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Time course of morphine withdrawal and preproenkephalin gene expression in the periaqueductal gray of rats
Molecular Brain Research 55 Ž1998. 221–231
Research report
Time course of morphine withdrawal and preproenkephalin gene expression in the periaqueductal gray of rats Yuko Fukunaga ) , Shigeru Nishida, Norihiro Inoue, Masahiko Miyamoto, Shiroh Kishioka, Hiroyuki Yamamoto Department of Pharmacology, Wakayama Medical College, 9-Bancho 27, Wakayama-City, Wakayama 640, Japan Accepted 9 December 1997
Abstract We have previously reported the increase of preproenkephalin ŽPPE. mRNA in the caudal periaqueductal gray ŽPAG. of rats during morphine withdrawal. In this study, it was further evidenced that PPE mRNA in the caudal PAG was not increased by various kinds of stressor, suggesting that the increase in PPE mRNA in the caudal PAG is specific to morphine withdrawal. In order to investigate the physiological significance of the increase of PPE mRNA in the caudal PAG, we compared the time course of the increase of PPE mRNA in the caudal PAG with that of naloxone-precipitated or spontaneous morphine withdrawal signs. The increase of plasma corticosterone ŽPCS: 52 and 52 m gr100 ml; control group, 18 and 15 m gr100 ml. and body weight loss Žy6 and y9%; control group, 0 and y1%. were observed but PPE mRNA increase was not detected 1 and 2 h after naloxone in morphine treated rats. PPE mRNA increased by 37 to 56%, while PCS elevation and body weight loss gradually diminished 4 h to 2 days after naloxone. A total of 12 h after spontaneous withdrawal, PCS was prominently increased Ž51 m gr100 ml; control group, 12 m gr100 ml., but body weight and PPE mRNA were not affected. One day after spontaneous withdrawal, PCS elevation Ž38 m gr100 ml; control group, 8 m gr100 ml. and body weight loss Žy5%; control group, q3%. were observed and PPE mRNA also increased by 42%. Two to 3 days after the final morphine injection, PCS recovered to control level and body weight loss gradually disappeared, while PPE mRNA was still increased by 74 to 46%. These results suggest that PPE gene expression in the caudal PAG is stimulated in the recuperative phase of these morphine withdrawal signs. q 1998 Elsevier Science B.V. Keywords: Morphine withdrawal; Body weight loss; Plasma corticosterone elevation; Preproenkephalin mRNA; Caudal periaqueductal gray; In situ hybridization; Rat
1. Introduction Many attempts have been made to investigate neuronal mechanisms involved in the opiate dependence and withdrawal processes. One possible mechanism is proposed to involve the alterations in opioid systems in the brain during opiate dependence and withdrawal w30x. The role of this opioidergic mechanism has not been fully elucidated, because of conflicting results on changes in endogenous opioids and their mRNA levels. Most of these reports have shown that the levels of enkephalins andror pre-
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0169-328Xr98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII S 0 1 6 9 - 3 2 8 X Ž 9 7 . 0 0 3 7 4 - 4
proenkephalin ŽPPE. mRNA are either unchanged or decreased after chronic morphine w22,26,27,32,58x, whereas a few reports have indicated an increase w3,26x. Morphine withdrawal has been reported to cause an increase w15,26,39,47 x, decrease w26,27,58 x or no change w19,26,27,32x of enkephalins andror PPE mRNA levels. These conflicting results may be from the differences in brain regions, duration of chronic morphine treatment, different types of morphine abstinence, i.e., spontaneous and antagonist-precipitated withdrawal, and different interval from the final morphine dose or opioid antagonist injection to sacrifice. The periaqueductal gray ŽPAG. is an important brain region for several biological responses to opioids, including analgesia w1,42,51,63x, reinforcement w7,33x, immuno-
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suppression w31,61x and intestinal motility w45,53x. A high density of m-opioid receptors w59x and its mRNA w17,24,43x has been detected in this site. There are several lines of evidence for the implication of the PAG in the development of morphine dependence and withdrawal. The severe physical dependence was developed after chronic administration of an enkephalin analog or morphine in this area w6,62x, and the severe withdrawal symptoms were precipitated by administrating opioid antagonists into the PAG w35,38,41x. Recently, the increase in c-fos expression, as a marker for neuronal activation, has been observed in the lateral and ventrolateral, particularly caudal ventrolateral, PAG neurons, during naloxone-precipitated withdrawal in rats chronically treated with morphine w11x. In spite of the importance of the PAG for physical dependence and withdrawal, there is little evidence on changes of endogenous opioid levels in the PAG during chronic morphine treatment and withdrawal. We have examined the role of endogenous enkephalin in the PAG in morphine withdrawal because the majority of peptidergic terminals into the PAG are the enkephalinergic type w25x. Previously, we reported that the level of PPE mRNA in the caudal PAG was increased 1 day after final morphine injection Žspontaneous withdrawal. and 4 h after naloxone Ž5 mgrkg. treatment Žnaloxone-precipitated withdrawal. in morphine dependent rats w23x. The change in the levels of PPE mRNA in the rostral PAG or the hypothalamic paraventricular nucleus ŽPVN. were induced only by naloxone-precipitated withdrawal but not by spontaneous withdrawal w23x. In addition, the administration of peptidase inhibitors in the PAG, which inhibit the degradation of endogenous enkephalins, attenuated naloxone-precipitated withdrawal w28,40x. These results suggest the importance of enkephalin in the PAG in morphine withdrawal. However, the findings on the level of PPE mRNA reported were obtained only at one time point after morphine withdrawal and insufficient for understanding of the physiological significance of enkephalins during morphine withdrawal. The aim of this study is to examine the physiological role of the increment of PPE mRNA in the caudal PAG during morphine withdrawal. In this study, we compared the time course of the change of PPE mRNA in the caudal PAG after spontaneous or naloxone-precipitated withdrawal with that of morphine withdrawal signs. We also estimated the time course of the change of PPE mRNA in the rostral PAG and the PVN, because we could not previously find the change of PPE mRNA of those brain regions 1 day after cessation of chronic morphine treatment in spontaneous withdrawal w23x. The expression of PPE mRNA in the PVN is facilitated by various kinds of stressor w34,37,67x. The estimation of the effects of various kinds of stressor on the PPE mRNA levels in the caudal PAG will give us information as to whether the increase in the expression of PPE mRNA in the caudal PAG is specific response to morphine withdrawal or nonspecific response to stress.
