- Email: [email protected]
Morphine tolerance and transcription factor expression in mouse spinal cord tissue
Neuroscience Letters 272 (1999) 79±82
Morphine tolerance and transcription factor expression in mouse spinal cord tissue Xiangqi Li, J. David Clark* Veterans Administration Palo Alto Health Care System and Stanford University Department of Anesthesiology, Palo Alto, CA 94304, USA Received 16 March 1999; received in revised form 24 June 1999; accepted 28 June 1999
Abstract Little is known about changes in gene expression responsible for the acquisition and maintenance of tolerance to the analgesic effects of opioids. In these studies we examine changes in the expression of several transcription factors in the spinal cords of morphine tolerant C57BL/6 mice. Western blots demonstrate a 1.9-fold increase in cyclic AMP response element binding protein (CREB) and a 2.4-fold increase phospho-CREB immunoreactivities in spinal cord homogenates from morphine tolerant animals. Likewise, Fos B and DFos B immunoreactivities were increased 2.2 and 2.3-fold, respectively. The expression of c-Fos remained unchanged. Immunohistological analysis showed the increase of phospho-CREB and Fos B/DFos B to be primarily in the dorsal horn region of the spinal cord. We conclude that chronic exposure to opioids causes changes in gene expression in sensory processing areas of the spinal cord. q 1999 Elsevier Science Ltd. All rights reserved. Keywords: C-Fos; Fos B; DFos B; CREB; Morphine; Tolerance; Spinal cord
Many approaches exist for the control of pain, but the most commonly used and ef®cacious treatment for the control of serious pain such as acute postoperative pain or cancer pain involves opioid pharmacotherapy. Unfortunately several factors limit the use of opioids for the treatment of pain including dose-related side effects, worry of opioid addiction and tolerance to the analgesic actions of opioids. Tolerance in particular is problematic since it appears even after the acute administration of opioids in clinical populations [17], and has been noted in populations of patients receiving intrathecal opioids chronically for pain [15]. Tolerance has been studied for decades in animal models, yet this research has yet to lead to clinically useful methods for limiting the appearance of tolerance or reversing tolerance once it appears. Opioid analgesia is mediated principally by opioid receptors located in the dorsal root ganglia, spinal cord and brain. The receptors expressed on the spinal cord's dorsal horn neurons are involved in mediating analgesia as opposed to respiratory depression, sedation, nausea or addiction which involve opioid receptors and mechanisms in other locations. * Corresponding author. Vapahcs Anesthesiology, 112A 3801 Miranda Avenue, Palo Alto, CA 94304, USA. Tel.: 11-650-4935000 ext. 67184; fax: +1-650-852-3423. E-mail address: [email protected] (J.D. Clark)
In fact, clinicians exploit these spinal cord receptors to provide patients analgesia when they use intrathecal, epidural and systemic opioids in the treatment of acute or chronic pain. The study of tolerance to the analgesic actions of opioids may therefore be appropriately studied by examining changes in the actions of opioids in the spinal cord after chronic administration of these drugs. It has been recognized that alterations of gene expression may underlie many of the physiological and behavioral changes which occur with chronic exposure to opioids and other substances of abuse like cocaine [12]. Experiments directed at understanding opioid and cocaine dependence and addiction have identi®ed changes in the levels of various transcription factors such as c-Fos, Fos B, DFos B (a splice variant of Fos B), CREB (cAMP response element binding protein) and phospho-CREB in CNS tissue with chronic exposure to these narcotics [2,4,6±8,13,16,19]. Unfortunately, these experiments have generally looked at changes in brain tissue in an effort to understand behavioral changes associated with addiction, and not spinal cord tissue in an effort to understand analgesic tolerance. In this report, we document signi®cant changes in expression of several but not all of these transcription factors in the spinal cords of animals rendered tolerant to the analgesic actions of morphine. For these studies C57BL/6 mice 14±18 weeks old were
