Introduction

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Several central nervous system physiological processes, including cardiovascular regulation, sedation, and analgesia are mediated by the ₂-adrenergic receptor (₂AR) family of G protein-coupled receptors. The ₂ARs are divided into three distinct, but highly homologous, subtypes, _2A, _2B, and _2C, which share common signal transduction pathways (Bylund et al., 1994). Discreet central nervous system localization of each subtype (Rosin et al., 1996; 1998; Talley et al., 1996; Stone et al., 1998; Rosin, 2000; Shi et al., 2000) implies that different subtypes may mediate different processes. Identification of separate physiological roles for different ₂AR subtypes could improve design of novel compounds for specific therapeutic goals. Resolution of the functions specific to each ₂AR subtype has been difficult due to lack of sufficiently selective pharmacological tools. However, genetic manipulation has yielded mouse lines with a dysfunctional _2AAR (MacMillan et al., 1996) or deleted _2BAR or _2CAR (Link et al., 1996), which has permitted improved evaluation of the specific physiological roles of each ₂AR subtype (see Discussion). Interestingly, clarification of the physiological roles of _2CAR has reportedly been difficult (MacDonald et al., 1997). Initially, only minimal differences in ₂AR agonist-induced responses (Link et al., 1996) were observed between _2CAR knockout (KO) and wild-type (WT) mice. Comparing the subtle differences among _2CAR-KO, _2CAR over-expresser (OE), and their respective WT control mice provided converging evidence that _2CARs contribute to cardiovascular function (MacDonald et al., 1997) and several physiological processes (see Discussion, Bjorklund et al., 1998; Sallinen et al., 1998a,b). A role for _2CAR in analgesia has been previously suggested (Takano and Yaksh, 1993; Guo et al., 1999; Fairbanks and Wilcox, 1999; Graham et al., 2000) but not clearly established. Identification of spinal _2CAR-mediated analgesia has been elusive, perhaps because most of the commonly used ₂AR agonists appear to require the _2AAR to achieve full antinociceptive potency; functional knockout of the _2AAR dramatically reduced the potency or efficacy of these agents (clonidine, norepinephrine, dexmedetomidine, UK-14,304) (Hunter et al., 1997; Lakhlani et al., 1997; Stone et al., 1997; Fairbanks and Wilcox, 1999). Unlike these agonists, spinally administered moxonidine produces ₂AR-mediated antinociception that appears largely _2AAR-independent (Fairbanks and Wilcox, 1999). The present study capitalized on the apparent _2AAR independence of moxonidine in spinal antinociception to test for an analgesic role for the _2CAR. The studies presented here apply gene substitution (_2AAR; MacMillan et al., 1996), gene knockout (_2BAR, _2CAR; Link et al., 1996), and gene knockdown (_2CAR) strategies to demonstrate a subtle but clear role for the _2CAR in spinal antinociception and ₂AR-opioid antinociceptive synergy in the mouse spinal cord.

	Materials and Methods

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Animals. Experimental subjects included several strains and lines of mice. First, male ICR mice (20-25 g; Harlan, Madison, WI) were used for the antisense experiments represented in Fig. 2. Second, three separate strains of mice, each mutated for one of the ₂ARs, were used for experiments represented in Figs. 1, 2, 3, and 5. In each experiment, equal numbers of male and female mice (15-20 g) were used for comparison with the corresponding WT line with the same genetic background. The genetic backgrounds of each mutated mouse line were as follows. For _2AAR, mice with a gene-targeted mutation (D79N) that renders the _2AAR dysfunctional (MacMillan et al., 1996; Stone et al., 1997) were used for the experiments represented in Figs. 1A and 4. Both the mutated mouse line (designated D79N-_2A) and the corresponding wild-type line (designated _2AWT) were generated on a combined 129Sv/J × C57BL/6 genetic background. For _2BAR, mice with a gene-targeted mutation that knocks out the _2BAR (Link et al., 1996) were used for the experiments represented in Fig. 1B. Both the mutated mouse line (_2BKO) and the corresponding WT line (WT-_2B) were generated on a combined 129Sv/J × C57BL/6J genetic background. For _2CAR, mice with a gene-targeted mutation that knocks out the _2CAR (Link et al., 1996) were used for the experiments represented in Figs. 1C, 3, and 5. Both the mutated mouse line (_2CKO) and the corresponding WT line (WT-_2C) were generated on a combined 129Sv/J × FVB/N genetic background. Subjects were housed in groups of 5 to 10 in a temperature- and humidity-controlled environment. Subjects were maintained on a 12-h light/dark cycle and had free access to food and water. Each animal was used only once. These experiments were approved by the University of Minnesota Institutional Animal Care and Use Committee.