2. Materials and methods 2.1. Morphine dependence and withdrawal Male Sprague–Dawley rats, weighing about 190 g at the beginning of chronic morphine or saline treatment, were used. In order to induce morphine dependence, morphine Žmorphine hydrochloride. treatment was carried out according to a previous study w23x. Morphine was injected daily s.c. with gradually increasing doses Ž40–160 mgrkg. for 10 days, being followed by a final dose of 40 mgrkg on the morning of the 11th day. Spontaneous and opioid antagonist-precipitated withdrawal were induced by cessation of morphine treatment and the injection of naloxone Ž5 mgrkg s.c.. 1 h after the final morphine dose, respectively. Control groups received saline instead of morphine or naloxone. Body weight loss were estimated and the animals were sacrificed 1, 2, 4, 12 h, and 1, 2 and 4 days after naloxone injection for naloxone-precipitated withdrawal and 12 h, and 1, 2, 3, 5 and 10 days after the final treatment of morphine for spontaneous withdrawal after the administration of a large dose of pentobarbital Ž600– 900 mgrkg i.p... Other morphine withdrawal signs Ždiarrhea and salivation. were estimated 2 h after naloxone injection for naloxone-precipitated withdrawal and 1 day after the final treatment of morphine for spontaneous withdrawal. The brain was rapidly removed, blocked and frozen with powdered dry ice. A total of 15 m m coronal sections were cut throughout the areas containing PVN and PAG at y208C on a cryostat, and mounted on gelatincoated glass slides. The sections were stored at y808C until used for in situ hybridization. Body weight change Ž%. was calculated as follows: wŽbody weight just before sacrificey body weight just before naloxone or the final morphine injection.rbody weight just before naloxone or the final morphine injectionx = 100. For estimation of plasma corticosterone ŽPCS. level, the trunk blood was collected into the heparinized test tube and was centrifuged at 2000 = g for 30 min at 48C. The plasma was stored at y208C until assayed. The PCS was estimated fluorometrically according to the method of Zenker and Bernstein w68x. 2.2. In situ hybridization The oligonucleotide probe, complementary to bases 322–360 of the rat PPE mRNA w66x, was labeled at the 3X end with w a- 35 SxdATP and terminal deoxynucleotidyl transferase. Specific activity of the labeled probe was 7–10 = 10 5 c.p.m.rng. In situ hybridization was performed as described previously w23x. Briefly, the sections were fixed at 48C in 4% paraformaldehyde in phosphate-buffered saline ŽPBS. and rinsed twice in PBS; they were treated with 0.25% acetic anhydrate in 0.1 M triethanolaminer0.9% saline at room temperature, dehydrated in a graded series of ethanol, dilapidated in chloroform and rinsed in absolute ethanol. A
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total of 100 m l of hybridization buffer consisting of 50% formamide, 4 = SSC ŽSSC s 0.15 M NaClr0.015 M sodium citrate, pH 7.4., 1 = Denhardt’s solution, 0.25 mgrml of tRNA, 0.4 mgrml of shared and denatured salmon sperm DNA, 10% dextran sulfate, 20 mM dithiothreitol and 5 = 10 6 c.p.m.rml of labeled oligonucleotide was applied to each slide and left overnight at 378C in a moist chamber. Following overnight hybridization, the slides were washed in 1 = SSC at room temperature, 1 = SSC and 0.5 = SSC at 558C and in 0.1 = SSC at room temperature. The slides were allowed to dry and then were exposed to Kodak XAR-5 film for 2 days. After film development, the slides were dipped in emulsion ŽKodak NTB-2, diluted 1:1 with distilled water., exposed for 10 to 14 days and then developed. The specificity of the in situ hybridization reaction for PPE mRNA were estimated. Pretreatment of the sections with RNase A Ž100 m grml. for 1 h at 378C or excess unlabeled probe overnight at 378C completely eliminated the hybridization signal. Furthermore, in the sections reacted with PPE sense probe no labelings were seen above background Ždata not shown..
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an equivalent volume of isotonic saline. The rats were put back in their home cages and sacrificed 5 h after injection. 2.4.3. Isolation Each rat was placed individually in a opaque plastic cage Ž35 = 30 = 17 cm. and kept in continuous isolation for 14 days. Control rats were housed in group of four in the same cage. The rats were sacrificed on the 14th day.
2.3. QuantitatiÕe analysis of mRNA Analysis of the autoradiographic films was performed using the Macintosh-based software program IMAGE ŽW. Rasband, National Institutes of Health, USA.. Because the signal of PPE mRNA was intense in the lateral and ventrolateral PAG, optical density in that area was estimated. Optical densities in an area of 1.0 = 0.8 mm ŽPAG. and 0.8 mm = 0.6 mm ŽPVN. were determined in the rostral PAG, caudal PAG and the PVN at levels of y6.3 mm, y8.3 mm and y1.8 mm from Bregma w46x, respectively. Measurements were performed on two and four fields per rat for the PAG and PVN, respectively. Values after subtracting background density were averaged to give an optical density for each animal. Background density was determined by averaging optical densities in five fields of the same areas of the cortex on the same sections. Optical density of each rat was expressed as percent of mean optical density of morphine naive group and optical density of each experimental group was calculated by averaging optical densities of four animals. Statistical analysis was performed using Student’s t-test. 2.4. Stress paradigms 2.4.1. Restraint Rats were introduced into a narrow perspex restrainer for 4 h. The animals were put back in their home cages and sacrificed 5 h after onset of stress. 2.4.2. Hypertonic saline Rats were stressed by a hypertonic saline Ž9% NaCl; 1.5 mlr100 g. i.p. injection, whereas control animals received
Fig. 1. Time course of naloxone-precipitated withdrawal signs. Body weight was measured and trunk blood was collected 1, 2, 4 and 12 h, and 1, 2 and 4 days Žd. after naloxone injection. Plasma corticosterone ŽPCS. was fluorometrically determined. Each value is the mean"s.e. Ž ns 4.. Differs from chronic salineqsaline, ) p- 0.05, )) p- 0.01, ))) p0.001.
