Comparison of opioid receptor binding in horse, guinea pig, and rat cerebral cortex and cerebellum

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1 Veterinary Anaesthesia and Analgesia, 2007, 34, doi: /j x RESEARCH PAPER Comparison of opioid receptor binding in horse, guinea pig, and rat cerebral cortex and cerebellum Sara M Thomasy DVM, PhD, Benjamin C Moeller BS & Scott D Stanley PhD K.L. Maddy Equine Analytical Chemistry Laboratory, California Animal Health and Food Safety Laboratory, School of Veterinary Medicine, University of California, Davis, CA, USA Correspondence: Scott D Stanley, K.L. Maddy Equine Analytical Chemistry Laboratory, School of Veterinary Medicine, University of California, West Health Sciences Dr, Davis, CA 95616, USA. sdstanley@ucdavis.edu Abstract Objective To compare the density and binding characteristics of opioid receptor subtypes in horse, rat, and guinea pig cerebral cortex and cerebellum. Study design Prospective receptor binding study. Animals Whole brains were obtained from four neurologically normal adult horses during necropsy. Rat and guinea pig brains were obtained commercially. Methods The cerebellum and cerebral cortex were dissected from each brain, and tissue homogenates prepared. A radioligand binding technique with the highly selective ligands [ 3 H]-DAMGO, [ 3 H]-U69593, and [ 3 H]-DPDPE was used to identify the mu- (l), kappa- (j) and delta- (d) opioid receptors, respectively. Competitive binding assays were performed with these ligands and varying concentrations of one of multiple unlabeled ligands. Results While there were marked species differences in relative densities of opioid receptors, all radioligands interacted with their binding sites with high, nanomolar affinity in both the cerebral cortex and cerebellum. In the horse cerebral cortex, the percentages of total opioid binding sites for the l-, j- and d-receptors were 71%, 14% and 15%, respectively. In the rat and guinea pig cerebral cortex, the corresponding values were 56% l-, 4% j- and 40% d-receptors, and 25% l-, 37% j- and 38% d-receptors, respectively. In horse and guinea pig cerebellum, the binding was 37% l-, 59% j- and 4% d-receptors, and 15% l-, 76% j- and 10% d-receptors, respectively. For competitive analysis, all competitors of the l-, j- and d-receptors completely displaced [ 3 H]-DAMGO, [ 3 H]-U69593, and [ 3 H]-DPDPE and had inhibitory constants in the nanomolar range. Conclusion and clinical relevance Horses used in this study had a greater density of l-receptors in the cerebral cortex compared with rats and guinea pigs but without further characterization of the functional role of these receptors it is impossible to determine the clinical significance of these data. Keywords brain, equine, guinea pig, opioid receptor, rat. Introduction Opioids, especially l-receptor agonists such as morphine exert a diverse array of behavioral effects in humans and animals, ranging from central nervous system (CNS) depression to stimulation. These effects appear to be species-dependent as the predominant behavioral response to opioids in dogs, monkeys, guinea pigs, and humans is sedation, while in cats, rats, mice, cows, and horses, excitement and increased locomotor activity are commonly observed (Simon 1978; Kamerling et al. 1989; Saito 1990; Brent & Bunn 1994; Sills & Vaccarino 1998). The reasons for species differences 351

