Withdrawal from dependence upon butorphanol uniquely increases κ1-opioid receptor binding in the rat brain

Brain Research Bulletin, Vol. 58, No. 2, pp. 149–160, 2002 Copyright © 2002 Published by Elsevier Science Inc. 0361-9230/02/$–see front matter

PII: S0361-9230(02)00760-8

Withdrawal from dependence upon butorphanol uniquely increases κ 1-opioid receptor binding in the rat brain Lir-Wan Fan,1 Sachiko Tanaka,1,2 Lu-Tai Tien,1 Tangeng Ma,1 Robin William Rockhold1 and Ing Kang Ho1∗ 1 Department of Pharmacology and Toxicology, University of Mississippi Medical Center, Jackson, MS, USA; and 2 Department of Biochemical Toxicology, School of Pharmaceutical Science, Showa University, Tokyo, Japan

[Received 15 October 2001; Revised 2 January 2002; Accepted 9 January 2002] agent [52,53] whose interaction with µ-, δ-, and κ-opioid receptors differs significantly from that of the classic opioid analgesic, morphine. Specifically, butorphanol has been shown to bind to µ-, δ-, and κ-opioid receptors with affinity ratio of 1:4:25 [4,34]. Its analgesic effects result primarily from actions on κ-opioid receptors [4,9,34]. Butorphanol exerts profound diuretic actions and is about 5–7 times more potent as an analgesic, and produces less respiratory depression, than morphine. In contrast, butorphanol produces a hyperthermic response similar to that caused by morphine [9,20,33]. When used within the therapeutic dose range, butorphanol is considered to have a lower dependence liability and abuse potential than morphine [15,52]. However, butorphanol has rewarding properties [40] and has been shown to cause dependence following chronic administration of higher doses in experimental animals [21,53,73]. Both parenteral and nasal spray formulations of butorphanol have been linked to abuse in humans [11,14]. Studies in our laboratory have demonstrated that continuous i.c.v. infusion with butorphanol (26 nmol/µl/h) for 72 h can produce physical dependence in the rat [21,28]. The degree of development of dependence upon an opioid is quantitated by evaluation of physiological and/or behavioral responses that occur shortly following termination of opioid administration or challenge with an opioid receptor antagonist. Administration of an opioid antagonist can precipitate a more dramatic withdrawal syndrome, when compared with the “natural” withdrawal syndrome that typically begins to appear 6–8 h after the cessation of chronic butorphanol administration [21,23,28]. The use of antagonist-precipitated withdrawal allows behavioral measurements to be restricted to a 30 min period immediately following antagonist administration. The degree of opioid dependence is evaluated by grading the incidence of 10 distinct behaviors (escape attempts, wet-dog shakes, teeth chattering, scratching, rearing, abnormal posturing, vocalization, ptosis, diarrhea, and penis licking) within that 30 min period [21,28]. Mortality is extremely rare in this model of withdrawal from opioid dependence. The roles for µ-, δ-, and κ-opioid receptors in mediation of physical dependence upon butorphanol had been systematically investigated using receptor-selective antagonists. Both

ABSTRACT: Changes in κ 1 -opioid receptor binding have been implicated in the development of dependence upon and withdrawal from butorphanol. Autoradiographic characterization of binding for brain κ 1 -([3 H]CI-977), µ-([3 H]DAMGO), and δ-([3 H]DPDPE) opioid receptors was performed in rats undergoing naloxone-precipitated withdrawal from dependence upon butorphanol or morphine. Dependence was induced by a 72 h i.c.v. infusion with either butorphanol or morphine (26 nmol/µl/h). Withdrawal was subsequently precipitated by i.c.v. challenge with naloxone (48 nmol/5 µl/rat), administered 2 h following cessation of butorphanol or morphine infusion. During withdrawal from butorphanol, but not morphine, κ 1 -opioid receptor binding was increased significantly in the frontal cortex, posterior basolateral amygdaloid nucleus, dorsomedial hypothalamus, hippocampus, posterior paraventricular thalamic nucleus, ventral tegmental area and locus coeruleus. In contrast, µ-opioid receptor binding decreased in these brain regions in naloxone-precipitated withdrawal from morphine, but not butorphanol, while binding for δ-opioid receptors was altered in both withdrawal groups. The brain κ 1 -opioid receptor appears to be more directly involved in the development of physical dependence upon, and the expression of withdrawal from, butorphanol, as opposed to the prototypical opioid analgesic, morphine. © 2002 Published by Elsevier Science Inc. KEY WORDS: κ 1 -, µ-, δ-Opioid receptors, Naloxone-precipitated butorphanol or morphine withdrawal, Autoradiography.