224 Y. Fukunaga et al.r Molecular Brain Research 55 (1998) 221–231 Fig. 2. Bright-field photomicrograms of typical transverse sections through the rostral PAG ŽA and D., caudal PAG ŽB and E. and PVN ŽC and F. hybridized with 35 S-labeled probe complementary to PPE mRNA. A, B and C: section from chronic salineqsaline treated rat. D, E and F: section from chronic morphineqnaloxone treated rat Žnaloxone-precipitated withdrawal.. Rats were decapitated 4 h after saline or naloxone challenge.
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3. Results 3.1. Effects of naloxone on the gene expression of PPE, body weight and PCS in morphine naiÕe rats PPE mRNA expression in the PAG and PVN remained unchanged 4 h after naloxone injection in chronic salinetreated rats Ždata not shown.. A single injection of naloxone also did not affect PCS levels Žchronic saline q saline:
Fig. 4. Time course of spontaneous withdrawal signs. Body weight was measured and trunk blood was collected 0.5, 1, 2, 3, 5 and 10 days Žd. after the final morphine injection. Plasma corticosterone ŽPCS. was determined by fluorometrical method. Each value is the mean"s.e. Ž ns 4.. Differs from chronic saline, )) p- 0.01, ))) p- 0.001.
5.3 " 1.7, chronic saline q naloxone: 7.7 " 1.1 m gr100 ml.. Rats treated with either naloxone or saline has a slight body weight loss of similar magnitude Žchronic saline q saline: y0.9 " 0.3, chronic saline q naloxone: y1.6 " 0.5%. 4 h after naloxone injection. Fig. 3. Effect of naloxone-precipitated morphine withdrawal on the level of PPE mRNA in the rostral and caudal PAG and the PVN. Each value Žmean"s.e., ns 4. is expressed as percent of mean value of chronic saline treated group. Differs from chronic salineqsaline, ) p- 0.05, )) p- 0.01.
3.2. Time course of naloxone-precipitated withdrawal syndromes PCS and body weight changes during naloxone-precipitated withdrawal were shown in Fig. 1. PCS was
226 Y. Fukunaga et al.r Molecular Brain Research 55 (1998) 221–231 Fig. 5. Bright-field photomicrograms of typical transverse sections through the rostral PAG ŽA and D., caudal PAG ŽB and E. and PVN ŽC and F. hybridized with 35 S-labeled probe complementary to PPE mRNA. A, B and C: section from chronic saline treated rat. D, E and F: section from chronic morphine treated rat Žspontaneous withdrawal.. Rats were decapitated 1 day after final injection.
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increased 1 h after naloxone injection, and this increase persisted until 1 day after naloxone injection and returned to base line 2 days after naloxone injection. Naloxone injection decreased body weight of chronic morphine treated rats from 1 h after naloxone injection. The maximum body weight loss was observed 2 to 4 h after naloxone injection. Then, body weight began to increase 12 h after naloxone injection. The incidence of diarrhea and salivation are 100% 2 h after naloxone injection. 3.3. Effects of naloxone-precipitated withdrawal on the gene expression of PPE Fig. 2 showed typical bright-field photomicrograms of transverse sections through the rostral PAG, caudal PAG and PVN in naloxone-precipitated withdrawal rats. PPE mRNA was decreased in the rostral PAG and increased in the caudal PAG and PVN 4 h after naloxone injections in morphine dependent rats. Fig. 3 showed the time course of the change of PPE mRNA levels in discrete brain regions. The levels of PPE mRNA in the rostral PAG returned to control values 12 h after naloxone injection. On the other hand, the stimulation of expression of PPE mRNA in the caudal PAG was sustained 2 days after naloxone injection. PPE mRNA in the PVN was significantly increased 2 h to 2 days after naloxone injection. 3.4. Time course of spontaneous withdrawal syndromes The time course of PCS elevation and body weight loss after the final morphine administration was shown in Fig. 4. PCS was increased 12 h to 1 day after the final morphine injection, and returned to control levels 2 days after the final morphine injection. One day after the final injection body weight was increased in control rats, but decreased by about 5% of initial body weight in spontaneous withdrawal rats. Then, body weight began to increase 2 days after the final morphine injection and returned to control level 10 days after the final morphine injection. The incidence of diarrhea and salivation are 50% and 0% 1 day after the final morphine injection, respectively. 3.5. Effects of spontaneous withdrawal on the gene expression of PPE Fig. 5 showed typical photomicrograms of transverse sections through the rostral PAG, caudal PAG and PVN in spontaneous withdrawal rats. Change of PPE mRNA in the rostral PAG, caudal PAG and PVN is determined 1 day after the final morphine injection in morphine-dependent rats. PPE mRNA in the caudal PAG increased during spontaneous withdrawal, whereas PPE mRNA in the rostral PAG and PVN did not change. Fig. 6 shows the time course of PPE mRNA levels after the final morphine administration. PPE mRNA in the rostral PAG and PVN
Fig. 6. Effect of spontaneous morphine withdrawal on the level of PPE mRNA in the rostral PAG, caudal PAG and the PVN. Each value Žmean"s.e., ns 4. is expressed as percent of mean value of chronic saline treated group. Differs from chronic saline, ) p- 0.05, )) p- 0.01.
did not change 12 h to 10 days after the final morphine dose. On the other hand, PPE mRNA levels in the caudal PAG was increased up to 3 days after the final morphine injection and returned to control levels 5 days after the final morphine injection. 3.6. Effects of Õarious kinds of stressor on the gene expression of PPE Fig. 7 showed the responses of PPE mRNA in the PAG and PVN to various kinds of stressor. PPE mRNA in the
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4. Discussion
Fig. 7. Effect of restraint for 4 h, hypertonic saline i.p. injection and isolation for 14 days on the level of PPE mRNA in the rostral PAG, caudal PAG and the PVN. Rats were decapitated 5 h after onset of restraint stress or i.p. injection of hypertonic saline and 14 days after onset of isolation stress. Each value Žmean"s.e., ns 4. is expressed as percent of mean value of control group. Differs from control, ) p- 0.05, )) p- 0.01.
rostral and caudal PAG did not change, whereas PPE mRNA in the PVN was increased in response to restraint stress, hypertonic saline stress and isolation stress.