2 in responses to opioids are unclear, but radioligand binding studies in the brain have demonstrated species differences in the numbers and relative proportions of mu- (l), kappa (j), and delta- (d) opioid receptors. In the cerebral cortex of dogs, guinea pigs, and humans, j- and d-opioid receptors predominate with only a small population of l-receptors (Pfeiffer et al. 1982; Robson et al. 1985; Sharif et al. 1990). In contrast, l-opioid receptors predominate in rat and mouse brain with smaller populations of d- and j-opioid receptors (Robson et al. 1985). The behavioral and physiologic responses of horses to high doses of opioids have been well documented and consist of CNS stimulation and subsequent increased spontaneous locomotor activity, tachycardia, increased arterial blood pressure, and hyperthermia (Kamerling et al. 1985, 1988; Pascoe et al. 1991; Mama et al. 1993). In contrast to cats, cows, rats, and mice, which experience both CNS stimulation and analgesia following opioid administration, the analgesic efficacy of opioids in horses has not been consistently documented and the use of l-receptor agonists such as fentanyl and morphine for analgesia or balanced anesthesia remains controversial (Bennett & Steffey 2002). The study of opioid receptor subtypes in the horse brain has been limited (Hellyer et al. 2003; Wetmore et al. 2003). An autoradiographic study demonstrated significantly higher binding to l-opioid receptors in the frontal cortex, somatosensory cortex, mid-brain, and cerebellum in horses in comparison with dogs and higher binding to j-opioid receptors in the frontal cortex in dogs and cerebellum in horses (Hellyer et al. 2003). However, autoradiography cannot distinguish whether an increase in binding is due to increased numbers of receptors or an increase in the affinity of the receptor for the ligand. In addition, this study did not evaluate d-opioid receptors in the horse or dog (Hellyer et al. 2003). The horse l-opioid receptor was recently partially cloned and sequenced and found to have high homology with the cat and pig (94%), and cow (93%) and lower homology with the mouse (88%) and rat (86%) (Wetmore et al. 2003). In addition, an atypical amino acid at residue AA214 (glutamine fi histidine) in the extracellular loop II of the horse l-opioid receptor was noted which was shared with the cat but no other species, and could influence the binding of opioid ligands to the receptor (Wetmore et al. 2003). In order to compare the presence and nature of opioid receptors in horse, rat and guinea pig cerebral cortex and cerebellum, the selective radioligands [ 3 H]-DAMGO, [ 3 H]-U-69593, and [ 3 H]- DPDPE were used to identify and characterize l-, j- and d-opioid receptors, respectively. In addition, [ 3 H]-DAMGO, [ 3 H]-U-69593, and [ 3 H]-DPDPE, together with a number of opioid ligands, were used to further compare the binding characteristics of l-, j-, and d-opioid receptors. Materials and methods Animals The University of California, Davis, Animal Care and Use Committee approved this study. Whole brains were obtained from four adult horses (two females, two castrated males, 3 12 years) following euthanasia, by an overdose of sodium pentobarbital, as a secondary use of tissue. All horses were neurologically normal at the time of euthanasia and had received no opioids in the past 48 hours. Following euthanasia, the brains were removed and frozen at )80 C until tissue preparation could be performed. Rat (Pel-Freeze Biologicals, Rogers, AR, USA) and guinea pig (Harlan Bioproducts, Indianapolis, IN, USA) brains were commercially obtained and frozen at )80 C until tissue preparation was performed. Tissue preparation Brain homogenates were prepared as previously described by Kosterlitz et al. (1980) with modifications by Paterson et al. (1990). Briefly, each brain was thawed in 10 mm Tris-HCl buffer (ph 7.4 at 0 C) and the cerebral cortex and cerebellum were dissected. Rat and guinea pig cerebelli were pooled from four separate brains due to the small amount of tissue available. The cerebral cortex and cerebellum were homogenized separately using a Brinkmann polytron (Brinkmann Instruments, Westbury, NY, USA) at setting 4 for 10 seconds, then centrifuged at 40,905 g for 10 minutes at 0 C (Sorvall RC5B Plus; Kendro Laboratory Products, Newton, CT, USA). The tissue pellets were separated from the supernatant and re-suspended in fresh buffer in a 5% weight to volume ratio. The re-suspended pellets were homogenized and incubated at 37 C for 45 minutes to degrade any endogenous opioids. The homogenates were centrifuged at 40,905 g for 10 minutes at 0 C, and 352 Ó 2007 The Authors. Journal compilation Ó 2007 Association of Veterinary Anaesthetists, 34,