View larger version (21K): [in this window] [in a new window]   Fig. 1.   Dose-dependent inhibition of SP-elicited behavior by moxonidine in D79N-2A, 2BKO, 2CKO, and their respective wild-type counterpart mice. A, 2A-WT and D79N-2A mice. The potency of moxonidine was significantly greater in 2AWT [ED50 = 0.25 nmol (0.19-0.32); squares] than in D79N-2A [ED50 = 0.96 nmol (0.57-1.6); circles] mice. B, 2B-WT and 2BKO mice. The potency of moxonidine did not differ between 2BWT [ED50 = 0.64 pmol (0.40-1.0); open squares] and 2BKO [ED50 = 0.92 pmol (0.52-1.6); circles] mice. C, 2C-WT and 2CKO mice. The potency of moxonidine was significantly greater in 2C WT [ED50 = 0.40 nmol (0.32-0.49); squares] versus 2C AR KO [ED50 = 0.83 nmol (0.61-1.1); circles] mice. When SP was given to animals in the absence of moxonidine, the number of behaviors did not differ between the D79N-2A (68 ± 8.7, n = 9) and 2AWT (52 ± 8.7, n = 8) mice or 2CKO (59 ± 1.7, n = 8) and 2CWT (51 ± 6.4, n = 8) mice (Student's t test, p > 0.05), but did differ significantly between the 2BKO (24 ± 1.3, n = 6) and 2BWT (48 ± 2.3, n = 4) mice (Student's t test, p < 0.05). Moxonidine-induced inhibition of SP-elicited behavior was normalized for each mouse line by using the mean number of SP behaviors observed in each line for the control value in the equation for percentage of inhibition.

Chemicals. Moxonidine [4-chloro-5-(2imidazolin-2-ylamino)-6-methoxy-2-methylpyrimidine] chloride was a generous gift of Solvay Pharma GmbH (Hannover, Germany). Substance P was purchased from Sigma Chemical (St. Louis, MO). SmithKline Beecham (King of Prussia, PA) generously donated [6-chloro-2,3,4,5-tetrahydro-3-methyl-1-H-3-benzazepine] (SK&F 86466). Efaroxan [2-(2-ethyl-2,3-dihydrobenzofuranyl)-2-imidazoline] hydrochloride and deltorphin II were purchased from Sigma/RBI (Natick, MA). Moxonidine was dissolved in 1% acetic acid and diluted with acidified saline (pH 3.2-4.0, 0.01 N acetic acid). All other drugs were dissolved in 0.9% saline. All drugs were administered intrathecally in a 5-µl volume in conscious mice according to the method of Hylden and Wilcox (1980) as modified by Wigdor and Wilcox (1987).

Substance P Nociceptive Test. Nociceptive responsiveness was tested in the substance P nociceptive test, a sensitive indicator of milder analgesics (Hylden and Wilcox, 1982). A constant dose of SP (10-20 ng) was injected intrathecally to produce approximately 40 to 60 behaviors (scratches and bites directed to the hindlimbs) in the first minute postinjection. The dose of SP required to produce this number of behaviors was confirmed with each new experiment. Coadministration of opioid or ₂-adrenergic analgesics dose dependently inhibits those behaviors (Hylden and Wilcox, 1981). To test the ability of moxonidine and deltorphin II to inhibit SP-induced behavior, the drugs were coadministered with SP and inhibition was expressed as a percentage of the mean response of the control group (determined with each new experiment) according to the following equation:
The ED₅₀ values and their 95% confidence limits (CL) were then calculated according to the method of Tallarida and Murray (1987) (see below). The ED₈₀ value was calculated from the regression line of the dose-inhibition curve to facilitate experiments using ₂AR-selective antagonists. In these experiments, antagonists were coadministered with the moxonidine-SP combinations. In the case of SK&F 86466 and efaroxan, dose-antagonism curves were determined for their respective abilities to antagonize the moxonidine-mediated inhibition of SP behavior by an approximately 80% effective dose of moxonidine. The percentage of antagonism was expressed by the following equation:
The ED₅₀ values for percentage of antagonism were then calculated according to the method of Tallarida and Murray (1987) (see below). Each ED₈₀ dose of moxonidine used in the percentage of antagonism experiments was retested to determine the actual inhibition on the day of the experiment. When the experimental dose of moxonidine did not produce an exact 80% inhibition, the equation was adjusted accordingly [e.g., 76% (_2CWT mice) or 67% (_2CKO mice)].