INTRODUCTION Opioid systems are believed to play a role in a variety of central nervous system (CNS) functions, including pain modulation, endocrine and immune function, ingestive and reproductive activities, learning, and motor behavior [8,37]. Opioid receptors are classified into at least three types [µ-(OP3 ), δ-(OP1 ), and κ-(OP2 )] based on pharmacological, anatomical, and molecular analysis [8,30,56]. Butorphanol (17-cyclobutylmethyl-3,14-dihydroxymorphinan) tartrate (stadol), a member of the phenanthrene class of opioids, is a relatively potent synthetic “agonist–antagonist” opioid analgesic

∗ Address for correspondence: Dr. Ing Kang Ho, Ph.D., Department of Pharmacology and Toxicology, University of Mississippi Medical Center, Jackson, MS 39216, USA. Fax: +1-(601)-984-1673; E-mail: [email protected]

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nor-binaltorphimine (nor-BNI, a κ-opioid receptor-selective antagonist) and naltrindole (a δ-opioid receptor-selective antagonist) have been shown to precipitate withdrawal signs similar to those precipitated by the nonselective opioid receptor antagonist, naloxone, in butorphanol-dependent rats [25–27]. However, β-funaltrexamine, a µ-opioid receptor-selective antagonist, failed to precipitate withdrawal in butorphanol-dependent rats [26,28]. Moreover, nor-BNI blocked the development of physical dependence upon butorphanol, but not on morphine, when given prior to or during opioid administration [27]. Administration of nor-BNI has been shown to selectively precipitate behavioral and neurochemical signs of withdrawal in rats treated with butorphanol, or the κ 1 -opioid receptor selective agonist, U69,593, but not in rats treated with morphine [13,22]. These results strongly implicate κ-, and/or δ-opioid receptors, and not µ-opioid receptors, in mediation of dependence upon butorphanol [25,27,28]. However, regionally specific changes in brain opioid receptor binding during the development of dependence upon and withdrawal from butorphanol have not yet been shown. Thus, the present experiments were designed to determine changes in opioid receptor bindings in different brain regions during naloxone-precipitated withdrawal from dependence upon butorphanol, as compared to, morphine. MATERIALS AND METHODS Animals All animal use procedures were in strict accordance with the NIH Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee of the University of Mississippi Medical Center. Male Sprague–Dawley rats (Harlan Sprague–Dawley, Indianapolis, IN) weighing 250–275 g were purchased and housed, prior to surgical intervention, in groups of three animals in plastic cages. They were acclimated for 1 wk under controlled conditions of temperature (22 ± 2◦ C), light cycle (12/12 h light/dark cycle), and humidity (50–55%) with free access to food and water. Surgical Procedures Rats were weighed and survival surgical procedures were performed under equithensin (4.25 g chloral hydrate, 2.23 g MgSO4 ·7H2 O, 0.972 g sodium pentobarbital, 44.4 ml propylene glycol, 10 ml 95% ethanol, and sterile water to make a final volume of 100 ml, 0.3 ml/100 g body weight, i.p.) anesthesia. The head of each rat was shaved and scrubbed with 10% providone–iodine solution. Each rat was then placed in a stereotaxic instrument (David Kopf Instruments, Tujunga, CA) and kept warm during surgical intervention. Postoperative analgesia was routinely provided by infiltration of the wound site with sensorcaine (0.5% bupivacaine with 1:200,000 epinephrine). A mid-saggital dorsal skull incision was made through the skin and the skin retracted. The soft tissues overlying the skull were removed. The landmarks of the skull, bregma and lambda, were identified and the skull was oriented such that both points were positioned at the same horizontal level. After clearing of the underlying fascia, three stainless steel anchor screws were placed in the skull. A burr-hole was then drilled through the skull over coordinates corresponding to the site of interest. The coordinates were estimated from the rat brain atlas of Paxinos and Watson [51]. A stainless steel guide cannula (21 gauge, 10 mm in length) was implanted into the right lateral cerebral ventricle (anterior–posterior (AP): −0.5 mm from bregma, lateral (LAT): +1.3 mm from bregma, and vertical (VERT): −4.5 mm from dura) with the bregma chosen as the stereotaxic reference point for i.c.v. infusion. The presence of cerebrospinal fluid (CSF) in the