0304-3940/99/$ - see front matter q 1999 Elsevier Science Ltd. All rights reserved. PII: S03 04 - 394 0( 9 9) 00 55 9- 5
80
X. Li, J.D. Clark / Neuroscience Letters 272 (1999) 79±82
obtained from our breeding colony and kept under standard conditions. Mice were brie¯y anesthetized with iso¯urane and a 1 cm incision was made in the skin over the lumbar spine. At this point either a 50 mg morphine (MSO4) tablet (NIDA) was inserted and the incision closed, or the incision was immediately closed with staples for control animals. All animals were used 5 days after surgery. Neither death nor gross behavioral changes were common in animals using this protocol. This protocol closely follows that described by Welch et al. [18]. After 5 days, animals were taken to a behavioral lab and allowed to acclimate for 30 min. Using a modi®cation of the method of Hargreaves as we described previously [3], hindpaw ¯ick latencies were measured. This was followed by the injection morphine sulfate subcutaneously, and repeated measurement of hindpaw ¯ick latency after 25 min when analgesia was at peak levels as established in preliminary experiments. Noxious stimulation was limited to not more than 20 s to minimize the chance of tissue damage. For Western analysis, spinal cord protein homogenates were ®rst made. Animals were rapidly sacri®ced using CO-2 asphyxiation, followed by extrusion of the spinal cords. Samples were rapidly homogenized in 2% SDS containing buffer, 1 ml buffer per 100 mg spinal cord tissue. After boiling, 10-min samples were subjected to a 13000 £ g centrifugation for 10 min. Supernatants were collected and stored at 2808C. An Aliquot of each sample was used for protein concentration determination using Bio-Rad DC Protein Assay (Bio-Rad, USA). Polyacrylamide gel electrophoreses was carried out using the indicated amounts of protein with 10% acrylamide as the resolving gel. After electrotransfer of proteins to nitrocellulose membranes, blots were blocked overnight in 5% dry milk. Primary antibody was applied for 3 h at room temperature with gentle agitation. The concentrations of primary antibodies were as follows: anti-CREB and phospho-CREB 1:1000 (New England Biolabs, USA), anti-Fos B and DFos B 1:500 (Santa Cruz Biotechnology, USA), anti-cFos 1:2000 (Santa Cruz Biotechnology, USA). The same antibody was used for Fos B and DFos B which is directed against a conserved N-terminal region. Fos B and DFos B are readily separable using gel electrophoresis [2]. After washing, blots were incubated 1 h in horseradish peroxidase (HRP) conjugated goat anti-rabbit antibody 1:2000 (Chemicon, USA). Bands were visualized using ECL 1 regents (Amersham, USA) and Kodak XAR ®lm. Films were digitized, and bands of interest were quanti®ed using UN-SCAN-IT software (Silk Scienti®c, USA). The data presented represent results from at least ®ve animals in each of the control and morphine tolerant groups. Protein samples from each animal were run on more than one gel, in duplicate or triplicate. Statistical analysis utilized paired t-testing for bands from control and tolerant samples run on the same gels. For immunohistochemical experiments, extruded spinal cords were ®xed in 4% paraformaldehyde in 0.9% saline for 4 h at room temperature followed by overnight incubation in