Antisense Oligonucleotide Treatment. Midland Certified Reagent (Midland, TX) generated the unmodified 18-base antisense oligodeoxynucleotide (ODN) directed against the 5' end of the coding sequence of _2CAR. Bases 1, 5, 11, and 16 were shuffled to create a mismatch control ODN sequence. The sequences were 5'-CCA-TTC-GCC-CGC-GTC-GCT-CC-3' (antisense) and 5'-GCA-TGC-GCC-CTC-GTC-CCT-CC-3' (mismatch). The ODNs were injected intrathecally (12.5 µg/5-µl injection) by direct lumbar puncture twice a day for 3 days before testing according to the method of Lai et al. (1996). On day 4, the animals received one more injection in the morning several hours before testing or perfusion. Each study included antisense ODN, mismatch ODN, and vehicle control groups. Several animals from each treatment group were chosen at random, anesthetized (75 mg/kg ketamine, 5 mg/kg xylazine, and 1 mg/kg acepromazine mixture i.m.), and perfused transcardially with 4% paraformaldehyde and 0.2% picric acid in 0.1 M phosphate-buffered saline, pH 6.9, by vascular perfusion as previously described (Wessendorf and Elde, 1985). Spinal cord tissue was processed for immunohistochemistry to confirm that knock down of receptor expression had occurred in a manner similar to that previously described (Lai et al., 1996). Full dose-response curves for moxonidine-induced inhibition of SP-elicited behavior were constructed for each treatment group.

Immunohistochemistry. Spinal cords were removed and rinsed overnight with 10% sucrose in phosphate-buffered saline. Spinal segments were frozen and thaw-mounted cryostat sections (14 µm) prepared for indirect immunofluorescence histochemistry. Cryostat sections were preincubated for 1 h at room temperature in diluent containing 1% normal donkey serum, 0.3% Triton X-100, 0.01% sodium azide, and 1% bovine serum albumin. Sections were then incubated overnight at 4°C in a humid chamber with primary antisera and rinsed several times with phosphate-buffered saline. Sections were then incubated with secondary antisera for 1 h at room temperature, rinsed, and coverslipped in glycerol and p-phenylenediamine in phosphate-buffered saline with sodium bicarbonate. The primary antisera used were rabbit-derived anti-_2AAR and guinea pig-derived anti-_2CAR was used at a dilution of 1:1000 (Stone et al., 1998). The sequences against which both the _2AAR and _2CAR antisera were directed are the same in mouse as in rat. Preparations were visualized with cyanine 3.18-conjugated secondary antisera 1:200 (Jackson Immunoresearch Laboratories, Inc., West Grove, PA) and examined with a Bio-Rad MRC-1000 confocal imaging system (Bio-Rad Microscience Division, Cambridge, MA). Micrographs used in plates were assembled using Photoshop 5.0 (Adobe Systems, Mountain View, CA).

Statistical Analysis. The ED₅₀ values and 95% confidence limits of drugs in nanomoles were calculated using the graded dose-response curve method of Tallarida and Murray (1987). A minimum of three doses was used for each drug or drug combination. To determine differences in agonist or antagonist potency between treatment groups, nonoverlapping 95% CL were considered to represent statistically significant differences. When evaluating the extent of a potency shift between treatment groups, a potency ratio representing the ratio of the respective ED₅₀ values was calculated.

View this table: [in this window] [in a new window]   TABLE 1 Summary of moxonidine-deltorphin II spinal antinociceptive interactions in 2AWT and D79N-2A mice

View this table: [in this window] [in a new window]   TABLE 2 Summary of moxonidine-deltorphin II spinal antinociceptive interactions in 2CWT and KO mice