3 tissue pellets were separated from the supernatant and stored at )80 C. Prior to performing the binding assays, the tissue pellets were resuspended in 10 mm Tris-HCl buffer (ph 7.4 at 20 C) to a protein concentration of approximately 5 10 mg ml )1, as determined by the Lowry protein assay using bovine serum albumin (Sigma Aldrich, St Louis, MO, USA) as a standard (Lowry et al. 1951). Mu-opioid receptor binding Binding experiments were based on the methods of Kosterlitz et al. (1980) with some modifications. Incubation times were determined from association rate and dissociation experiments performed for each species and tissue. The incubation times for horse, rat, and guinea pig cerebral cortex and cerebellum were 3.5, 3.0, and 4.5 hours, respectively. Nonspecific and total binding of [ 3 H]-DAMGO ([ 3 H]-[D-Ala 2, MePhe 4, Gly 5 -ol]-enkephalin, Ci mmol )1 ; Perkin Elmer Life Sciences, Boston, MA, USA) were determined in the presence and absence, respectively, of 1 lm unlabeled naloxone (Sigma Aldrich). Each experiment was performed in duplicate using a concentration range for [ 3 H]- DAMGO of nm and incubated at 25 C in polypropylene tubes. The experiment was terminated by rapid vacuum filtration over Whatman GF/ B glass filters (Whatman Inc., Florham Park, NJ, USA) soaked in a 0.1% polyethyleneimine solution (Sigma Aldrich). Each filter was washed three times with 3 ml of ice-cold 10 mm Tris-HCl buffer (ph 7.4). The filter-bound radioactivity was counted by liquid scintillation spectrometry (Beckman LS 6000IC; Beckman Instruments, Fullerton, CA, USA) with 40% efficiency for tritium. Specific binding was defined as the amount of binding obtained by subtracting nonspecific binding from total binding. The specific binding for [ 3 H]-DAMGO was 70% of the total binding. Delta-opioid receptor binding [ 3 H]-DPDPE ([ 3 H]-[D-Pen2, 5]-enkephalin, Ci mmol )1 ; Perkin Elmer Life Sciences) binding was performed under identical conditions to the [ 3 H]- DAMGO assay described previously, except that incubation times were 6.5, 4.5, and 4.5 hours for horse, rat, and guinea pig cerebral cortex and cerebellum, respectively. In addition, the concentration range for [ 3 H]-DPDPE was nm, and nonspecific binding was determined in the presence of 10 lm naloxone. Specific binding for [ 3 H]-DPDPE was 40% of the total binding. Kappa-opioid receptor binding [ 3 H]-U69593 ([ 3 H]-(5a,7a,8b))(+)-N-Methyl-N-7- [1-pyrrolidinyl]-1-oxaspiro [4.5] dec-80yl) benzeneacetamide, Ci mmol )1 ; Perkin Elmer Life Sciences) binding was performed under identical conditions to the [ 3 H]-DAMGO assay described previously, except that incubation times were 0.7, 1.0, and 0.8 hours for horse, rat, and guinea pig cerebral cortex, respectively, and 2.0, 2.0, and 0.8 hours for horse, rat, and guinea pig cerebellum, respectively. In addition, the concentration range for [ 3 H]-U69593 was nm. Specific binding for [ 3 H]-U69593 was 80% of the total binding. Competitive binding studies Binding assays were carried out with 1.2 nm [ 3 H]- DAMGO, [ 3 H]-U69593, or [ 3 H]-DPDPE in the absence (control) or presence of varying concentrations of one of several unlabeled ligands including DAMGO, DPDPE, U69593, morphine, fentanyl, levorphanol, trans- (±))3,4-dichloro-N-methyl- N-92- [1-pyrrolidinyl] cyclohexyl)-benzenacetamide (U50488), Tyr-Gly-Gly-Phe-Leu-Arg-Arg-Ile-Arg- Pro-Lys-Leu-Lys-Trp-Asp-Asn-Gln (Dynorphin A), [D-Ala 2, D-Leu 5 ]-enkephalin (DADLE), and [D-Ser 2, Leu 5 ]-enkephalin-thr (DSLET) (Sigma Aldrich). Assay conditions were identical to those described previously for each