We have previously reported that expression of PPE mRNA in the caudal PAG was stimulated by both naloxone-precipitated withdrawal Ž4 h after naloxone injection. and spontaneous withdrawal Ž1 day after final morphine., while PPE mRNA in the rostral PAG was decreased and PVN was stimulated 4 h after naloxone injection, but not change 1 day after cessation of chronic morphine treatment in morphine dependent rats w23x. There is a possibility that the change of PPE mRNA in the PAG could be the nonspecific response to stress during morphine withdrawal. In the present study, it was evidenced that the expression of PPE mRNA in the PAG was not affected by various kinds of stress tested. In previous study, we assessed PPE mRNA levels at only one time point, i.e., 1 day after spontaneous morphine withdrawal. In this study, changes in PPE mRNA in the rostral PAG and PVN were not observed 12 h to 10 days after cessation of chronic morphine treatment. These results indicate that the increase of PPE mRNA in the caudal PAG is specific response to morphine withdrawal and suggest some role of endogenous enkephalins in the caudal PAG in morphine withdrawal process. Time course of increase in PPE mRNA in the caudal PAG during morphine withdrawal was compared with that of morphine withdrawal syndromes to elucidate the physiological significance of increase in PPE mRNA in the caudal PAG in morphine withdrawal process. The present studies show that PPE mRNA increased in the course of improvement of morphine withdrawal signs and returned to control value after disappearing of morphine withdrawal signs. Peptidase inhibitors microinjected into the PAG inhibited naloxone-precipitated withdrawal in morphine dependent rats w28,40x. In addition, the systemic and intracerebroventricular treatment of enkephalins reduced morphine withdrawal w4,5x. These findings suggest that increase in PPE mRNA in the caudal PAG responded to morphine withdrawal may contribute to the recovery from morphine withdrawal. There are two possibilities for increase in the expression of PPE mRNA. One possibility is that the decrease of methionine ŽMet. –enkephalin content, a result of increased release during morphine withdrawal, may drive an increment of synthesis of PPE mRNA. The increase of PPE mRNA may therefore compensate for decreased Met–enkephalin content. Another possibility is the change of a signal transduction pathway linking membrane receptors to gene expression w12x. It is reported that m-type opioid receptors in rat brain are coupled to guanine–nucleotide binding proteins ŽG proteins. w20x and inhibit adenylate cyclase activity w9,18x. Chronic morphine increased levels of adenylate cyclase in several brain regions w18,44,57x. Spontaneous withdrawal or naloxone-precipitated withdrawal leads to further change of G a mRNA and an increase in basal and stimulated adenylate cyclase activity,
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as observed in vitro Žin NG108-15 cells. w54x and in vivo Žin rat locus coeruleus. w48x. Upstream of the PPE gene includes the cAMP responsive element and its transcription can be activated by an increase of cAMP w21x. In addition, the 5X region of the PPE genes has the binding site of AP-1 which is dimer of Fos and Jun family protein w16x. Expression of PPE mRNA is regulated by Fos and Jun w55x. Fos like immunoreactivity in the PAG w14,56x, particularly dorsolateral caudal PAG w11x, was increased during morphine withdrawal. These results suggest that Fos and Jun increase during morphine withdrawal and facilitate the synthesis of PPE mRNA. The mechanism by which increased enkephalin in the PAG reduces morphine withdrawal was remained to be elucidated. There are excitatory amino acid ŽEAA. nerves in the PAG w13x. EAA antagonists have been reported to reduce morphine withdrawal symptoms w36,49,57x, suggesting that EAA might be related to the occurrence of morphine withdrawal. An electrophysiological study showed that the glutamatergic component of fast postsynaptic potentials in single PAG neurons in rat brain slices were inhibited by Met–enkephalin w10x. These reports suggest that enkephalin in the PAG might inhibit morphine withdrawal via an inhibition of EAA action. There are axo-somatic synapses from enkephalinergic axon terminals to g-aminobutyric acid ŽGABA. neurons in the PAG w60x. Therefore, there is also a possibility that enkephalin might inhibit morphine withdrawal via GABA neurons in the PAG. It has been reported that PPE mRNA in the PVN is increased during naloxone-precipitated withdrawal w29,39x and this is confirmed by the present results. In spite of the marked increase in PPE mRNA in the PVN after naloxone-precipitated withdrawal, we could not find any change of PPE mRNA in the PVN after spontaneous withdrawal. Because the expression of PPE mRNA in the PVN is facilitated by various kinds of stressor, as shown in the present results and previous reports w34,37,67x, this gene expression could nonspecifically respond to some kinds of stressor. In this study, naloxone-precipitated withdrawal symptoms were more severe than spontaneous withdrawal symptoms. These results suggest that the naloxone-precipitated withdrawal paradigm induced a sufficient strength of stress to change PPE mRNA in the PVN, whereas spontaneous withdrawal in this study might induce a milder stress. The increase of PPE mRNA in the caudal PAG was induced after both naloxone-precipitated and spontaneous morphine withdrawal and long lasting. On the other hand, PPE mRNA in the rostral PAG was reduced transiently after naloxone-precipitated morphine withdrawal and not affected by spontaneous withdrawal. The PAG is not homogeneous, not only in anatomical terms w2,8,52x, but also in functional terms w2,50,65x. The caudal PAG is more sensitive to morphine analgesia rather than the rostral PAG w50,64,65x. Further study is required to elucidate the differ-
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ence in response of caudal PAG and rostral PAG to morphine withdrawal and the role of the enkephalins in the rostral PAG in precipitated withdrawal. In conclusion, it was demonstrated in both naloxoneprecipitated and spontaneous morphine withdrawal study that PPE mRNA in the caudal PAG was not affected at the onset of morphine withdrawal syndromes but increased thereafter during improvement of morphine withdrawal, being restored when these withdrawal syndromes were disappeared. These results suggest that the increase in PPE mRNA, consequent increase in enkephalins, in the caudal PAG may attribute to the recovery from morphine withdrawal.