guide cannula was examined as verification of proper placement. Rapidly polymerizing dental acrylic cement (Lang Dental MFG Co. Inc., Chicago, IL) was applied to the surface of the skull, and a protective aluminum cap was placed around the cannula. After the acrylic had hardened, each animal was removed from the stereotaxic frame. A stylet (26 gauge stainless steel, 10 mm in length) was placed into the guide cannula to allow the guide cannula to maintain patency. After surgery, rats were given 300,000 units of procaine penicillin G (Pfizerpen-AS, Pfizer, New York, NY), s.c., to prevent infection. Rats were placed in an individual plastic cage and kept warm till consciousness recovered. Wound sites were inspected and cleaned on a regular basis. Rats were allowed 7 d following surgical intervention to recover before drug administration. Rats with any neurological deficit were excluded from the study. Administration Schedule and Induction of Butorphanol or Morphine Dependence Rats were randomly divided into three groups of seven rats per group. Two of these groups were rendered physically dependent upon opioids by i.c.v. infusion of butorphanol tartrate (26 nmol/µl/h, Bristol–Myers Corporation, Syracuse, NY) or morphine (26 nmol/µl/h, Sigma Chemical Corp., St. Louis, MO) for 72 h through osmotic minipumps (Alzet 2001, Alza Corp., Palo Alto, CA). Both drugs were dissolved in sterile physiological saline. Both the infusion period and dose paradigm were determined by previous studies [21,27], and the dose and the period of treatment selected have successfully produced opioid dependence. A third (control) group of rats received an i.c.v. infusion of saline (1 µl/h) for the same period of time. A 5 cm piece of tygon tubing (0.38 mm i.d., Cole–Palmer, Chicago, IL) was applied to connect the minipump to a piece of “double L-shaped” stainless steel injector tubing (26 gauge, 30 mm in length) with one end having the same length as the guide cannula. Before introduction into pump, solutions were passed through 0.2 µm sterile Acrodisk filters (Gelman Science, Ann Arbor, MI) to trap bacteria and particles. The minipumps were primed overnight at 35◦ C by immersion in sterile normal saline so that the nominal pumping rate (1 µl/h) was achieved prior to implantation. Under halothane (2.5% halothane in medical grade oxygen) anesthesia, rats were each implanted with an osmotic minipump into a subcutaneous pocket through a midline skin incision between the scapulae. Each wound was closed and rats permitted to recover. Precipitation of Butorphanol and Morphine Withdrawal by Challenge of Naloxone Rats were placed individually in a plastic cage and acclimated for at least 1 h prior to behavioral experiments. After 72 h of i.c.v. infusion, the connecting tube between the i.c.v. cannula and the outlet of the minipump were disconnected followed by a 5 µl saline flush to clear the tube and cannula. The successful infusion was determined by checking the amount of the drug left in the pump. Naloxone (48 nmol/5 µl/rat, dissolved in sterile physiological saline, Sigma Chemical Corp., St. Louis, MO) was injected i.c.v. through a hand-driven microliter syringe into each animal 2 h following termination of the opioid infusion. A 5 µl saline flush was followed to clear the tube and cannula, and a stylet was inserted in the tube to seal the cannula. Ten distinct behaviors (escape attempts, wet-dog shakes, teeth chattering, scratching, rearing, abnormal posturing, vocalization, ptosis, diarrhea, and penis licking) were scored during the 30 min period as behavioral signs of withdrawal. The dose and time point selected is based on the previous studies in this laboratory [21,26,28].

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Preparation of Brain Tissue Sections