	Results

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Moxonidine Produces Antinociception in D79N-_2A Mutant and _2CKO Mice. To determine the relative importance of _2AAR vis a vis _2CAR activation in moxonidine-mediated spinal antinociception, we evaluated the ability of moxonidine to inhibit SP-evoked behavior in mice mutated for _2A-, _2B-, and _2CAR and their respective wild-type counterparts. Moxonidine produced dose-dependent inhibition of SP-evoked behavior and was significantly (3.8-fold; CL, 2.1-6.8) less potent in D79N-_2A mice (Fig. 1A) than in their respective wild-type counterparts (WT-_2A). We have previously demonstrated that moxonidine-induced antinociception produced in the D79N-_2A mice is reversed by a selective ₂AR antagonist (SK&F 86466) and therefore requires ₂AR activation (Fairbanks and Wilcox, 1999). These results (retention of efficacy with small rightward shift) implicate the participation of either _2B- or _2CAR in moxonidine-mediated antinociception. To test for the involvement of those receptors, we evaluated antinociception produced by moxonidine in both _2BAR and _2CAR knockout mice in comparison with the respective wild-type counterparts. Moxonidine inhibited SP-evoked behavior with equal potency in _2B-WT and _2BKO mice (Fig. 1B), suggesting that _2B-adrenergic receptors do not participate in moxonidine-mediated antinociception. The potency of moxonidine was decreased in _2CKO mice (Fig. 1C) as evidenced by a moderate (2-fold; CL, 1.5-3.1) but significant parallel rightward shift in dose-response curve compared with that of their wild-type counterparts (WT-_2C). This result was confirmed in the repeat experiment represented in Fig. 5A where moxonidine showed significantly decreased potency (2.8-fold; CL, 1.4-5.4) in _2CKO versus _2C-WT mice. These results show that the _2CAR contributes to, but is not absolutely required for, moxonidine-mediated antinociception. These data indicate that activation of both the _2AAR and _2CAR (but not _2BAR) contributes to the expression of moxonidine's full antinociceptive potency.

_2CAR Participates in Moxonidine-Mediated Antinociception. To determine whether compensatory changes accompanying _2CAR knockout accounted for KO/WT differences, we evaluated the analgesic potency of moxonidine in ICR mice treated with antisense ODN directed against the _2CAR (Fig. 2A). Moxonidine inhibited SP-evoked behavior with significantly lower potency in _2CAR antisense-treated mice relative to control mice treated with vehicle (5.8-fold difference; CL, 3.8-11) or mismatch ODN (5.1-fold difference; CL, 3.0-8.5). To confirm the integrity of the knockdown, immunohistochemistry was performed on treated tissue by using subtype-selective antisera directed against the _2CAR and the _2AAR. _2CAR immunoreactivity was observed as previously reported (Stone et al., 1999) in the superficial dorsal horn of spinal cords extracted from vehicle-treated animals (Fig. 2B). In contrast, tissue from mice given _2CAR antisense showed a substantial decrease in _2CAR-immunoreactivity (Fig. 2C); such a decrease in _2CAR-immunoreactivity was not observed in tissue from mismatch-treated (Fig. 2D) controls. Furthermore, the antisense-mediated knockdown appears to be specific because no change was observed in _2AAR-ir (Stone et al., 1998) after _2CAR antisense (Fig. 2E) or mismatch treatment (Fig. 2F). These results confirm the involvement of the _2CAR in antinociception.

View larger version (85K): [in this window] [in a new window]   Fig. 2.   Reduction in moxonidine-mediated antinociception and 2CAR-ir after chronic treatment with 2CAR-antisense ODN. A, moxonidine dose-response curves in mice treated with vehicle [ED50 = 0.29 nmol (0.019-0.045); circles], mismatch ODN [ED50 = 0.034 nmol (0.025-0.047); squares], and 2CAR-antisense ODN [ED50 = 0.17 nmol (0.11-0.26); triangles]. Error bars represent mean ± S.E.M. for each dose point (n = 6-8 mice/dose). When SP was given to mice in the absence of moxonidine, the number of behaviors did not differ between saline-treated (48 ± 2.4, n = 8), mismatch-treated (41 ± 4.9, n = 6), and 2CAR-antisense-treated (42 ± 4.7, n = 6) mice (ANOVA, p > 0.05). B, 2CAR-ir was present in the superficial dorsal horn of mice treated with vehicle. C) 2CAR-ir was absent in dorsal horn of the mice treated with 2C AR-antisense ODN. D, 2CAR-ir was present in the dorsal horn of mice treated with mismatch ODN. E, 2AAR-ir was present in the dorsal horn of mice treated with 2CAR-antisense ODN. F, 2AAR-ir was present in the dorsal horn of mice treated with mismatch ODN.