30% sucrose at 48C. The lumbar region of the spinal cords was embedded in OCT medium, and 30 um sections made on a cryostat with sections then mounted on slides. Blocking again took place overnight at 48C with 5% dry milk, followed by overnight exposure to the primary antibody anti-phospho-CREB 1:500. Next, ABC reagents (Vector Labs) were used to visualize immunoreactive structures. Control studies included incubations without primary antibody, or, in other experiments, secondary antibody. Examination of slides was done using standard light microscopy using an Olympus BH-2 microscope. Chronic exposure to opioids renders mice tolerant to morphine. We ®rst sought to establish that mice implanted with subcutaneous morphine pellets became tolerant to the analgesic actions of morphine, as had been showed by others [18]. In sham operated animals the baseline paw ¯ick of 3:35 ^ 0:6 s was extended to 12:5 ^ 3:7 s after a 10 mg/kg MSO4 challenge dose (P , 0:01), while in mice with morphine pellets the baseline of 3:20 ^ 0:4 s increased to only 4:7 ^ 0:8 (P , 0:05). Intermediate doses of morphine caused intermediate prolongation of paw ¯ick latency. Chronic exposure to morphine results in the increased expression of multiple transcription factors in mouse spinal cords. Fig. 1 shows the results of Western blot analysis of spinal cord protein for expression of various transcription factors and transcription factor proteins. Levels of c-Fos, a transcription factor whose expression is increased by a number of acute and chronic treatments in a variety of tissues was expressed in equal amounts in control and tolerant animal's spinal cords. Expression of other transcription factors like Fos B and DFos B were, however, increased 2.2 and 2.3-fold, respectively. Furthermore, both the transcription factors CREB and the active phosphorylated form phospho-CREB were increased in abundance, 1.9- and 2.4-fold, respectively (Fig. 2). Thus, the expression of some but not all transcription factor proteins implicated in opioid depen-
Fig. 1. Western analysis of spinal cord homogenates from opioid naõÈve, 2, and opioid tolerant, 1, mice. Data represent results from homogenates made from at least ®ve mice in each group. Examples of bands from blots incubated with anti-CREB or anti-phospho-CREB are displayed above. Desitometry results are displayed beneath ^ SD. *P , 0:05.
X. Li, J.D. Clark / Neuroscience Letters 272 (1999) 79±82
Fig. 2. Western analysis of spinal cord homogenates from opioid naõÈve, 2, and opioid tolerant, 1, mice. Data represent results from homogenates made from at least ®ve mice in each group. Examples of bands from blots incubated with anti-cFos, Fos B and DFos B are displayed above. Densitometry results are displayed beneath ^ SD. *P , 0:05, **P , 0:01.
dence and addiction is increased in spinal cord tissue of opioid tolerant mice. Our next experiments sought to identify spinal cord regions where these transcription factors are expressed. Immunohistochemical studies were performed in an attempt to identify particular spinal cord regions where phospho-CREB was expressed. We selected phosphoCREB for additional study because it is believed to be the active form of CREB [5]. Analysis of FosB vs. DFos B distribution was not possible because of the cross-reactivity of our antibody. The data presented are from sections made in the lumbar area, a major sensory and motor signaling center where opioid-mediated effects on gene expression might be expected to be relevant to pain and analgesia in the animal's hind limbs. Analysis of phospho-CREB staining revealed most staining to be nuclear in distribution. Furthermore, cells in the super®cial, layers I-II, dorsal horn were most likely to contain positive nuclei. (Fig. 3). Ventral horn tissue contained little phospho-CREB immunoreactivity (data not
Fig. 3. Spinal cord tissue sections from mice made tolerant to morphine. Sections were incubated with polyclonal antibody for phospho-CREB. The image presented was photographed under 40£ magni®cation.