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Research report
Time course of morphine withdrawal and preproenkephalin gene expression in the periaqueductal gray of rats Yuko Fukunaga ) , Shigeru Nishida, Norihiro Inoue, Masahiko Miyamoto, Shiroh Kishioka, Hiroyuki Yamamoto Department of Pharmacology, Wakayama Medical College, 9-Bancho 27, Wakayama-City, Wakayama 640, Japan Accepted 9 December 1997
Abstract We have previously reported the increase of preproenkephalin ŽPPE. mRNA in the caudal periaqueductal gray ŽPAG. of rats during morphine withdrawal. In this study, it was further evidenced that PPE mRNA in the caudal PAG was not increased by various kinds of stressor, suggesting that the increase in PPE mRNA in the caudal PAG is specific to morphine withdrawal. In order to investigate the physiological significance of the increase of PPE mRNA in the caudal PAG, we compared the time course of the increase of PPE mRNA in the caudal PAG with that of naloxone-precipitated or spontaneous morphine withdrawal signs. The increase of plasma corticosterone ŽPCS: 52 and 52 m gr100 ml; control group, 18 and 15 m gr100 ml. and body weight loss Žy6 and y9%; control group, 0 and y1%. were observed but PPE mRNA increase was not detected 1 and 2 h after naloxone in morphine treated rats. PPE mRNA increased by 37 to 56%, while PCS elevation and body weight loss gradually diminished 4 h to 2 days after naloxone. A total of 12 h after spontaneous withdrawal, PCS was prominently increased Ž51 m gr100 ml; control group, 12 m gr100 ml., but body weight and PPE mRNA were not affected. One day after spontaneous withdrawal, PCS elevation Ž38 m gr100 ml; control group, 8 m gr100 ml. and body weight loss Žy5%; control group, q3%. were observed and PPE mRNA also increased by 42%. Two to 3 days after the final morphine injection, PCS recovered to control level and body weight loss gradually disappeared, while PPE mRNA was still increased by 74 to 46%. These results suggest that PPE gene expression in the caudal PAG is stimulated in the recuperative phase of these morphine withdrawal signs. q 1998 Elsevier Science B.V. Keywords: Morphine withdrawal; Body weight loss; Plasma corticosterone elevation; Preproenkephalin mRNA; Caudal periaqueductal gray; In situ hybridization; Rat
1. Introduction Many attempts have been made to investigate neuronal mechanisms involved in the opiate dependence and withdrawal processes. One possible mechanism is proposed to involve the alterations in opioid systems in the brain during opiate dependence and withdrawal w30x. The role of this opioidergic mechanism has not been fully elucidated, because of conflicting results on changes in endogenous opioids and their mRNA levels. Most of these reports have shown that the levels of enkephalins andror pre-
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0169-328Xr98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII S 0 1 6 9 - 3 2 8 X Ž 9 7 . 0 0 3 7 4 - 4
proenkephalin ŽPPE. mRNA are either unchanged or decreased after chronic morphine w22,26,27,32,58x, whereas a few reports have indicated an increase w3,26x. Morphine withdrawal has been reported to cause an increase w15,26,39,47 x, decrease w26,27,58 x or no change w19,26,27,32x of enkephalins andror PPE mRNA levels. These conflicting results may be from the differences in brain regions, duration of chronic morphine treatment, different types of morphine abstinence, i.e., spontaneous and antagonist-precipitated withdrawal, and different interval from the final morphine dose or opioid antagonist injection to sacrifice. The periaqueductal gray ŽPAG. is an important brain region for several biological responses to opioids, including analgesia w1,42,51,63x, reinforcement w7,33x, immuno-
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suppression w31,61x and intestinal motility w45,53x. A high density of m-opioid receptors w59x and its mRNA w17,24,43x has been detected in this site. There are several lines of evidence for the implication of the PAG in the development of morphine dependence and withdrawal. The severe physical dependence was developed after chronic administration of an enkephalin analog or morphine in this area w6,62x, and the severe withdrawal symptoms were precipitated by administrating opioid antagonists into the PAG w35,38,41x. Recently, the increase in c-fos expression, as a marker for neuronal activation, has been observed in the lateral and ventrolateral, particularly caudal ventrolateral, PAG neurons, during naloxone-precipitated withdrawal in rats chronically treated with morphine w11x. In spite of the importance of the PAG for physical dependence and withdrawal, there is little evidence on changes of endogenous opioid levels in the PAG during chronic morphine treatment and withdrawal. We have examined the role of endogenous enkephalin in the PAG in morphine withdrawal because the majority of peptidergic terminals into the PAG are the enkephalinergic type w25x. Previously, we reported that the level of PPE mRNA in the caudal PAG was increased 1 day after final morphine injection Žspontaneous withdrawal. and 4 h after naloxone Ž5 mgrkg. treatment Žnaloxone-precipitated withdrawal. in morphine dependent rats w23x. The change in the levels of PPE mRNA in the rostral PAG or the hypothalamic paraventricular nucleus ŽPVN. were induced only by naloxone-precipitated withdrawal but not by spontaneous withdrawal w23x. In addition, the administration of peptidase inhibitors in the PAG, which inhibit the degradation of endogenous enkephalins, attenuated naloxone-precipitated withdrawal w28,40x. These results suggest the importance of enkephalin in the PAG in morphine withdrawal. However, the findings on the level of PPE mRNA reported were obtained only at one time point after morphine withdrawal and insufficient for understanding of the physiological significance of enkephalins during morphine withdrawal. The aim of this study is to examine the physiological role of the increment of PPE mRNA in the caudal PAG during morphine withdrawal. In this study, we compared the time course of the change of PPE mRNA in the caudal PAG after spontaneous or naloxone-precipitated withdrawal with that of morphine withdrawal signs. We also estimated the time course of the change of PPE mRNA in the rostral PAG and the PVN, because we could not previously find the change of PPE mRNA of those brain regions 1 day after cessation of chronic morphine treatment in spontaneous withdrawal w23x. The expression of PPE mRNA in the PVN is facilitated by various kinds of stressor w34,37,67x. The estimation of the effects of various kinds of stressor on the PPE mRNA levels in the caudal PAG will give us information as to whether the increase in the expression of PPE mRNA in the caudal PAG is specific response to morphine withdrawal or nonspecific response to stress.