Binding Assays for Opioid Receptors by Autoradiography

Rats were sacrificed by decapitation. Brains were removed rapidly, put on tissue object holders with one drop of Tissue-Tek O.C.T compound (Sakura Finetek USA Inc., Torrance, CA) and frozen in liquid nitrogen for 20 s. The mounted brains were each wrapped with parafilm to prevent dehydration and then stored at −80◦ C for a maximum period of 1 mth until sectioning. The frozen brains were placed in a cryostat microtome (Leica CM3050S, Leica Microsystems Nussloch GmbH, Nussloch, Germany) for 1 h to reach an optimal temperature (−20◦ C) for sectioning. For autoradiographic mapping, 20 µm thick frozen coronal sections were cut according to the atlas of Paxinos and Watson [51] using a cryostat, thaw-mounted onto precleaned and precoated microscope slides (Allegiance Healthcare Corporation, McGaw Park, IL). The selected coronal sections of the rat brain from rostal to caudal brain regions included the following (according to the atlas of Paxinos and Watson [51]): (A) forebrain sections I: sections correspond to approximately bregma 4.20 mm, (B) forebrain sections II: sections correspond to approximately bregma 1.20 mm, (C) diencephalon sections: sections correspond to approximately bregma −3.80 mm, (D) midbrain sections: sections correspond to approximately bregma −5.80 mm, (E) pons (with cerebellum) sections: sections correspond to approximately bregma −9.68 mm. These figures of brain sections are adapted from the atlas of Paxinos and Watson [51] and presented in Fig. 1 (with major structures labeled). Adjacent sections were air-dried at −20◦ C and stored at −80◦ C for a maximum period of 1 mth until used.

Receptor autoradiography of rat brain opioid receptor binding was performed according to the methods of Kitchen et al. [29] and Mansour et al. [41] with modifications. A concentration of approximately 3–4 times Kd was used for labeling receptors in the mapping study. Ligand concentrations of 5 nM [3 H]DAMGO for µ-opioid receptors, 15 nM [3 H]DPDPE for δ-opioid receptors, and 2.5 nM [3 H]CI-977 for κ 1 -opioid receptors, respectively, were used. Nonspecific binding for µ-, δ-, and κ 1 -opioid receptor radioligands was defined by the addition of 1 µM of unlabeled DAMGO, DPDPE, or U-69,593, respectively, to the solution containing the radiolabeled ligand. One slide for nonspecific binding was chosen for each radioligand-labeled section from each animal. The radioligand-labeled slides for each brain region from all three treatment groups plus the same number of slides to determine nonspecific binding were placed in the X-ray cassettes with a set of 3 H-impregnated plastic standards ([3 H]microscale RPA 510, Amersham Life Science) and juxtaposed to a tritium-sensitive film ([3 H]hyperfilm, Amersham Life Science). Exposure times of 8, 16, and 12 wks for the µ-([3 H]DAMGO), δ-([3 H]DPDPE), and κ 1 -([3 H]CI-977) opioid receptor radioligand, respectively, were used at 4◦ C. Films were developed in standard Kodak D19 developer (Eastman Kodak, Rochester, NY) at room temperature for 5 min, rinsed in 2% acetic acid, and fixed in Rapid-Fix fixer (Eastman Kodak, Rochester, NY) for 10 min.

FIG. 1. The selected coronal sections of the rat brain from rostal to caudal brain regions included the following: (A) forebrain section I, (B) forebrain section II, (C) diencephalon section, (D) midbrain section, and (E) pons (with cerebellum) section. The anterior–posterior level of each figure, in millimeter relative to the skull landmark, bregma, is noted in each figure.

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FIG. 2. Autoradiograms of [3 H]CI-977 (2.5 nM) binding to κ 1 -opioid receptors, [3 H]DAMGO (5 nM) binding to µ-opioid receptors, and [3 H]DPDPE (15 nM) binding to δ-opioid receptors in the following: (A) coronal forebrain section I, (B) forebrain section II, (C) diencephalon sections, (D) midbrain sections, and (E) pons (with cerebellum) sections of control rats and rats undergoing naloxone-precipitated withdrawal from dependence upon butorphanol and morphine.

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FIG. 2. (Continued ).

Quantification of Autoradiograms Autoradiograms were analyzed using a digital scanning densitometer (Personal Densitometer, Molecular Dynamics, Sunnyvale, CA) operating on the image acquisition and analysis program, ImageQuant 3.3 (Molecular Dynamics). The anatomical structures of interest were identified by reference to the rat atlas of Paxinos and Watson [51]. For each region, identical areas were selected for quantification from both right and left sides of relevant brain sections. Readings from three adjacent sections for each region of each animal were averaged, and the n value in each table refers to the number of animals analyzed. For each area examined in the autoradiograms, the optical density values were converted to nanoCurie/milligram tissue equivalents using the equation obtained from the linear regression of data for the 3 H-impregnated plastic standards ([3 H]microscale RPA 510, Amersham Life Science). Data were obtained from seven rats and expressed as the mean ± SEM, in nanoCurie/milligram wet brain tissue. Data Analysis The data obtained were analyzed by microcomputer-based statistical packages available in Sigmastat (V2.03, Jandel Corporation, San Rafael, CA). Data from the measurements of autoradiographic binding were compared by one-way ANOVA. The Student-Newman–Keuls method was used for all post hoc comparisons. Differences were considered statistically significant when p < 0.05.