radiolabeled ligand, and were performed on cerebral cortex only for each species. Data and statistical analysis All data were analyzed using Graphpad Prism software (version 3.02; San Diego, CA, USA). For saturation binding experiments, the binding capacity (B max ) and equilibrium dissociation constant (K D ) were determined using one-site models for l, j and d binding sites in all species. These values were compared to those calculated from Scatchard analysis. The values for the percentage of total binding sites were calculated from the B max concentrations assuming that the molecular weights of the three opioid receptors are similar. For competitive studies, the IC 50 was determined and the inhibition constant (K I ) was calculated from the equation K I ¼ IC 50 / (1+[L]/K D ), where [L] is the concentration of the labeled ligand. Ó 2007 The Authors. Journal compilation Ó 2007 Association of Veterinary Anaesthetists, 34,

4 All statistical analyses were performed using SAS (version 9.1; Cary, NC, USA). Data were natural log transformed to satisfy tests of normality. Statistical differences between two groups were tested by the use of an unpaired Student s t-test. When multiple comparisons were made, values were analyzed by one-way model analysis of variance (ANOVA), using the Tukey Kramer method as a post-hoc test. Significance was set at p < Data are presented as mean ± SD. Results Specific binding of [ 3 H]-DAMGO, [ 3 H]-U69593, and [ 3 H]-DPDPE was saturable and of high, nanomolar affinity to cerebral cortex in all three species (Fig. 1). For saturation analyses, both species and tissue type had a significant effect on K D and B max (Table 1). In order to further characterize and verify the types of opioid binding sites, numerous opioids competed with [ 3 H]-DAMGO, [ 3 H]-U69593, and [ 3 H]-DPDPE for l-, j- and d-receptor binding sites in horse, rat, and guinea pig cerebral cortex homogenates (Table 2). For competitive analyses, species had a significant effect on K I for DPDPE and DADLE competition with [ 3 H]-DPDPE (Fig. 2). However, species did not significantly affect the K I for any ligands competing for [ 3 H]-DAMGO and [ 3 H]- U Discussion Analysis of saturation curves at selectively labeled opioid binding sites revealed that the horse, rat, and guinea pig cerebral cortex contain opioid receptors of the l-, j-, and d- subtypes. There were marked species differences in the relative proportions of the l-, j-, and d-receptors in both the cerebral cortex and cerebellum. In the horse cerebral cortex, 71% of the total opioid binding sites are l-receptors, with d- and j-receptors accounting for just 15% and 14%, respectively. In the rat cerebral cortex the majority of the opioid binding sites are l- (56%) and d-receptors (40%), with a very small population of j-receptors (4%). In contrast, the guinea pig cerebral cortex has a more even distribution of all three Figure 1 Representative specific binding curves for [ 3 H]-DAMGO, [ 3 H]-U69593 and [ 3 H]-DPDPE in homogenates of horse, rat, and guinea pig cerebral cortex. 354 Ó 2007 The Authors. Journal compilation Ó 2007 Association of Veterinary Anaesthetists, 34,

5 Table 1 Binding characteristics of tritiated opioids in homogenates of horse, rat, and guinea pig cerebral cortex and cerebellum Horse Rat Guinea pig K D (nm) B max (fmol mg )1 protein) K D (nm) B max (fmol mg )1 protein) K D (nm) B max (fmol mg )1 protein) Cerebral cortex [ 3 H]-DAMGO (l) 0.55 ± 0.28 a,1 91 ± 51 a, ± 0.08 a 84 ± 31 a 0.39 ± 0.10 a,1 28 ± 3.4 b,1 [ 3 H]-U69593 (j) 0.33 ± 0.05 a,1 18 ± 9.3 a, ± 0.25 a 6.0 ± 2.8 a 0.50 ± 0.17 a,1 42 ± 8.6 b,1 [ 3 H]-DPDPE (d) 1.33 ± 0.73 a,1 19 ± 11 a, ± 1.55 b 59 ± 19 b 1.22 ± 0.28 a,1 43 ± 5.3 b,1 Cerebellum [ 3 H]-DAMGO (l) 1.02 ± 0.48 a,1 30 ± 13 a,2 NB 0.47 ± 0.37 a,1 6.5 ± 3.0 b,2 [ 3 H]-U69593 (j) 0.60 ± 0.47 a,1 48 ± 17 a,2 NB 0.60 ± 0.09 a,1 34 ± 15 a,1 [ 3 H]-DPDPE (d) 1.18 ± 0.85 a,1 2.9 ± 0.8 a,2 NB 0.46 ± 0.27 a,2 4.3 ± 1.8 a,2 NB, no binding. Values are mean ± SD of four experiments. a,b For each variable, different superscripts mean significant effect of species (p < 0.05). 