2. Materials and methods 2.1. Morphine dependence and withdrawal Male Sprague–Dawley rats, weighing about 190 g at the beginning of chronic morphine or saline treatment, were used. In order to induce morphine dependence, morphine Žmorphine hydrochloride. treatment was carried out according to a previous study w23x. Morphine was injected daily s.c. with gradually increasing doses Ž40–160 mgrkg. for 10 days, being followed by a final dose of 40 mgrkg on the morning of the 11th day. Spontaneous and opioid antagonist-precipitated withdrawal were induced by cessation of morphine treatment and the injection of naloxone Ž5 mgrkg s.c.. 1 h after the final morphine dose, respectively. Control groups received saline instead of morphine or naloxone. Body weight loss were estimated and the animals were sacrificed 1, 2, 4, 12 h, and 1, 2 and 4 days after naloxone injection for naloxone-precipitated withdrawal and 12 h, and 1, 2, 3, 5 and 10 days after the final treatment of morphine for spontaneous withdrawal after the administration of a large dose of pentobarbital Ž600– 900 mgrkg i.p... Other morphine withdrawal signs Ždiarrhea and salivation. were estimated 2 h after naloxone injection for naloxone-precipitated withdrawal and 1 day after the final treatment of morphine for spontaneous withdrawal. The brain was rapidly removed, blocked and frozen with powdered dry ice. A total of 15 m m coronal sections were cut throughout the areas containing PVN and PAG at y208C on a cryostat, and mounted on gelatincoated glass slides. The sections were stored at y808C until used for in situ hybridization. Body weight change Ž%. was calculated as follows: wŽbody weight just before sacrificey body weight just before naloxone or the final morphine injection.rbody weight just before naloxone or the final morphine injectionx = 100. For estimation of plasma corticosterone ŽPCS. level, the trunk blood was collected into the heparinized test tube and was centrifuged at 2000 = g for 30 min at 48C. The plasma was stored at y208C until assayed. The PCS was estimated fluorometrically according to the method of Zenker and Bernstein w68x. 2.2. In situ hybridization The oligonucleotide probe, complementary to bases 322–360 of the rat PPE mRNA w66x, was labeled at the 3X end with w a- 35 SxdATP and terminal deoxynucleotidyl transferase. Specific activity of the labeled probe was 7–10 = 10 5 c.p.m.rng. In situ hybridization was performed as described previously w23x. Briefly, the sections were fixed at 48C in 4% paraformaldehyde in phosphate-buffered saline ŽPBS. and rinsed twice in PBS; they were treated with 0.25% acetic anhydrate in 0.1 M triethanolaminer0.9% saline at room temperature, dehydrated in a graded series of ethanol, dilapidated in chloroform and rinsed in absolute ethanol. A
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total of 100 m l of hybridization buffer consisting of 50% formamide, 4 = SSC ŽSSC s 0.15 M NaClr0.015 M sodium citrate, pH 7.4., 1 = Denhardt’s solution, 0.25 mgrml of tRNA, 0.4 mgrml of shared and denatured salmon sperm DNA, 10% dextran sulfate, 20 mM dithiothreitol and 5 = 10 6 c.p.m.rml of labeled oligonucleotide was applied to each slide and left overnight at 378C in a moist chamber. Following overnight hybridization, the slides were washed in 1 = SSC at room temperature, 1 = SSC and 0.5 = SSC at 558C and in 0.1 = SSC at room temperature. The slides were allowed to dry and then were exposed to Kodak XAR-5 film for 2 days. After film development, the slides were dipped in emulsion ŽKodak NTB-2, diluted 1:1 with distilled water., exposed for 10 to 14 days and then developed. The specificity of the in situ hybridization reaction for PPE mRNA were estimated. Pretreatment of the sections with RNase A Ž100 m grml. for 1 h at 378C or excess unlabeled probe overnight at 378C completely eliminated the hybridization signal. Furthermore, in the sections reacted with PPE sense probe no labelings were seen above background Ždata not shown..
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an equivalent volume of isotonic saline. The rats were put back in their home cages and sacrificed 5 h after injection. 2.4.3. Isolation Each rat was placed individually in a opaque plastic cage Ž35 = 30 = 17 cm. and kept in continuous isolation for 14 days. Control rats were housed in group of four in the same cage. The rats were sacrificed on the 14th day.
2.3. QuantitatiÕe analysis of mRNA Analysis of the autoradiographic films was performed using the Macintosh-based software program IMAGE ŽW. Rasband, National Institutes of Health, USA.. Because the signal of PPE mRNA was intense in the lateral and ventrolateral PAG, optical density in that area was estimated. Optical densities in an area of 1.0 = 0.8 mm ŽPAG. and 0.8 mm = 0.6 mm ŽPVN. were determined in the rostral PAG, caudal PAG and the PVN at levels of y6.3 mm, y8.3 mm and y1.8 mm from Bregma w46x, respectively. Measurements were performed on two and four fields per rat for the PAG and PVN, respectively. Values after subtracting background density were averaged to give an optical density for each animal. Background density was determined by averaging optical densities in five fields of the same areas of the cortex on the same sections. Optical density of each rat was expressed as percent of mean optical density of morphine naive group and optical density of each experimental group was calculated by averaging optical densities of four animals. Statistical analysis was performed using Student’s t-test. 2.4. Stress paradigms 2.4.1. Restraint Rats were introduced into a narrow perspex restrainer for 4 h. The animals were put back in their home cages and sacrificed 5 h after onset of stress. 2.4.2. Hypertonic saline Rats were stressed by a hypertonic saline Ž9% NaCl; 1.5 mlr100 g. i.p. injection, whereas control animals received
Fig. 1. Time course of naloxone-precipitated withdrawal signs. Body weight was measured and trunk blood was collected 1, 2, 4 and 12 h, and 1, 2 and 4 days Žd. after naloxone injection. Plasma corticosterone ŽPCS. was fluorometrically determined. Each value is the mean"s.e. Ž ns 4.. Differs from chronic salineqsaline, ) p- 0.05, )) p- 0.01, ))) p0.001.