RESULTS Figure 2 shows representative autoradiographs of [3 H]CI-977 (for κ 1 -opioid receptors), [3 H]DAMGO (for µ-opioid receptors), and [3 H]DPDPE (for δ-opioid receptors) binding to coronal sections of brains from control rats and rats undergoing naloxone-precipitated withdrawal from dependence upon butorphanol (butorphanol-withdrawal) or morphine (morphine-withdrawal). The patterns of distinct anatomical distributions for each of the κ 1 -, µ-, and δ-opioid receptors in rat brain are similar to those previously reported in the literature [29,41]. In butorphanol-withdrawal rats, quantitative analysis of κ 1 -opioid receptors from brain sections of forebrain (I and II), diencephalon, midbrain and pons (with cerebellum) showed significant increases in [3 H]CI-977 binding in all brain regions examined including, the frontal cortex, nucleus accumbens, claustrum, dorsal endopiriform nucleus, caudate putamen, parietal cortex, posterior basolateral amygdaloid nucleus, dorsomedial hypothalamus, hippocampus (granular and molecular layers of the dentate gyrus, pyramidal cell layer in the CA3 field of Ammon’s horn and the stratum radiatum), posterior paraventricular thalamic nucleus, periaqueductal gray, substantia nigra, superficial gray layer of the superior colliculus, ventral tegmental area, and locus coeruleus (n = 7, p < 0.05) (Table 1). The κ 1 -opioid receptor binding data for the control group are from 0.21 to 1.86 nCi/mg, those for the butorphanol-withdrawal group are from 0.36 to 2.42 nCi/mg, and those for the morphine-withdrawal group are from 0.25 to 1.98 nCi/mg. Especially, there were more marked increases of

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BUTORPHANOL, OPIOID RECEPTOR, DEPENDENCE WITHDRAWAL binding in certain brain regions, such as the parietal cortex, posterior basolateral amygdaloid nucleus, dorsomedial hypothalamus, hippocampus (granular and molecular layers of the dentate gyrus, pyramidal cell layer in the CA3 field of Ammon’s horn and the stratum radiatum), and the locus coeruleus (>40%). However, similar increases in [3 H]CI-977 binding were not observed in rats undergoing naloxone-precipitated withdrawal from morphine dependence. Furthermore, brain sections from morphine-withdrawal rats showed significant decreases in [3 H]DAMGO (for µ-opioid receptors) binding in many brain regions examined including, the frontal cortex, posterior basolateral amygdaloid nucleus, dorsomedial hypothalamus, hippocampus (granular and molecular layers of the dentate gyrus, pyramidal cell layer in the CA3 field of Ammon’s horn and the stratum radiatum), posterior paraventricular thalamic nucleus, ventral tegmental area, and locus coeruleus (n = 7, p < 0.05) (Table 2). The µ-opioid receptor binding data for the control group are from 1.83 to 8.47 nCi/mg, those for the butorphanol-withdrawal group are from 1.01 to 8.25 nCi/mg, and those for the morphine-withdrawal group are from 1.35 to 6.94 nCi/mg. Particularly marked decreases of binding were noted in the posterior paraventricular thalamic nucleus, ventral tegmental area, and locus coeruleus (>34%). However, except in the ventral tegmental area, such decreases in binding were not observed in withdrawal from butorphanol dependence. In addition, binding increased in the periaqueductal gray and substantia nigra of the brain of rats undergoing withdrawal from butorphanol dependence. Furthermore, there were no significant changes in the coronal forebrain II sections, which included the nucleus accumbens, claustrum, dorsal endopiriform nucleus, caudate putamen, and parietal cortex, in rats undergoing naloxone-precipitated withdrawal from dependence upon either butorphanol or morphine. In both butorphanol- and morphine-withdrawal groups, quantitative analysis of δ-opioid receptors from coronal sections of forebrain I, diencephalon and pons (with cerebellum) showed significant increases above control in [3 H]DPDPE binding in the frontal cortex, posterior basolateral amygdaloid nucleus, dorsomedial hypothalamus, hippocampus (granular and molecular layers of the dentate gyrus, pyramidal cell layer in the CA3 field of Ammon’s horn and the stratum radiatum), posterior paraventricular thalamic nucleus, and locus coeruleus (n = 7, p < 0.05) (Table 3). The δ-opioid receptor binding data for the control group are from 0.12 to 3.20 nCi/mg, those for the butorphanol-withdrawal group are from 0.14 to 3.43 nCi/mg, and those for the morphine-withdrawal group are from 0.21 to 3.41 nCi/mg. However, at the level of the midbrain, binding decreased in the periaqueductal gray, substantia nigra, superficial gray layer of the superior colliculus, and ventral tegmental area in rats undergoing naloxone-precipitated withdrawal from dependence upon butorphanol (n = 7, p < 0.05). Such decreases were not observed in rats undergoing naloxone-precipitated withdrawal from dependence upon morphine. DISCUSSION The present study shows, on the basis of quantitative receptor autoradiography, that naloxone-precipitated withdrawal from dependence upon butorphanol or morphine is associated with regionally specific changes in brain κ 1 -, µ-, and δ-opioid receptors. Particularly striking was the fact that increases in κ 1 -opioid receptor binding were observed in butorphanol-dependent rats and that these were not present in morphine-dependent rats. These data are similar to the finding in previous studies that κ 1 -opioid receptor binding was significantly increased both in butorphanol-dependent (without a precipitated withdrawal) and butorphanol-withdrawal rats [12]. In contrast, µ-opioid receptor binding was noted to de-