Moxonidine-Mediated Antinociception Is ₂AR-Dependent in _2CKO Mice. Moxonidine's high affinity for the imidazoline (I₁) receptor raises the possibility that the I₁ receptor mediates moxonidine-induced antinociception in mice with disrupted _2AAR or deleted _2CAR. To address this question in a previous study (Fairbanks and Wilcox, 1999), we compared the abilities of the ₂AR-selective antagonist SK&F 86466 (Hieble et al., 1986) and the mixed I₁/₂AR antagonist efaroxan (Haxhiu et al., 1994) to antagonize the effects of moxonidine in D79N-_2A mice (Fairbanks and Wilcox, 1999). We observed that moxonidine-mediated antinociception in D79N-_2A mice was dose dependently reversed by both antagonists and concluded that moxonidine produced an _2AAR-independent but ₂AR-dependent antinociception. In the present study, we used these same antagonists to test for ₂AR dependence of moxonidine-induced antinociception in _2CKO mice and their wild-type counterparts. In _2CWT mice, a dose of moxonidine (1 nmol) was used that provided a 76 ± 3.3% antinociceptive response (n = 8 mice); SK&F 86466 dose dependently antagonized moxonidine's antinociceptive effect with a comparable but 2-fold higher potency than that of efaroxan (Fig. 3A). The comparable potency of these antagonists to inhibit moxonidine-mediated antinociception confirms the requirement for ₂AR activation in this mouse line. In the _2CKO mice, the magnitude of the antinociceptive response (67 ± 5.4%) to moxonidine (1.5 nmol) did not differ from that of WT-_2C mice (Student's t test, p > 0.05). Similar to the results observed in the WT-_2C mice, SK&F 86466 and efaroxan dose dependently antagonized moxonidine with comparable ED₅₀ values (Fig. 3B); these results confirm the requirement of ₂AR activation for moxonidine-mediated antinoception in _2CAR KO mice. Unambiguous demonstration of a role of the imidazoline receptor in this process is not possible in the absence of more selective antagonists for that receptor.

View larger version (26K): [in this window] [in a new window]   Fig. 3.   SK&F 86466 and efaroxan antagonize moxonidine-induced inhibition in 2C-WT and 2CKO mice. A, 2C-WT: dose-antagonism curves were generated for SK&F 86466 [ED50 = 1.7 nmol (1.1-2.4); circles] and efaroxan [ED50 = 3.8 nmol (3.3-4.4); squares] against a constant dose of moxonidine (1 nmol) that provided a 76 ± 3.3% antinociceptive response (n = 8 mice). B, 2CKO: dose-antagonism curves were generated to SK&F 86466 [ED50 = 2.2 nmol (1.1-4.0); circles] and efaroxan [ED50 = 3.2 nmol (2.3-4.3); squares] against a constant dose of moxonidine (1.5 nmol) that provided a 67 ± 5.4% analgesic response (n = 8 mice). The magnitude of this response did not statistically differ (Student's t test, p > 0.05) from that (76 ± 3.3%) used in the WT mice in A.

Moxonidine and Deltorphin II Produce Antinociceptive Synergy in _2AAR-Mutant Mice. When agonists to both ₂AR and opioid receptors are coadministered with SP, they act synergistically to inhibit SP-elicited behavior (Roerig et al., 1992). Although _2AAR mediates opioid synergy with UK-14,304, the _2CAR involvement of moxonidine-induced antinociception raised the possibility that _2CAR contributes to ₂AR-opioid receptor synergy. Intrathecally administered moxonidine and deltorphin II both dose dependently inhibited SP-evoked behavior in _2AAR WT mice (Fig. 4A). The moxonidine-deltorphin II equi-effective dose ratio used (1:6) was based on their ED₅₀ values. Combination of moxonidine and deltorphin II at this dose ratio resulted in significant leftward shifts in the dose-response curves (i.e., increased potency) compared with those of each agonist administered separately (Fig. 4A), with ED₅₀ values significantly less than the calculated theoretical additive values (Fig. 4B; Table 1). This result indicates a synergistic interaction. Intrathecally administered moxonidine and deltorphin II both inhibited SP-evoked behavior (Fig. 4C) in D79N-_2A mice. The moxonidine-deltorphin equi-effective dose ratio used was 1:1.5. Combination of moxonidine and deltorphin at this dose ratio resulted in increased potency compared with that of each agonist administered separately (Fig. 4C; Table 1). The coadministration of moxonidine-deltorphin II combinations in mice resulted in antinociceptive dose-response curves with ED₅₀ values significantly less than the calculated theoretical additive values (Fig. 4D; Table 1), confirming a synergistic interaction in mice with dysfunctional _2AAR. The dependence of moxonidine-mediated antinociception on ₂AR activation (Fairbanks and Wilcox, 1999; Fig. 3) together with the observation of moxonidine-deltorphin II synergism in D79N-_2A mice suggests an important role for _2CAR in ₂AR-opioid receptor antinociceptive synergy.