224 Y. Fukunaga et al.r Molecular Brain Research 55 (1998) 221–231 Fig. 2. Bright-field photomicrograms of typical transverse sections through the rostral PAG ŽA and D., caudal PAG ŽB and E. and PVN ŽC and F. hybridized with 35 S-labeled probe complementary to PPE mRNA. A, B and C: section from chronic salineqsaline treated rat. D, E and F: section from chronic morphineqnaloxone treated rat Žnaloxone-precipitated withdrawal.. Rats were decapitated 4 h after saline or naloxone challenge.
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3. Results 3.1. Effects of naloxone on the gene expression of PPE, body weight and PCS in morphine naiÕe rats PPE mRNA expression in the PAG and PVN remained unchanged 4 h after naloxone injection in chronic salinetreated rats Ždata not shown.. A single injection of naloxone also did not affect PCS levels Žchronic saline q saline:
Fig. 4. Time course of spontaneous withdrawal signs. Body weight was measured and trunk blood was collected 0.5, 1, 2, 3, 5 and 10 days Žd. after the final morphine injection. Plasma corticosterone ŽPCS. was determined by fluorometrical method. Each value is the mean"s.e. Ž ns 4.. Differs from chronic saline, )) p- 0.01, ))) p- 0.001.
5.3 " 1.7, chronic saline q naloxone: 7.7 " 1.1 m gr100 ml.. Rats treated with either naloxone or saline has a slight body weight loss of similar magnitude Žchronic saline q saline: y0.9 " 0.3, chronic saline q naloxone: y1.6 " 0.5%. 4 h after naloxone injection. Fig. 3. Effect of naloxone-precipitated morphine withdrawal on the level of PPE mRNA in the rostral and caudal PAG and the PVN. Each value Žmean"s.e., ns 4. is expressed as percent of mean value of chronic saline treated group. Differs from chronic salineqsaline, ) p- 0.05, )) p- 0.01.
3.2. Time course of naloxone-precipitated withdrawal syndromes PCS and body weight changes during naloxone-precipitated withdrawal were shown in Fig. 1. PCS was
226 Y. Fukunaga et al.r Molecular Brain Research 55 (1998) 221–231 Fig. 5. Bright-field photomicrograms of typical transverse sections through the rostral PAG ŽA and D., caudal PAG ŽB and E. and PVN ŽC and F. hybridized with 35 S-labeled probe complementary to PPE mRNA. A, B and C: section from chronic saline treated rat. D, E and F: section from chronic morphine treated rat Žspontaneous withdrawal.. Rats were decapitated 1 day after final injection.
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increased 1 h after naloxone injection, and this increase persisted until 1 day after naloxone injection and returned to base line 2 days after naloxone injection. Naloxone injection decreased body weight of chronic morphine treated rats from 1 h after naloxone injection. The maximum body weight loss was observed 2 to 4 h after naloxone injection. Then, body weight began to increase 12 h after naloxone injection. The incidence of diarrhea and salivation are 100% 2 h after naloxone injection. 3.3. Effects of naloxone-precipitated withdrawal on the gene expression of PPE Fig. 2 showed typical bright-field photomicrograms of transverse sections through the rostral PAG, caudal PAG and PVN in naloxone-precipitated withdrawal rats. PPE mRNA was decreased in the rostral PAG and increased in the caudal PAG and PVN 4 h after naloxone injections in morphine dependent rats. Fig. 3 showed the time course of the change of PPE mRNA levels in discrete brain regions. The levels of PPE mRNA in the rostral PAG returned to control values 12 h after naloxone injection. On the other hand, the stimulation of expression of PPE mRNA in the caudal PAG was sustained 2 days after naloxone injection. PPE mRNA in the PVN was significantly increased 2 h to 2 days after naloxone injection. 3.4. Time course of spontaneous withdrawal syndromes The time course of PCS elevation and body weight loss after the final morphine administration was shown in Fig. 4. PCS was increased 12 h to 1 day after the final morphine injection, and returned to control levels 2 days after the final morphine injection. One day after the final injection body weight was increased in control rats, but decreased by about 5% of initial body weight in spontaneous withdrawal rats. Then, body weight began to increase 2 days after the final morphine injection and returned to control level 10 days after the final morphine injection. The incidence of diarrhea and salivation are 50% and 0% 1 day after the final morphine injection, respectively. 3.5. Effects of spontaneous withdrawal on the gene expression of PPE Fig. 5 showed typical photomicrograms of transverse sections through the rostral PAG, caudal PAG and PVN in spontaneous withdrawal rats. Change of PPE mRNA in the rostral PAG, caudal PAG and PVN is determined 1 day after the final morphine injection in morphine-dependent rats. PPE mRNA in the caudal PAG increased during spontaneous withdrawal, whereas PPE mRNA in the rostral PAG and PVN did not change. Fig. 6 shows the time course of PPE mRNA levels after the final morphine administration. PPE mRNA in the rostral PAG and PVN
Fig. 6. Effect of spontaneous morphine withdrawal on the level of PPE mRNA in the rostral PAG, caudal PAG and the PVN. Each value Žmean"s.e., ns 4. is expressed as percent of mean value of chronic saline treated group. Differs from chronic saline, ) p- 0.05, )) p- 0.01.
did not change 12 h to 10 days after the final morphine dose. On the other hand, PPE mRNA levels in the caudal PAG was increased up to 3 days after the final morphine injection and returned to control levels 5 days after the final morphine injection. 3.6. Effects of Õarious kinds of stressor on the gene expression of PPE Fig. 7 showed the responses of PPE mRNA in the PAG and PVN to various kinds of stressor. PPE mRNA in the
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4. Discussion
Fig. 7. Effect of restraint for 4 h, hypertonic saline i.p. injection and isolation for 14 days on the level of PPE mRNA in the rostral PAG, caudal PAG and the PVN. Rats were decapitated 5 h after onset of restraint stress or i.p. injection of hypertonic saline and 14 days after onset of isolation stress. Each value Žmean"s.e., ns 4. is expressed as percent of mean value of control group. Differs from control, ) p- 0.05, )) p- 0.01.
rostral and caudal PAG did not change, whereas PPE mRNA in the PVN was increased in response to restraint stress, hypertonic saline stress and isolation stress.