81
shown). Consideration was given to quanti®cation of the staining differences in sections from control and tolerant animals, but given the degree of section-to-section and slide-to-slide variability in staining, it was clear that Western analysis was the more objective way to analyze and present the data. The data presented above support the conclusion that the acquisition tolerance to opioid analgesics is accompanied by changes in expression of speci®c transcription factors in the spinal cords of mice. These transcription factors include CREB, phospho-CREB, Fos B and DFos B. Opioid tolerance does not cause a `global' increase in transcription factor expression, as we have found that c-Fos, a transcription factor whose expression is elevated after many types of stimuli in many tissues, is not increased under these conditions. Interestingly, c-Fos levels are increased in spinal cord tissue if opioid withdrawal is precipitated [16]. Furthermore, these transcription factors seem to be expressed in spinal cord dorsal horn tissue, an area of the spinal cord densely populated by opioid receptors, and clearly involved in nociceptive signaling. The observation that the expression of these transcription factors is elevated is important for two reasons. First, we might now begin to search for target genes for these transcription factors. Second, we may be able to use changes in the expression or activity of these transcription factors as indexes of the regulation of gene expression in opioid tolerance. The observations of changes in transcription factor levels raise the question of which genes are the targets for regulation. To date the expression of only a few genes has been shown to increase in spinal cord tissue of opioid tolerant animals. These genes code for the potassium ion channels Kv1.5 and Kv1.6, and synapsin I [10,11]. Whether these changes have functional consequences has not been demonstrated. Future efforts might be directed at identifying other genes regulated during opioid tolerance, and establishing whether or not the gene products are involved in the acquisition or maintenance of analgesic tolerance. Tools such as antisense oligonucleotides to generate `knock-down' models or homologous recombination strategies to generate `knock-out' models will likely be helpful in answering these questions. The continuing development of technologies such as differential display and cDNA subtraction should help to identify genes differentially expressed in basal and opioid tolerant states. Independent of knowing which genes constitute targets for these transcription factors, we may be able to use transcription factor expression as an index of gene regulation. With this sort of index we could develop strategies limiting changes in gene expression with chronic exposure to opioids. Future studies could, for example, determine if speci®c opiates or dosing schedules were more likely than others to cause the changes in phospho-CREB or Fos B expression. Likewise, we may be able to determine if agents which limit or reverse tolerance to the analgesic effects of opioids do so by in¯uencing transcription factor expression.
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Such agents limiting or reversing tolerance include NMDA antagonists [1,9], and nitric oxide synthase inhibitors [14,20]. Such efforts would hopefully lead to strategies which limit the extent of opioid tolerance itself, and render opioids more useful for the treatment of chronic pain. [1] Bilsky, E.J., Inturrisi, C.E., Sadee, W., Hruby, V.J. and Porreca, F., Competitive and non-competitive NMDA antagonists block the development of antinociceptive tolerance to morphine, but not to selective mu or delta opioid agonists in mice. Pain, 68 (1996) 229±237. [2] Chen, J., Kelz, M.B., Hope, B.T., Nakabeppu, Y. and Nestler, E.J., Chronic Fos-related antigens: stable