View larger version (42K): [in this window] [in a new window]   Fig. 4.   Intrathecally administered moxonidine produces spinal antinociceptive synergy with deltorphin II in both 2A-WT and D79N-2A mice. SP-elicited baseline responses did not differ between 2A-WT WT (41 ± 2.6, n = 8) and D79N-2A (42 ± 2.1, n = 8) mice (Student's t test, p > 0.05). A, 2A-WT: dose-response curves of the spinal antinociceptive effect of moxonidine [ED50 = 270 pmol (220-240); open circles], deltorphin II [ED50 = 1600 pmol (1000-2600); open triangles], moxonidine in the presence of deltorphin II ([ED50 = 27 pmol (21-34); closed circles], and deltorphin II in the presence of moxonidine [ED50 = 160 pmol (120-200); closed triangles]. B, isobolographic representation of the interaction between moxonidine and deltorphin II. In these graphs the ED50 values of moxonidine and deltorphin II are plotted as the y- and x-axis intercepts, respectively. The thicker lines directed from each ED50 value toward zero represent the lower 95% confidence limit of each ED50 value. The straight line connecting these two points is the theoretical additive line. The open circle that lies on or near the theoretical additive line represents the calculated theoretical ED50 value of the combination, were the interaction additive. The closed circle represents the experimentally observed ED50 value of the combination of moxonidine-deltorphin II. In this experiment, the ED50 value of the combination of moxonidine-deltorphin II falls below, and outside the lower confidence limits of, the theoretical additive, suggesting the interaction is synergistic. Consistent with this, the experimental ED50 value differed significantly from the theoretical additive ED50 value (Student's t test, p < 0.05), indicating that the interaction was synergistic. C) D79N-2A: dose-response curves of the spinal antinociceptive effect of moxonidine [ED50 = 3900 pmol (2500-5400); open circles], deltorphin II [ED50 = 4500 pmol (2800-6900); open triangles], moxonidine in the presence of deltorphin II [ED50 = 10 pmol (1-19); closed circles], and deltorphin II in the presence of moxonidine [ED50 = 26 pmol (30-480); closed triangles]. D, isobolographic representation of the antinociceptive effect of the combination of moxonidine-deltorphin II in D79N-2A mice. Isobolographic analysis was applied to the data in C as described in 4B. The experimental ED50 value differed significantly from the theoretical additive ED50 value indicating that the interaction is synergistic.

Deletion of _2CAR Impairs Analgesic Synergism between Moxonidine and Deltorphin II _2CWT. Intrathecally administered moxonidine and deltorphin II both dose dependently inhibited SP-evoked behavior in _2CAR WT mice (Fig. 5A). The moxonidine-deltorphin II equi-effective dose ratio used was 24:1. Combination of moxonidine and deltorphin II at this dose ratio resulted in increased potency compared with that of each agonist administered separately (Fig. 5A). The coadministration of moxonidine-deltorphin II in mice resulted in antinociceptive dose-response curves with ED₅₀ values significantly less than the calculated theoretical additive values (Fig. 5B; Table 2). This result indicates a synergistic interaction in _2CAR WT mice.

View larger version (42K): [in this window] [in a new window]   Fig. 5.   Intrathecally administered moxonidine produces spinal antinociceptive synergy with deltorphin II in 2C-WT but not in 2C AR KO mice. SP-elicited (15-ng) baseline responses did not differ between 2C-WT (46 ± 4.9, n = 6) and 2CKO (53 ± 3.2, n = 8) mice (Student's t test, p > 0.05). A, 2C-WT: dose-response curves of the spinal antinociceptive effect of moxonidine [ED50 = 84 pmol (47-148); open circles], deltorphin II [ED50 = 2.5 pmol (1.0-6.2); open triangles], moxonidine in the presence of deltorphin II [ED50 = 3.6 pmol (0.29-6.8); closed circles], and deltorphin II in the presence of moxonidine [ED50 = 0.15 pmol (0.012-0.25); closed triangles]. B, isobolographic representation of the antinociceptive (% inhibition) effect of the combination of moxonidine-deltorphin II. Isobolographic analysis was applied to the data in 5A as described in 4B. The experimental ED50 value differed significantly from the theoretical additive ED50 value confirming that the interaction is synergistic. C, 2CKO: dose-response curves of the spinal antinociceptive effect of moxonidine [ED50 = 235 pmol (164-337); open circles], deltorphin II [ED50 = 5.2 pmol (2.0-13); open triangles], moxonidine in the presence of deltorphin II [ED50 = 72 pmol (44-100); closed circles], and deltorphin II in the presence of moxonidine [ED50 = 1.6 pmol (0.96-2.2); closed triangles]. D isobolographic representation of the antinociceptive effect of the combination of moxonidine-deltorphin II in 2CKO mice. Isobolographic analysis was applied to the data in 5C as described in 4B. The experimental ED50 value did not differ from the theoretical additive ED50 value demonstrating that the interaction is additive.