We have previously reported that expression of PPE mRNA in the caudal PAG was stimulated by both naloxone-precipitated withdrawal Ž4 h after naloxone injection. and spontaneous withdrawal Ž1 day after final morphine., while PPE mRNA in the rostral PAG was decreased and PVN was stimulated 4 h after naloxone injection, but not change 1 day after cessation of chronic morphine treatment in morphine dependent rats w23x. There is a possibility that the change of PPE mRNA in the PAG could be the nonspecific response to stress during morphine withdrawal. In the present study, it was evidenced that the expression of PPE mRNA in the PAG was not affected by various kinds of stress tested. In previous study, we assessed PPE mRNA levels at only one time point, i.e., 1 day after spontaneous morphine withdrawal. In this study, changes in PPE mRNA in the rostral PAG and PVN were not observed 12 h to 10 days after cessation of chronic morphine treatment. These results indicate that the increase of PPE mRNA in the caudal PAG is specific response to morphine withdrawal and suggest some role of endogenous enkephalins in the caudal PAG in morphine withdrawal process. Time course of increase in PPE mRNA in the caudal PAG during morphine withdrawal was compared with that of morphine withdrawal syndromes to elucidate the physiological significance of increase in PPE mRNA in the caudal PAG in morphine withdrawal process. The present studies show that PPE mRNA increased in the course of improvement of morphine withdrawal signs and returned to control value after disappearing of morphine withdrawal signs. Peptidase inhibitors microinjected into the PAG inhibited naloxone-precipitated withdrawal in morphine dependent rats w28,40x. In addition, the systemic and intracerebroventricular treatment of enkephalins reduced morphine withdrawal w4,5x. These findings suggest that increase in PPE mRNA in the caudal PAG responded to morphine withdrawal may contribute to the recovery from morphine withdrawal. There are two possibilities for increase in the expression of PPE mRNA. One possibility is that the decrease of methionine ŽMet. –enkephalin content, a result of increased release during morphine withdrawal, may drive an increment of synthesis of PPE mRNA. The increase of PPE mRNA may therefore compensate for decreased Met–enkephalin content. Another possibility is the change of a signal transduction pathway linking membrane receptors to gene expression w12x. It is reported that m-type opioid receptors in rat brain are coupled to guanine–nucleotide binding proteins ŽG proteins. w20x and inhibit adenylate cyclase activity w9,18x. Chronic morphine increased levels of adenylate cyclase in several brain regions w18,44,57x. Spontaneous withdrawal or naloxone-precipitated withdrawal leads to further change of G a mRNA and an increase in basal and stimulated adenylate cyclase activity,
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as observed in vitro Žin NG108-15 cells. w54x and in vivo Žin rat locus coeruleus. w48x. Upstream of the PPE gene includes the cAMP responsive element and its transcription can be activated by an increase of cAMP w21x. In addition, the 5X region of the PPE genes has the binding site of AP-1 which is dimer of Fos and Jun family protein w16x. Expression of PPE mRNA is regulated by Fos and Jun w55x. Fos like immunoreactivity in the PAG w14,56x, particularly dorsolateral caudal PAG w11x, was increased during morphine withdrawal. These results suggest that Fos and Jun increase during morphine withdrawal and facilitate the synthesis of PPE mRNA. The mechanism by which increased enkephalin in the PAG reduces morphine withdrawal was remained to be elucidated. There are excitatory amino acid ŽEAA. nerves in the PAG w13x. EAA antagonists have been reported to reduce morphine withdrawal symptoms w36,49,57x, suggesting that EAA might be related to the occurrence of morphine withdrawal. An electrophysiological study showed that the glutamatergic component of fast postsynaptic potentials in single PAG neurons in rat brain slices were inhibited by Met–enkephalin w10x. These reports suggest that enkephalin in the PAG might inhibit morphine withdrawal via an inhibition of EAA action. There are axo-somatic synapses from enkephalinergic axon terminals to g-aminobutyric acid ŽGABA. neurons in the PAG w60x. Therefore, there is also a possibility that enkephalin might inhibit morphine withdrawal via GABA neurons in the PAG. It has been reported that PPE mRNA in the PVN is increased during naloxone-precipitated withdrawal w29,39x and this is confirmed by the present results. In spite of the marked increase in PPE mRNA in the PVN after naloxone-precipitated withdrawal, we could not find any change of PPE mRNA in the PVN after spontaneous withdrawal. Because the expression of PPE mRNA in the PVN is facilitated by various kinds of stressor, as shown in the present results and previous reports w34,37,67x, this gene expression could nonspecifically respond to some kinds of stressor. In this study, naloxone-precipitated withdrawal symptoms were more severe than spontaneous withdrawal symptoms. These results suggest that the naloxone-precipitated withdrawal paradigm induced a sufficient strength of stress to change PPE mRNA in the PVN, whereas spontaneous withdrawal in this study might induce a milder stress. The increase of PPE mRNA in the caudal PAG was induced after both naloxone-precipitated and spontaneous morphine withdrawal and long lasting. On the other hand, PPE mRNA in the rostral PAG was reduced transiently after naloxone-precipitated morphine withdrawal and not affected by spontaneous withdrawal. The PAG is not homogeneous, not only in anatomical terms w2,8,52x, but also in functional terms w2,50,65x. The caudal PAG is more sensitive to morphine analgesia rather than the rostral PAG w50,64,65x. Further study is required to elucidate the differ-
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ence in response of caudal PAG and rostral PAG to morphine withdrawal and the role of the enkephalins in the rostral PAG in precipitated withdrawal. In conclusion, it was demonstrated in both naloxoneprecipitated and spontaneous morphine withdrawal study that PPE mRNA in the caudal PAG was not affected at the onset of morphine withdrawal syndromes but increased thereafter during improvement of morphine withdrawal, being restored when these withdrawal syndromes were disappeared. These results suggest that the increase in PPE mRNA, consequent increase in enkephalins, in the caudal PAG may attribute to the recovery from morphine withdrawal.
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