of exogenously applied OFQ/N is primarily determined by the status of NOP₁ receptors, not endogenous peptide levels. No data have thus far been collected as to whether NOP₁ receptors are altered after injury.

In addition to their effects in the CNS, opioids can produce analgesia in the periphery, especially in the presence of inflammation (Stein et al., 1990; Kolesnikov et al., 1996). This fact, along with the ability of OFQ/N to affect transmitter release in the peripheral nervous system (see Giuliani et al., 2000), suggests that OFQ/N may modulate nociception directly at the site of pain and/or injury. A small number of studies have investigated this possibility, again with somewhat conflicting results. Two elegant studies by Inoue and colleagues (1998, 1999) demonstrated the ability of OFQ/N at remarkably low doses, up to 10,000-fold lower than substance P and 1000-fold lower than bradykinin, to elicit the nociceptive flexor reflex after intraplantar injection into the hindpaw. This effect appears to be secondary to local substance P release in the paw, since the phenomenon can be blocked by inhibition of transmitter release by botulinum toxin, depletion of substance P by capsaicin, by NK₁ receptor antagonists, and is abolished in tachykinin-1 gene knockout mice (Inoue et al., 1998). In the second study, however, a higher OFQ/N dose (1 nmol) was analgesic, producing a complete blockade of substance P-induced flexor reflexes and SBL (Inoue et al., 1999) (see Section VIII.D.5.). Another group, also using higher doses, demonstrated the analgesic efficacy of OFQ/N applied subcutaneously to the tail (Kolesnikov and Pasternak, 1999). This analgesia was naloxone-reversible, but insensitive to antagonism by either µ- or -specific antagonists.

The modulatory effects of OFQ/N on rat knee joint afferents were very recently studied by McDougall et al. (2000). They found a sensitizing effect of OFQ/N in normal joints, and a desensitizing effect during hyper-rotation in acutely inflamed knees. Interestingly, both these effects may be explained by the OFQ/N-substance P interactions described above (Inoue et al., 1998; Lecci et al., 2000b). However, Kumar and colleagues (1999) were unable to demonstrate [³H]OFQ/N binding in human synovial joint fluid or tissue. OFQ/N has been implicated in fibromyalgia where female sufferers display decreased plasma levels of the peptide (Anderberg et al., 1998).

The preceding descriptions of OFQ/N effects on nociceptive phenomena at the supraspinal, spinal, and peripheral levels (see Tables 2 and 3) illustrate the considerable uncertainty that still surrounds the simplest of questions: What are the actions of OFQ/N when injected? The next sections will address a number of factors that may be relevant to reconciling the divergent results found in the literature and thus to illuminating the endogenous role of OFQ/N.

1. Noxious Stimulus Modality. There is a large literature demonstrating differential processing of different types of pain by neurochemically distinct circuitries (for reviews, see Mogil et al., 1996c, 1999b). It is possible, therefore, that activities of OFQ/N may be dependent upon the nociceptive assay used. The majority of the studies to date have used thermal assays (tail-flick/withdrawal, hot-plate tests) (Tables 2 and 3), which is to be expected since these assays are easily performed and commonly used in the opioid field. Supraspinal OFQ/N anti-analgesia is a robust response and has been demonstrated against thermal, electrical, and chemical assays, of varying durations (acute to chronic). Spinal hyperalgesia/allodynia and analgesia have been demonstrated against thermal, chemical, and mechanical assays. Even some of the less common findings, such as spinal anti-analgesia or anti-hyperalgesia/anti-allodynia, have been observed using multiple nociceptive assays. Overall, there does not appear to be strong evidence at the present time for modality-specific effects of the pronociceptive actions of OFQ/N.

2. Robustness of Various Phenomena. Not all of the reported phenomena are equally robust. For example, of the 16 studies reporting supraspinal hyperalgesia listed in Table 6, half featured latency decreases, or formalin rating increases, of <40% compared with baseline and/or vehicle values. By contrast, virtually all studies reporting anti-opioid analgesic actions of OFQ/N demonstrated a complete blockade of even profound analgesia. Also, the supraspinal hyperalgesic actions of OFQ/N are quite transient compared with the anti-analgesic actions, with the former lasting only 15 to 30 min in virtually all cases. One can easily point to degradation of the peptide as an explanation of transient effects, but such degradation does not prevent long-lasting anti-analgesic actions (see especially Candeletti and Ferri, 2000). Particularly weak is the phenomenon of supraspinal OFQ/N analgesia defined as a quantal doubling of the baseline tail-flick latency, which can be demonstrated in only 40% of CD-1 mice and was not seen in two other strains (see Section VIII.D.3.) (Rossi et al., 1996b, 1997). It should be noted that blockade of the anti-opioid ₁ receptor system with the  receptor antagonist, haloperidol, dramatically enhanced the analgesic actions of OFQ/N and its fragments in all strains tested (Rossi et al., 1997). Supraspinal OFQ/N analgesia does seem to be more robust in the rat (Rossi et al., 1998b).

3. Influence of Stress. The original investigations of the supraspinal OFQ/N quantified the effect of the peptide relative to a control group receiving an isovolumetric injection of vehicle (Meunier et al., 1995; Reinscheid et al., 1995). Although a reasonable control, it alone is not sufficient since mice receiving intracerebroventricular injections are not at "baseline". This is particularly evident when dealing with nociceptive assays capable of detecting stress analgesia. Employing either "no injection" and/or preinjection baseline control groups, depending on the nociceptive assay, Mogil and colleagues (1996a) demonstrated that the apparent hyperalgesia noted previously could be explained by the reversal of stress-induced analgesia by OFQ/N. In these studies, the apparent hyperalgesia compared with the vehicle group was actually reversal of stress-induced analgesia related to the injection when compared with the no-injection group (Mogil et al., 1996a).