_2CKO. Intrathecally administered moxonidine and deltorphin II both inhibited substance P-evoked behavior (Fig. 5C) in _2CAR KO mice. The moxonidine-deltorphin equi-effective dose ratio used was 46:1. Although the combination of moxonidine and deltorphin II shifted each dose-response curve significantly, the ED₅₀ values did not differ significantly from the theoretical additive ED₅₀ values (Fig. 5D; Table 2). This result indicates that the interaction between moxonidine and deltorphin II was additive in mice with deleted _2CAR, which contrasts with the synergistic interaction shown in the corresponding WT mice. This result suggests that _2CAR activation is required for moxonidine-deltorphin II synergy; in contrast, although moxonidine may produce antinociception through _2AAR receptors, it appears that _2AAR activation is insufficient for moxonidine-deltorphin II antinociceptive synergy. These observations confirm a role for _2CAR in ₂AR-opioid antinociceptive synergy.

	Discussion

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We have previously demonstrated substantial _2AAR dependence for the antinociceptive action of a panel of ₂AR agonists [Fairbanks and Wilcox, 1999 (52.5°C warm water tail immersion and substance P tests); Stone et al., 1997 (substance P test)]. Functional knockout of _2A-adrenergic receptors in the D79N-_2A mouse line decreased potency or efficacy of these agonists with the following rank order from most to least affected: clonidine > dexmedetomidine > norepinephrine > UK-14,304. This mutation also blocked synergy of the least _2A-dependent agonist, UK-14,304, with the opioid receptor agonist deltorphin II and DAMGO. These two findings suggested that spinal ₂AR-opioid receptor synergy, as well as adrenergic antinociception itself, relies on intact _2A-receptor function (Stone et al., 1997). However, our recent characterization of the spinal analgesic action produced by a novel imidazoline₁/₂AR receptor agonist, moxonidine, suggested that another ₂AR subtype must participate in spinal ₂AR antinociception. Unlike the other ₂AR-selective agonists, moxonidine-induced antinociception demonstrated minimal decrease (2-3-fold) in antinociceptive potency in the D79N-_2A mice (Fairbanks and Wilcox, 1999). However, moxonidine-mediated analgesia in the D79N-_2A mice was fully reversed by the ₂AR-selective antagonist SK&F 86466, confirming an antinociceptive role of another ₂-receptor subtype. Therefore, we tested for changes in moxonidine potency in _2BAR and _2CAR KO mice. In the present study, moxonidine potency decreased moderately but significantly in _2CAR KO mice compared with their wild-type counterparts; no such change was seen with the _2BAR KO. To further probe this apparent contribution of _2CAR to moxonidine-induced antinociception, we applied an antisense strategy (Lai et al., 1996) to knock down _2CAR expression. Multiple intrathecal injections of antisense ODN reduced the _2CAR immunoreactivity and significantly decreased the antinociceptive potency of moxonidine relative to saline-treated and mismatch-treated controls. These complementary knockout and knockdown observations unequivocally demonstrate that the _2CAR must have an analgesic function. The participation of _2AAR and _2CAR in moxonidine-induced antinociception in mice with dysfunctional (D79N-_2A) or deleted (_2CKO) appears to be equivalent: small decreases in the potency of moxonidine are observed in both mouse lines, suggesting that both receptors participate in the functional outcome of moxonidine treatment. However, the studies of moxonidine-deltorphin synergy in these two lines indicate an important distinction. Unlike the UK-14,304-deltorphin II combination (Stone et al., 1997), moxonidine-deltorphin antinociceptive synergy is present in the D79N-_2A mice but not in the _2CAR KO mice (Fig. 5). These results extend our previous studies on the role of _2AAR in spinal analgesia by demonstrating that the _2CAR also contributes to ₂AR opioid synergy induced by certain agonists and identifies the potential of the _2CAR--opioid receptor pair as a participant in spinal analgesia.