1,2 For each variable, different superscripts mean significant effect of tissue type (p < 0.05). Table 2 Comparison of the inhibitory effects of opioids on the binding of [ 3 H]-DAMGO, [ 3 H]-U69593, and [ 3 H]- DPDPE in homogenates of horse, rat, and guinea pig cerebral cortex K I (nm) Horse Rat Guinea pig [ 3 H]-DAMGO Levorphanol 0.42 ± 0.15 a 0.32 ± 0.17 a 0.57 ± 0.39 a Fentanyl 0.83 ± 0.36 a 0.48 ± 0.18 a 0.59 ± 0.06 a Morphine 0.84 ± 0.36 a 0.70 ± 0.18 a 0.85 ± 0.14 a DAMGO 1.19 ± 0.40 a 0.77 ± 0.19 a 0.99 ± 0.19 a [ 3 H]-U69593 U ± 0.09 a 0.65 ± 0.08 a 0.62 ± 0.06 a U ± 0.25 a 1.15 ± 0.18 a 1.06 ± 0.09 a Levorphanol 1.84 ± 0.29 a 1.59 ± 0.58 a 3.18 ± 0.84 a Dynorphin A 1.89 ± 0.59 a 1.96 ± 0.92 a 1.73 ± 0.56 a [ 3 H]-DPDPE DADLE 1.07 ± 0.38 a 2.18 ± 0.77 b 0.95 ± 0.18 a DSLET 1.79 ± 1.31 a 2.43 ± 1.10 a 1.30 ± 0.12 a DPDPE 1.16 ± 0.34 a 5.58 ± 0.79 b 1.73 ± 0.22 c Levorphanol 2.81 ± 2.10 a 2.89 ± 2.10 a 5.00 ± 1.80 a Values are mean ± SD of four experiments. For each variable, different superscripts mean significant effect of species (p < 0.05). receptors, (l- 25%, d-38%, and j-37%). These results are consistent with a previous study that noted that the percentage of opioid receptors were 46% l-, 42% d- and 12% j-receptors in rat brain, and 24% l-, 32% d- and 44% j-receptors in guinea pig brain (Robson et al. 1985). In comparison with other species, the receptor density profile of the horse cerebral cortex is similar to that of the mouse brain (51% l-, 20% j-, and 29% d-receptors, Robson et al. 1985) but differs markedly from the dog (5% l-, 58% j-, and 37% d-receptors, Sharif et al. 1990) and human cerebral cortex (16% l-, 57% j-, and 26% d-receptors, Pfeiffer et al. 1982). The present study also confirms previous studies noting the lack of opioid receptors in the rat cerebellum (Mansour et al. 1988). In the horse cerebellum, the primary receptor populations are j-receptors (59%) and l-receptors (37%) with only a small population of d-receptors (4%). The guinea pig cerebellum also has the majority of its opioid binding sites as j-receptors (76%), with l- and d-receptors accounting for just 15% and 10%, respectively. This is similar to previous studies reporting that j-receptors are the predominant subtype in guinea pig cerebellum (Sim & Childers 1997). While there were marked species differences in relative density of opioid binding sites, all radioligands interacted with their binding sites with high, nanomolar affinity in both cerebral cortex and cerebellum. No significant species or tissue type differences were noted in the K d for [ 3 H]-DAMGO and [ 3 H]-U69593 at the l and j-opioid receptors, respectively. Therefore, the atypical amino acid at residue AA214 (glutamine fi histidine) in the extracellular loop II of the horse l opioid receptor Ó 2007 The Authors. Journal compilation Ó 2007 Association of Veterinary Anaesthetists, 34,

6 Figure 2 Representative displacement of d-receptor binding of [ 3 H]-DPDPE by DPDPE and DADLE in homogenates of horse, rat, and guinea pig cerebral cortex. does not appear to influence the binding of opioid ligands to the receptor nor does its relatively low sequence homology (86%) with the rat l-opioid receptor (Wetmore et al. 2003). [ 3 H]-DPDPE had a significantly higher affinity to d-opioid receptors in guinea pigs and horses in comparison with rats. This is similar to previous studies that have reported the K d for [ 3 H]-DPDPE to be approximately three to four times higher in rats versus guinea pig brain (Yeadon & Kitchen 1988; Goldstein & Naidu 1989; Yoburn et al. 1991; Mignat et al. 1995). [ 3 H]-DPDPE also had a significantly higher affinity to d-opioid receptors in guinea pig cerebellum versus