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crease significantly, a phenomenon that could be observed only in the brain of morphine-dependent rats. These data are similar to observations reported by other researchers [17,69]. Of specific note are the significant decreases in certain regions of the diencephalon and the lack of significant differences in the nucleus accumbens, striatum [69] and midbrain (except the ventral tegmental area) in chronic morphine administration rats (without a precipitated withdrawal) [17]. While changes in the binding of brain δ-opioid receptors were noted, these changes appeared to have similar distributions in both groups of dependent rats. However, compared with the results reported in a previous study [69], the present data did not show any long-lasting down-regulations, by morphine, in the density of δ-opioid receptors in the nucleus accumbens and striatum. These differences in the pattern of binding to brain κ 1 -, µ-, and δ-opioid receptors may be the result of differing affinities of butorphanol and morphine to each of the opioid receptors. Butorphanol acts primarily as an agonist at the κ 1 -opioid receptor [4,34], while morphine acts mainly on the µ-opioid receptor, with some additional effects at the δ-opioid receptor [1,42]. The opioid physical dependence and withdrawal syndromes are associated with long-lasting [23,67] drug-induced molecular and behavioral plasticity [19,45,69]. An early cellular adaptation to chronic opioid treatment is alteration in the number and characteristics of opioid receptors. Many G-protein coupled receptors, including opioid receptors, show adaptive responses to agonists after prolonged or repeated activation. Three processes have been demonstrated to occur, over a time scale ranging from seconds to days, in G-protein coupled receptors in response to agonists: (i) acute desensitization (seconds to hours), (ii) internalization (minutes to hours), and (iii) long-term desensitization and down-regulation (hours to days) [32,36,55]. The regionally specific decreases in µ-opioid receptor binding observed in rats undergoing naloxone-precipitated withdrawal from dependence upon morphine are consistent with results of other studies showing long-term desensitization and down-regulation to morphine administration [3,35,63]. These data may explain the diminished effect of chronic morphine administration on µ-opioid receptor-stimulated [35 S]GTP␥S in rat brain [61]. On the other hand, while it has been reported that µ-opioid receptors undergo endosomal internalization following stimulation by the selective µ-opioid receptor agonist, DAMGO, morphine sulfate was essentially and suprisingly incapable of causing endosomal internalization [70]. Hence, differences between morphine and butorphanol in the propensity to induced µ-opioid receptor internalization could explain some of the observed differences in receptor number. Endosomal internalization may involve a variety of relatively poorly understood phenomena, including receptor recycling, rephosphorylation, a process of redesensitization and mitogen-activated protein kinase (MAPK) activation, any or all of which have the potential to regulate µ-opioid receptor function [70,71]. In this study, morphine was noted to uniformly decrease the signal for µ-opioid receptors in 10 of 17 regions examined, while butorphanol had little effect, expect to cause increases in receptor number in both the periaqueductal gray and substantia nigra and a decrease only in the ventral tegmental area. The known differences in opioid receptor stimulation profiles and the fact that butorphanol is an “agonist–antagonist” at one or more of the opioid receptors [52,53] could also present explanations for the divergent results. However, a distinctly different pattern of change was noted in κ 1 -opioid receptor binding following dependence upon, and withdrawal from, chronic butorphanol administration. Many studies in our laboratory have demonstrated that rats continuously infused with i.c.v. butorphanol for 72 h can produce physical dependence. Such chronic administration of butorphanol has been shown to alter the activity or expression of diverse cellular