variants of deltaFosB induced in brain by chronic treatments. J. Neurosci., 17 (1997) 4933±4941. [3] Clark, J.D. and Tempel, B.L., Hyperalgesia in mice lacking the Kv1.1 potassium channel gene. Neurosci. Lett., 251 (1998) 121±124. [4] Curran, E.J., Akil, H. and Watson, S.J., Psychomotor stimulant- and opiate-induced c-fos mRNA expression patterns in the rat forebrain: comparisons between acute drug treatment and a drug challenge in sensitized animals. Neurochem. Res., 21 (1996) 1425±1435. [5] Gonzalez, G.A. and Montminy, M.R., Cyclic AMP stimulates somatostatin gene transcription by phosphorylation of CREB at serine 133. Cell, 59 (1989) 675±680. [6] Guitart, X., Thompson, M.A., Mirante, C.K., Greenberg, M.E. and Nestler, E.J., Regulation of cyclic AMP response element-binding protein (CREB) phosphorylation by acute and chronic morphine in the rat locus coeruleus. J. Neurochem., 58 (1992) 1168±1171. [7] Lane-Ladd, S.B., Pineda, J., Boundy, V.A., Pfeuffer, T., Krupinski, J., Aghajanian, G.K. and Nestler, E.J., CREB (cAMP response element-binding protein) in the locus coeruleus: biochemical, physiological, and behavioral evidence for a role in opiate dependence. J. Neurosci., 17 (1997) 7890±7901. [8] Liu, J., Nickolenko, J. and Sharp, F.R., Morphine induces cfos and junB in striatum and nucleus accumbens via D1 and N-methyl-d-aspartate receptors. Proc. Natl. Acad. Sci. USA, 91 (1994) 8537±8541. [9] Mao, J., Price, D.D., Lu, J. and Mayer, D.J., Antinociceptive tolerance to the muopioid agonist DAMGO is dose-depen-
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[19]
[20]
dently reduced by MK-801 in rats (In Process Citation). Neurosci. Lett., 250 (1998) 193±196. Matus-Leibovitch, N., Ezra-Macabee, V., Saya, D., Attali, B., Avidor-Reiss, T., Barg, J. and Vogel, Z., Increased expression of synapsin I mRNA in de®ned areas of the rat central nervous system following chronic morphine treatment. Brain Res. Mol. Brain Res., 34 (1995) 221±230. Matus-Leibovitch, N., Vogel, Z., Ezra-Macabee, V., Etkin, S., Nevo, I. and Attali, B., Chronic morphine administration enhances the expression of Kv1.5 and Kv1.6 voltagegated K 1 channels in rat spinal cord. Brain Res. Mol. Brain Res., 40 (1996) 261±270. Nestler, E.J., Molecular mechanisms of opiate and cocaine addiction. Curr. Opin. Neurobiol., 7 (1997) 713±719. Nye, H.E. and Nestler, E.J., Induction of chronic Fos-related antigens in rat brain by chronic morphine administration. Mol. Pharmacol., 49 (1996) 636±645. Pataki, I. and Telegdy, G., Further evidence that nitric oxide modi®es acute and chronic morphine actions in mice. Eur. J. Pharmacol., 357 (1998) 157±162. Quaicoe, S., McLaughlin, R. and Hassenbusch, S., Intrathecal opiate therapy and implantable devices. In R.P.R Payne and C. Stratton Hill (Eds.), Assessment and Treatment of Cancer Pain, 12, IASP Press, Houston, TX, 1998, pp. 223± 256. Rohde, D.S. and Basbaum, A.I., Activation of coeruleospinal noradrenergic inhibitory controls during withdrawal from morphine in the rat. J. Neurosci., 18 (1998) 4393±4402. Vinik, H.R. and Kissin, I., Rapid development of tolerance to analgesia during remifentanil infusion in humans. Anesth. Analg, 86 (1998) 1307±1311. Welch, S.P., Smith, F.L. and Dewey, W.L., Morphine tolerance-induced modulation of [ 3H]glyburide binding to mouse brain and spinal cord. Drug Alcohol Depend., 45 (1997) 47±53. Widnell, K.L., Self, D.W., Lane, S.B., Russell, D.S., Vaidya, V.A., Miserendino, M.J., Rubin, C.S., Duman, R.S. and Nestler, E.J., Regulation of CREB expression: in vivo evidence for a functional role in morphine action in the nucleus accumbens. J. Pharmacol. Exp. Ther., 276 (1996) 306±315. Zhao, G.M. and Bhargava, H.N., Nitric oxide synthase inhibition attenuates tolerance to morphine but not to [D-Ala2, Glu4] deltorphin II, a delta 2-opioid receptor agonist in mice. Peptides, 17 (1996) 619±623.