cerebral cortex. These species and tissue type differences may be due to different populations of d-opioid receptor subtypes as [ 3 H]-DPDPE is not only a preferential d 1 agonist but also a partial d 2 agonist (Govitrapong et al. 2002). A previous autoradiographic study noted higher binding by [ 3 H]-DAMGO to l-opioid receptors in cerebral cortex and [ 3 H]-U69593 to j-opioid receptors in the cerebellum of horses in comparison with dogs (Hellyer et al. 2003). The present study confirms that the l-receptor is the predominant subtype in the cerebral cortex and the j-receptor is the predominant subtype in the cerebellum of the horse. However, species and tissue type had no significant effect on the K d for [ 3 H]-DAMGO and [ 3 H]-U69593 at the l and j-opioid receptors, respectively. Therefore, this study showed that the increase in binding noted in the previous autoradiographic study is most likely due to an increased number of receptors rather than an increase in affinity of the receptor for the ligand (Hellyer et al. 2003). All competitors at the l-receptor completely displaced [ 3 H]-DAMGO and had inhibitory constants in the nanomolar range. For the three species, the rank order of potency for the competitors at the l-receptor was identical: levorphanol > fentanyl morphine > DAMGO. The rank order of potency previously reported in the guinea pig for three of these ligands was levorphanol > DAM- GO > morphine (Goldstein & Naidu 1989). The K I values previously reported in the rat for fentanyl and morphine are three to four times higher than the present study (Yeadon & Kitchen 1988). These differences may reflect differences in experimental conditions as temperature, type and concentration of buffer, and incubation time have been shown to influence opioid receptor binding (Simantov et al. 1976; Goldstein & Naidu 1989; Paterson et al. 1990). In addition, the present study was performed on the cerebral cortex for all three species while the previous studies were performed using whole rat or guinea pig brain (Yeadon & Kitchen 1988; Goldstein & Naidu 1989). At the j-receptor, all competitors completely displaced [ 3 H]-U69593 and had inhibitory constants in the nanomolar range. For the horse and rat, the rank order of potency for the competitors at the j-receptor was identical: U69593 > U50488 > levorphanol dynorphin A. The rank order of potency for the guinea pig was similar with only the last two compounds reversed. No significant differences in the K I values for each competitor were noted between the three species. In contrast to the present study, the rank order of potency previously reported in the guinea pig was dynorphin A > U50488 > U69593 > levorphanol (Goldstein & Naidu 1989; Lahti et al. 1985). This difference 356 Ó 2007 The Authors. Journal compilation Ó 2007 Association of Veterinary Anaesthetists, 34,

7 in rank order may be due to the competitors at the j-receptor being equipotent with only a three to fourfold difference separating the least and most potent competitor in the present and previous studies (Lahti et al. 1985). All competitors at the d-receptor completely displaced [ 3 H]-DPDPE and had inhibitory constants in the nanomolar range. However, the rank order of potency differed between all three species with DADLE > DPDPE > DSLET > levorphanol for the horse, DADLE > DSLET > levorphanol > DPDPE for the rat, and DADLE > DSLET > DPDPE > levorphanol for the guinea pig. Species did not have a significant effect on the K I values for DSLET and levorphanol. However, the K I for DADLE in the rat was twofold higher than the horse and the guinea pig. In addition, the K I for DPDPE was significantly different for all three species. The three to fivefold increase in K I for DPDPE in the rat in comparison with the guinea pig and horse reflects the similar threefold increase in K D for [ 3 H]-DPDPE in the rat versus the guinea pig and horse noted