158 proteins in specific target neurons within the CNS. Examples include signaling proteins, such as κ-opioid receptors [27,72], G-proteins [38,49], second-messenger synthetic enzymes [24], protein kinases [24,44,68], as well as transcription factors [17], and the associated alterations in gene expression [48]. Moreover, the roles of µ-, δ-, and κ-opioid receptors in the development of physical dependence upon butorphanol had been systematically investigated using receptor-specific antagonists. Both nor-BNI, a κ-opioid receptor selective antagonist and naltrindole, a δ-opioid receptor selective antagonist have been shown to precipitate withdrawal signs similar to those precipitated by the nonselective opioid receptor antagonist, naloxone, in butorphanol-dependent rats [25,27,28]. This is in contrast to the failure of β-funaltrexamine, a µ-opioid receptor selective antagonist, to precipitate withdrawal in butorphanol-dependent rats [26,28]. Moreover, nor-BNI blocked the development of physical dependence upon butorphanol, but not on morphine, when given prior to or during opioid administration [27]. It was further demonstrated that nor-BNI selectively precipitated behavioral and neurochemical signs of withdrawal in rats treated with butorphanol or the κ 1 -opioid receptor selective agonist, U69,593, but not in rats treated with morphine [12,22]. Therefore, the present finding of different patterns of change in binding, particularly of κ 1 -opioid receptors, is not surprising. The results strongly implicate a substantive and largely unique role for κ 1 -opioid receptors, but not µ-opioid receptors in mediation of withdrawal symptoms in butorphanol-dependent rats. In contrast to the observation of regionally specific decreases in µ-opioid receptor binding in acutely precipitated withdrawal from dependence upon morphine, increases in κ 1 -opioid receptor binding were noted in similarly treated, butorphanol-dependent rats. The mechanism by which the increase in κ 1 -opioid receptors is mediated may be similar to the increase observed in β 2 -adrenergic receptors, that has been noted following the exposure of cells to β-adrenergic agonists. Such exposure results in a transient increase in β 2 -adrenergic receptor gene expression mediated by an increase in cAMP response element binding protein (CREB) activation [6]. CREB proteins consist of a family of related transcription factors that mediate many of the effects of cAMP and calcium on gene expression. An involvement of CREB proteins has been demonstrated clearly in opioid dependence and during withdrawal. For example, acute opioid administration has been shown to lower the extent of CREB phosphorylation within the locus coeruleus, the magnitude of which was lessened in animals that received chronic opioid treatment [18,39]. Furthermore, opioid withdrawal increases CREB phosphorylation in the locus coeruleus [16,43,57]. It has been suggested that transcription factors play a role in triggering and maintaining intracellular adaptations to the opioid, and that increased levels of the transcription factors might be involved in returning the changes in intracellular messengers back to pretreatment levels during withdrawal [45,46]. It has been reported that increases in the cellular mRNA content coding for κ-opioid receptors were significantly increased in the midbrain including thalamus and pons when animals developed dependence upon butorphanol and subsequently, during withdrawal from such dependence [62]. Therefore, the increases in κ 1 -opioid receptor binding in precipitated withdrawal from dependence upon butorphanol may result from a de novo increase in κ 1 -opioid receptor number caused by cellular and molecular counteradaptations [12,62]. The apparent increase in expression of κ 1 -opioid receptors might be expected to increase suppression of presynaptic neuronal excitatory activity. For example, activation of κ 1 -opioid receptors has been shown to cause inhibition of adenylyl cyclase, activation of a potassium conductance, inhibition of calcium conductance and an inhibition of transmitter release [7]. However, the results of other studies suggest that following chronic butorphanol treat-