Morphine tolerance and transcription factor expression in mouse spinal cord tissue Xiangqi Li, J. David Clark* Veterans Administration Palo Alto Health Care System and Stanford University Department of Anesthesiology, Palo Alto, CA 94304, USA Received 16 March 1999; received in revised form 24 June 1999; accepted 28 June 1999
Abstract Little is known about changes in gene expression responsible for the acquisition and maintenance of tolerance to the analgesic effects of opioids. In these studies we examine changes in the expression of several transcription factors in the spinal cords of morphine tolerant C57BL/6 mice. Western blots demonstrate a 1.9-fold increase in cyclic AMP response element binding protein (CREB) and a 2.4-fold increase phospho-CREB immunoreactivities in spinal cord homogenates from morphine tolerant animals. Likewise, Fos B and DFos B immunoreactivities were increased 2.2 and 2.3-fold, respectively. The expression of c-Fos remained unchanged. Immunohistological analysis showed the increase of phospho-CREB and Fos B/DFos B to be primarily in the dorsal horn region of the spinal cord. We conclude that chronic exposure to opioids causes changes in gene expression in sensory processing areas of the spinal cord. q 1999 Elsevier Science Ltd. All rights reserved. Keywords: C-Fos; Fos B; DFos B; CREB; Morphine; Tolerance; Spinal cord
Many approaches exist for the control of pain, but the most commonly used and ef®cacious treatment for the control of serious pain such as acute postoperative pain or cancer pain involves opioid pharmacotherapy. Unfortunately several factors limit the use of opioids for the treatment of pain including dose-related side effects, worry of opioid addiction and tolerance to the analgesic actions of opioids. Tolerance in particular is problematic since it appears even after the acute administration of opioids in clinical populations [17], and has been noted in populations of patients receiving intrathecal opioids chronically for pain [15]. Tolerance has been studied for decades in animal models, yet this research has yet to lead to clinically useful methods for limiting the appearance of tolerance or reversing tolerance once it appears. Opioid analgesia is mediated principally by opioid receptors located in the dorsal root ganglia, spinal cord and brain. The receptors expressed on the spinal cord's dorsal horn neurons are involved in mediating analgesia as opposed to respiratory depression, sedation, nausea or addiction which involve opioid receptors and mechanisms in other locations. * Corresponding author. Vapahcs Anesthesiology, 112A 3801 Miranda Avenue, Palo Alto, CA 94304, USA. Tel.: 11-650-4935000 ext. 67184; fax: +1-650-852-3423. E-mail address: [email protected] (J.D. Clark)
In fact, clinicians exploit these spinal cord receptors to provide patients analgesia when they use intrathecal, epidural and systemic opioids in the treatment of acute or chronic pain. The study of tolerance to the analgesic actions of opioids may therefore be appropriately studied by examining changes in the actions of opioids in the spinal cord after chronic administration of these drugs. It has been recognized that alterations of gene expression may underlie many of the physiological and behavioral changes which occur with chronic exposure to opioids and other substances of abuse like cocaine [12]. Experiments directed at understanding opioid and cocaine dependence and addiction have identi®ed changes in the levels of various transcription factors such as c-Fos, Fos B, DFos B (a splice variant of Fos B), CREB (cAMP response element binding protein) and phospho-CREB in CNS tissue with chronic exposure to these narcotics [2,4,6±8,13,16,19]. Unfortunately, these experiments have generally looked at changes in brain tissue in an effort to understand behavioral changes associated with addiction, and not spinal cord tissue in an effort to understand analgesic tolerance. In this report, we document signi®cant changes in expression of several but not all of these transcription factors in the spinal cords of animals rendered tolerant to the analgesic actions of morphine. For these studies C57BL/6 mice 14±18 weeks old were
0304-3940/99/$ - see front matter q 1999 Elsevier Science Ltd. All rights reserved. PII: S03 04 - 394 0( 9 9) 00 55 9- 5
80
X. Li, J.D. Clark / Neuroscience Letters 272 (1999) 79±82