previously. In conclusion, the horse cerebral cortex contains a high concentration of high-affinity l-receptors, but a lower density of d- and j-receptors. This receptor density closely resembles that of the rat cerebral cortex, as well as other species in which opioidinduced CNS stimulation occurs, such as the mouse. In contrast, the horse cerebellum contains a high concentration of high-affinity j- and l-receptors, with a lower density of d-receptors, and most closely resembles that of the guinea pig cerebellum. In addition, this study demonstrates similar binding affinities of several l- and j-receptor agonists in three different species. Therefore, it is unlikely that drug-receptor affinity has an effect on the behavioral response of horses to opioids, but opioid receptor density may still play a role. Additional autoradiographic studies in horses are required to determine the distribution of opioid receptors subtypes in brain regions responsible for pain perception and regulation. Further investigation of downstream effectors, including G-proteins, may also better characterize the functional differences in opioid receptor subtypes and observed physiologic response to opioids in horses in comparison with other species. Acknowledgements This study was supported by the Veterinary Scientist Training Program Grant and Achievement Rewards for College Scientists Scholarship. References Bennett RC, Steffey EP (2002) Use of opioids for pain and anesthetic management in horses. Vet Clin North Am Equine Pract 18, Brent PJ, Bunn SJ (1994) In vivo treatment with mu and delta, but not kappa-selective opioid agonists reduces [3H]spiperone binding to the guinea-pig striatum: autoradiographic evidence. Brain Res 654, Goldstein A, Naidu A (1989) Multiple opioid receptors: ligand selectivity profiles and binding site signatures. Mol Pharmacol 36, Govitrapong P, Sawlom S, Ebadi M (2002) The presence of delta and mu-, but not kappa or ORL(1) receptors in bovine pinealocytes. Brain Res 951, Hellyer PW, Bai L, Supon J et al. (2003) Comparison of opioid and alpha-2 adrenergic receptor binding in horse and dog brain using radioligand autoradiography. Vet Anaesth Analg 30, Kamerling SG, DeQuick DJ, Weckman TJ et al. (1985) Dose-related effects of fentanyl on autonomic and behavioral responses in performance horses. Gen Pharmacol 16, Kamerling S, Weckman T, Donahoe J et al. (1988) Dose related effects of the kappa agonist U-50,488H on behaviour, nociception and autonomic response in the horse. Equine Vet J 20, Kamerling S, Wood T, DeQuick D et al. (1989) Narcotic analgesics, their detection and pain measurement in the horse: a review. Equine Vet J 21, Kosterlitz HW, Lord JA, Paterson SJ et al. (1980) Effects of changes in the structure of enkephalins and of narcotic analgesic drugs on their interactions with mu- and delta-receptors. Br J Pharmacol 68, Lahti RA, Mickelson MM, McCall JM et al. (1985) [3H]U a highly selective ligand for the opioid kappa receptor. Eur J Pharmacol 109, Lowry OH, Rosebrough NJ, Farr AL et al. (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193, Mama KR, Pascoe PJ, Steffey EP (1993) Evaluation of the interaction of mu and kappa opioid agonists on locomotor behavior in the horse. Can J Vet Res 57, Mansour A, Khachaturian H, Lewis ME et al. (1988) Anatomy of CNS opioid receptors. Trends Neurosci 11, Mignat C, Wille U, Ziegler A (1995) Affinity profiles of morphine, codeine, dihydrocodeine and their glucuronides at opioid receptor subtypes. Life Sci 56, Pascoe PJ, Black WD, Claxton JM et al. (1991) The pharmacokinetics and locomotor activity of alfentanil in the horse. J Vet Pharmacol Ther 14, Paterson SJ, Robson LE, Kosterlitz HW et al. (1990) Effect of Tris, HEPES, and TES buffers on binding at Ó 2007 The Authors. Journal compilation Ó 2007 Association of Veterinary Anaesthetists, 34,

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