FAN ET AL. ment, the structure or amount of κ 1 -opioid receptors may be changed and uncoupling of κ 1 -opioid receptors from their corresponding G-proteins may occur. Studies of [35 S]GTP␥S binding have demonstrated that chronic administration of butorphanol induced tolerance and abolished U-50,488 activation of G-proteins in many brain areas [49]. Chronic butorphanol administration also leads to a relative decrease in the degree of activation of the potassium channels, and increased activation of the calcium channels [65,66]. Consequently, the intrinsic excitability of neurons is increased. Chronic butorphanol regulates the cAMP pathway in the locus coeruleus at several levels of signal transduction, producing increased adenylyl cyclase and cAMP-dependent kinase activity [24,44,64] and decreased the G-protein coupling [49]. Moreover, it was found that κ 1 -opioid receptor binding were significantly increased both in butorphanol-dependent (without withdrawal) and butorphanol-withdrawal rats in previous studies [12]. Therefore, the increases in κ 1 -opioid receptor binding observed in the present study may not indicate that functional κ 1 -opioid receptors have been increased. It is possible that the apparent increase in κ 1 -opioid receptor number shown here could reflect an increase in receptors that are stored in the vesicles of the presynaptic neuronal terminal, rather than those fused to the presynaptic plasma membrane. It has been reported that κ 1 -opioid receptors and dynorphin coexist in the vesicles of the presynaptic neuronal terminal, but not observed in µ-opioid receptors and β-endorphine [60]. Further, κ 1 -opioid receptors have been shown to associate with structures resembling large dense-core vesicles in the dorsal root ganglia, the spinal cord, and the hippocampus [10,59,74]. The vesicles containing κ 1 -opioid receptors were reported to fuse with the presynaptic plasma membrane when the presynaptic neuron was stimulated. It has been concluded that presynaptic κ 1 -opioid receptors are transported in the regulatory secretory pathway and require nerve stimulation to be inserted in the plasma membrane [59]. Hence, the κ 1 -opioid receptor may be an autoreceptor whose activation can directly modulate the release of neurotransmitters [60]. The reasons for different patterns of opioid receptor binding in butorphanol-dependent as opposed to morphine-dependent rats may be related to internalization of κ 1 -opioid receptors into the vesicles of the presynaptic neuronal terminal. Another possibility is that this phenomenon may be caused by the increased afferent inhibition of locus coeruleus neurons from the other brain stem sites, such as the GABA-ergic neurons from the nucleus prepositus hypoglossi (PrH, in the dorsomedial rostral medulla) and adrenergic neurons from the nucleus paragigantocellularis (PGi) [2,50]. In addition, activation of presynaptic κ 1 -opioid receptors inhibits GABA release in the ventral tegmental area, thus reducing inhibitory postsynaptic currents (IPSCs) mediated by binding of GABA to postsynaptic GABAB -receptors [58]. Moreover, activation of κ 1 -opioid receptors directly hyperpolarizes terminals, thus inhibiting GABA release from striatopallidal and intrapallidal terminals of the globus pallidus [47]. It has been suggested that alterations in multiple brain areas contribute to opioid dependence and withdrawal, such as the locus coeruleus, PGi, periaqueductal gray matter, the central nucleus of the amygdala, nucleus raphe magnus, the anterior hypothalamus, the medial thalamus, the hippocampus, the nucleus accumbens, and the neocortex [19,31,54]. Many, although not necessarily all [5] of the behavioral activating effects associated with opioid withdrawal may be mediated through the locus coeruleus. Some signs, such as wet-dog shakes, may involve sites in the hypothalamus important for temperature regulation, while other signs, such as diarrhea and lacrimation, may be dependent upon peripheral opioid receptors [31]. The motivational aspects of opioid withdrawal may involve the nucleus accumbens [5,31,69]. The present

BUTORPHANOL, OPIOID RECEPTOR, DEPENDENCE WITHDRAWAL autoradiographic studies observed that significant increases in κ 1 -opioid receptor binding occurred in the parietal cortex, posterior basolateral amygdaloid nucleus, dorsomedial hypothalamus, subregions of the hippocampus, and the locus coeruleus of rats undergoing withdrawal from dependence upon butorphanol. Therefore, these brain regions may be particularly responsible for mediation of withdrawal from dependence upon butorphanol. The present experiments clearly demonstrate that κ 1 -opioid receptors are differentially and preferentially altered during withdrawal from dependence upon butorphanol, as opposed to morphine. Both changes in the number of internalized receptors or changes as a result of de novo synthesis in response to exposure to butorphanol could present explanations for the producing differences between the morphine- and butorphanol-exposed animals. These data should contribute to the understanding of the mechanisms of physical dependence upon butorphanol. ACKNOWLEDGEMENTS

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