obtained from our breeding colony and kept under standard conditions. Mice were brie¯y anesthetized with iso¯urane and a 1 cm incision was made in the skin over the lumbar spine. At this point either a 50 mg morphine (MSO4) tablet (NIDA) was inserted and the incision closed, or the incision was immediately closed with staples for control animals. All animals were used 5 days after surgery. Neither death nor gross behavioral changes were common in animals using this protocol. This protocol closely follows that described by Welch et al. [18]. After 5 days, animals were taken to a behavioral lab and allowed to acclimate for 30 min. Using a modi®cation of the method of Hargreaves as we described previously [3], hindpaw ¯ick latencies were measured. This was followed by the injection morphine sulfate subcutaneously, and repeated measurement of hindpaw ¯ick latency after 25 min when analgesia was at peak levels as established in preliminary experiments. Noxious stimulation was limited to not more than 20 s to minimize the chance of tissue damage. For Western analysis, spinal cord protein homogenates were ®rst made. Animals were rapidly sacri®ced using CO-2 asphyxiation, followed by extrusion of the spinal cords. Samples were rapidly homogenized in 2% SDS containing buffer, 1 ml buffer per 100 mg spinal cord tissue. After boiling, 10-min samples were subjected to a 13000 £ g centrifugation for 10 min. Supernatants were collected and stored at 2808C. An Aliquot of each sample was used for protein concentration determination using Bio-Rad DC Protein Assay (Bio-Rad, USA). Polyacrylamide gel electrophoreses was carried out using the indicated amounts of protein with 10% acrylamide as the resolving gel. After electrotransfer of proteins to nitrocellulose membranes, blots were blocked overnight in 5% dry milk. Primary antibody was applied for 3 h at room temperature with gentle agitation. The concentrations of primary antibodies were as follows: anti-CREB and phospho-CREB 1:1000 (New England Biolabs, USA), anti-Fos B and DFos B 1:500 (Santa Cruz Biotechnology, USA), anti-cFos 1:2000 (Santa Cruz Biotechnology, USA). The same antibody was used for Fos B and DFos B which is directed against a conserved N-terminal region. Fos B and DFos B are readily separable using gel electrophoresis [2]. After washing, blots were incubated 1 h in horseradish peroxidase (HRP) conjugated goat anti-rabbit antibody 1:2000 (Chemicon, USA). Bands were visualized using ECL 1 regents (Amersham, USA) and Kodak XAR ®lm. Films were digitized, and bands of interest were quanti®ed using UN-SCAN-IT software (Silk Scienti®c, USA). The data presented represent results from at least ®ve animals in each of the control and morphine tolerant groups. Protein samples from each animal were run on more than one gel, in duplicate or triplicate. Statistical analysis utilized paired t-testing for bands from control and tolerant samples run on the same gels. For immunohistochemical experiments, extruded spinal cords were ®xed in 4% paraformaldehyde in 0.9% saline for 4 h at room temperature followed by overnight incubation in
30% sucrose at 48C. The lumbar region of the spinal cords was embedded in OCT medium, and 30 um sections made on a cryostat with sections then mounted on slides. Blocking again took place overnight at 48C with 5% dry milk, followed by overnight exposure to the primary antibody anti-phospho-CREB 1:500. Next, ABC reagents (Vector Labs) were used to visualize immunoreactive structures. Control studies included incubations without primary antibody, or, in other experiments, secondary antibody. Examination of slides was done using standard light microscopy using an Olympus BH-2 microscope. Chronic exposure to opioids renders mice tolerant to morphine. We ®rst sought to establish that mice implanted with subcutaneous morphine pellets became tolerant to the analgesic actions of morphine, as had been showed by others [18]. In sham operated animals the baseline paw ¯ick of 3:35 ^ 0:6 s was extended to 12:5 ^ 3:7 s after a 10 mg/kg MSO4 challenge dose (P , 0:01), while in mice with morphine pellets the baseline of 3:20 ^ 0:4 s increased to only 4:7 ^ 0:8 (P , 0:05). Intermediate doses of morphine caused intermediate prolongation of paw ¯ick latency. Chronic exposure to morphine results in the increased expression of multiple transcription factors in mouse spinal cords. Fig. 1 shows the results of Western blot analysis of spinal cord protein for expression of various transcription factors and transcription factor proteins. Levels of c-Fos, a transcription factor whose expression is increased by a number of acute and chronic treatments in a variety of tissues was expressed in equal amounts in control and tolerant animal's spinal cords. Expression of other transcription factors like Fos B and DFos B were, however, increased 2.2 and 2.3-fold, respectively. Furthermore, both the transcription factors CREB and the active phosphorylated form phospho-CREB were increased in abundance, 1.9- and 2.4-fold, respectively (Fig. 2). Thus, the expression of some but not all transcription factor proteins implicated in opioid depen-
Fig. 1. Western analysis of spinal cord homogenates from opioid naõÈve, 2, and opioid tolerant, 1, mice. Data represent results from homogenates made from at least ®ve mice in each group. Examples of bands from blots incubated with anti-CREB or anti-phospho-CREB are displayed above. Desitometry results are displayed beneath ^ SD. *P , 0:05.