_2C-Adrenergic Receptor in Antinociception. Identification of a role for the _2CAR in analgesia is consistent with its localization and with physiological responses to adrenergic agonists. Adrenergic agonists inhibit release of peptides from spinal cord slices (Ono et al., 1991) and inhibit dorsal horn nociceptive neurons (Fleetwood-Walker et al., 1985). These actions suggest both presynaptic localization on primary afferent terminals in dorsal horn and postsynaptic localization on spinal neurons. In agreement with this physiological deduction, the _2AAR has been shown to be primarily localized on SP-containing primary afferent neurons (presynaptic sites), whereas the _2CAR appears to reside primarily in spinal dorsal horn neurons (postsynaptic sites; Stone et al., 1998). In situ hybridization studies have detected _2AAR mRNA in both dorsal root ganglion (DRG) and spinal cord neurons (Gold et al., 1997; Nicholas et al., 1993; Shi et al., 1999, 2000). Immunohistochemical studies by several independent groups have clearly demonstrated _2AAR-ir in the superficial dorsal horn (Rosin et al., 1993) and in DRG neurons (Gold et al., 1997; Birder and Perl, 1999). Stone et al. (1998) extended these results to show that spinal _2AAR-ir is primarily localized to the terminals of substance P-expressing, capsaicin-sensitive primary afferent terminals. There is, therefore, a strong case for both transcription and translation of the _2AAR gene in primary afferent neurons.

Other Functions of _2CAR Receptors. Physiological studies using the mouse lines with dysfunctional _2AAR (MacMillan et al., 1996) or deleted _2A-, _2B-, or _2CAR (Link et al., 1996; Altman et al., 1999) have provided strong evidence for discrete physiological functions for the respective ₂AR subtypes and have been recently comprehensively reviewed (Kable et al., 2000). Collectively, studies originally indicated that the _2AAR primarily mediated centrally mediated hypotension (MacMillan et al., 1996), anesthesia (Lakhlani et al., 1997), analgesia (Hunter et al., 1997; Lakhlani et al., 1997; Stone et al., 1997), sedation (Hunter et al., 1997; Lakhlani et al., 1997; Sallinen et al., 1997), antiepileptogenesis (Janumpalli et al., 1998), and ₂AR agonist-mediated inhibition of monoamine release and metabolism in brain (MacDonald et al., 1997). The _2BAR appears to be required for the initial peripheral hypertensive responses to ₂AR agonists (Link et al., 1996), salt-induced hypertension (Makaritsis et al., 1999) and possibly development or reproduction (Makaritsis et al., 1999). Clarification of the physiological role of _2CAR has reportedly been difficult (MacDonald et al., 1997). Despite widespread central nervous system distribution, it was notable that the _2CAR did not prove critical for the cardiovascular effects mediated by a reportedly nonselective ₂AR agonist, dexmedetomidine (Link et al., 1996). Further evaluation of small differences between _2CKO and _2CWT mice suggested _2CAR participation in dexmedetomidine-induced hypothermia, dopamine metabolism, and d-amphetamine-induced hyperlocomotion (Rohrer and Kobilka, 1998). Confirmation of these subtle physiological differences was greatly aided through comparison of ₂AR agonist-mediated effects in _2CAR KO and _2CAR OE mice (Bjorklund et al., 1998; Sallinen et al., 1998a,b). These studies of subtle differences between _2CAR KO and _2CAR OE mice also revealed participation by the _2CAR in cardiovascular function (MacDonald et al., 1997), the startle reflex and aggression (Sallinen et al., 1998a,b), and complex navigation behavior (Bjorklund et al., 1999). The present study extends those findings to illuminate (through rigorous examination of moderate, but significant effects in _2CKO and _2CWT mice as well as _2CAR antisense-treated mice) a role for the _2CAR in antinociception.

Significance. The present study directly demonstrates a requirement for _2CAR to mediate an antinociceptive action of an exogenously administered imidazoline₁/₂AR agonist (moxonidine). These observations provide strong evidence that the _2CAR subtype can contribute to ₂AR agonist-mediated analgesia and synergy with opioids in the mouse spinal cord. Based on the potential involvement of _2CAR in the action of endogenously released norepinephrine (Guo et al., 1999) we speculate that synergy between _2CAR and opioid receptors may mediate, at least in part, analgesia induced by systemic morphine. An interaction between morphine action at spinal sites and norepinephrine released spinally as a consequence of supraspinal morphine-mediated activation of descending noradrenergic pathways has been proposed (Wigdor and Wilcox, 1987). The present results support the assertion (Guo et al., 1999) that targeting the _2CAR for analgesic therapy may represent an improvement over _2AAR-selective agonists, because the latter receptor subtype likely mediates adrenergic agonist-induced sedation (Mizobe et al., 1996; Lakhlani et al., 1997). The validity of this target is further supported by clinical observations that antihypertensive doses of moxonidine produce significantly fewer side effects (sedation, dry mouth, rebound withdrawal) than clonidine, a strongly _2AAR-dependent agent (Fairbanks and Wilcox, 1999). The present study provides strong support for development of moxonidine or other _2CAR-selective agonists to be used either separately or in combination with opioid analgesics for the treatment of pain.