X. Li, J.D. Clark / Neuroscience Letters 272 (1999) 79±82
Fig. 2. Western analysis of spinal cord homogenates from opioid naõÈve, 2, and opioid tolerant, 1, mice. Data represent results from homogenates made from at least ®ve mice in each group. Examples of bands from blots incubated with anti-cFos, Fos B and DFos B are displayed above. Densitometry results are displayed beneath ^ SD. *P , 0:05, **P , 0:01.
dence and addiction is increased in spinal cord tissue of opioid tolerant mice. Our next experiments sought to identify spinal cord regions where these transcription factors are expressed. Immunohistochemical studies were performed in an attempt to identify particular spinal cord regions where phospho-CREB was expressed. We selected phosphoCREB for additional study because it is believed to be the active form of CREB [5]. Analysis of FosB vs. DFos B distribution was not possible because of the cross-reactivity of our antibody. The data presented are from sections made in the lumbar area, a major sensory and motor signaling center where opioid-mediated effects on gene expression might be expected to be relevant to pain and analgesia in the animal's hind limbs. Analysis of phospho-CREB staining revealed most staining to be nuclear in distribution. Furthermore, cells in the super®cial, layers I-II, dorsal horn were most likely to contain positive nuclei. (Fig. 3). Ventral horn tissue contained little phospho-CREB immunoreactivity (data not
Fig. 3. Spinal cord tissue sections from mice made tolerant to morphine. Sections were incubated with polyclonal antibody for phospho-CREB. The image presented was photographed under 40£ magni®cation.
81
shown). Consideration was given to quanti®cation of the staining differences in sections from control and tolerant animals, but given the degree of section-to-section and slide-to-slide variability in staining, it was clear that Western analysis was the more objective way to analyze and present the data. The data presented above support the conclusion that the acquisition tolerance to opioid analgesics is accompanied by changes in expression of speci®c transcription factors in the spinal cords of mice. These transcription factors include CREB, phospho-CREB, Fos B and DFos B. Opioid tolerance does not cause a `global' increase in transcription factor expression, as we have found that c-Fos, a transcription factor whose expression is elevated after many types of stimuli in many tissues, is not increased under these conditions. Interestingly, c-Fos levels are increased in spinal cord tissue if opioid withdrawal is precipitated [16]. Furthermore, these transcription factors seem to be expressed in spinal cord dorsal horn tissue, an area of the spinal cord densely populated by opioid receptors, and clearly involved in nociceptive signaling. The observation that the expression of these transcription factors is elevated is important for two reasons. First, we might now begin to search for target genes for these transcription factors. Second, we may be able to use changes in the expression or activity of these transcription factors as indexes of the regulation of gene expression in opioid tolerance. The observations of changes in transcription factor levels raise the question of which genes are the targets for regulation. To date the expression of only a few genes has been shown to increase in spinal cord tissue of opioid tolerant animals. These genes code for the potassium ion channels Kv1.5 and Kv1.6, and synapsin I [10,11]. Whether these changes have functional consequences has not been demonstrated. Future efforts might be directed at identifying other genes regulated during opioid tolerance, and establishing whether or not the gene products are involved in the acquisition or maintenance of analgesic tolerance. Tools such as antisense oligonucleotides to generate `knock-down' models or homologous recombination strategies to generate `knock-out' models will likely be helpful in answering these questions. The continuing development of technologies such as differential display and cDNA subtraction should help to identify genes differentially expressed in basal and opioid tolerant states. Independent of knowing which genes constitute targets for these transcription factors, we may be able to use transcription factor expression as an index of gene regulation. With this sort of index we could develop strategies limiting changes in gene expression with chronic exposure to opioids. Future studies could, for example, determine if speci®c opiates or dosing schedules were more likely than others to cause the changes in phospho-CREB or Fos B expression. Likewise, we may be able to determine if agents which limit or reverse tolerance to the analgesic effects of opioids do so by in¯uencing transcription factor expression.
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