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Effect of Morphine on Proenkephalin Gene Expression in the Rat Brain
Brain Research Bulletin, Vol. 43, No. 3, pp. 349–356, 1997 Copyright q 1997 Elsevier Science Inc. Printed in the USA. All rights reserved 0361-9230/97 $17.00 / .00
PII S0361-9230(97)00019-1
Effect of Morphine on Proenkephalin Gene Expression in the Rat Brain RUSTAM Y. YUKHANANOV 1 AND ROBERT J. HANDA 2 Department of Cell Biology, Neurobiology and Anatomy, Loyola University Medical Center, 2160 S. First Ave., Maywood, IL 60153, USA [Received 20 June 1996; Revised 22 October 1996; Accepted 2 December 1996] ABSTRACT: Previous studies indicate that an acute injection of morphine does not effect the level of opioid peptides and their mRNA in the brain. However, due to the presence of a large pool of mRNA and possible opposing changes in turnover rate it is often difficult to visualize the transitory and relatively small alterations in gene transcription by examining mRNA level. Therefore, in situ hybridization with probes directed against intronic sequences to measure the primary transcript of proenkephalin (PPE) mRNA (heteronucleic RNA, hnRNA) in the rat brain following morphine administration was used in this study. The distribution of the hybridization signal of probes against both the A and B intron of the PPE gene were identical and coincide with the distribution PPE mRNA. Thus, to increase the sensitivity of this assay both probes were concurrently hybridized. Female and male Sprague–Dawley rats were gonadectomized and injected with morphine (10 mg/kg, SC). We detected no changes in PPE mRNA levels in the striatum, olfactory tubercle (OT) and n. accumbens core (NAC) at any time following morphine administration. However, from 0.5 h until 24 h following morphine injection, the levels of PPE hnRNA in NAC and OT but not in the dorsal striatum were significantly decreased. The level of c-fos mRNA was increased only in the dorsal striatum following morphine injections. These data show that morphine administration can acutely change opioid peptide gene transcription. The observed decrease of PPE hnRNA levels for 24 h following a single morphine injection may indicate its importance for the development of acute and chronic dependence. However, the significance of these alterations in PPE gene transcription in term of the acute effect of morphine is not clear, because the steady-state level of mRNA was not changed. Q 1997 Elsevier Science Inc.
lowing chronic morphine treatment, which results in a deficiency of inhibitory enkephalinergic control following narcotics withdrawal. Because the tonic activity of enkephalinergic neurons are low and injections of opioid antagonists, for example, naloxone, produce sparse or no effect on intact animals [33], this deficit may be hidden, but following stress or other activating stimuli, the deficits in enkephalinergic pathways may provoke narcotic intake by addicts. Earlier studies of opioid peptide levels in different brain areas failed to detect any changes following the development of morphine tolerance and dependence [6,8,17,34,39]. The use of more specific antisera and the focus on specific brain areas revealed altered proopiomelanocortin synthesis and processing in the pituitary and hypothalamus following morphine administration [2,11,23]. It has also been shown that the implantation of a morphine pellet decreased the levels of PPE mRNA in the striatum [37]. However, the levels of opioid peptides were unchanged in the rat rendered tolerant to morphine and during a naloxoneprecipitated withdrawal reaction [23]. The results of studies examining the effect of morphine on the levels of prodynorphin and prodynorphin-derived peptides is also contradictory. Chronic morphine administration reduces the level of prodynorphin mRNA in the striatum [30], but may increase [36] or decrease [43] the concentration of dynorphin immunoreactivity in striatum and may increase it in the spinal cord [26]. These data do not provide sufficient information to formulate a coherent conclusion concerning the role of mu, kappa, and delta opioid receptors on opioid peptide expression in the brain following chronic morphine administration. However, there were consistently no changes in the opioid peptide and PPE mRNA levels following a single morphine injection [2,6,8,11,17,23,34,39]. Similarly, changes in PPE mRNA and enkephalin immunoreactivity following treatment with dopamine antagonists or 6-OHDA lesions required at least 7 days of treatment [24,28,35]. These data can be explained by a large pool of mRNA buffering small changes in the level of transcription. There are presently two acceptable methods to detect the rate of transcription. Nuclear run-on assay, and hybridization with probes directed against intronic sequences of a gene. Run-on assay directly reflects the level of transcription but cannot be used for anatomical mapping of transcription alterations. Hybridiza-
KEY WORDS: Morphine, Proenkephalin, in situ hybridization, n. Accumbens, c-fos, Striatum, Intronic probe, Heteronuclear RNA.
INTRODUCTION The role of opioid peptides in the regulation of emotion and mood makes it feasible that deficits in enkephalinergic pathways promote the long-term craving in the morphine addict. Opioid peptide participation in the development of morphine dependence, as proposed by Kosterlitz and Huges [19], is based on the hypothetical feedback inhibition of enkephalinergic pathways fol1 2
Present address: Department of Anesthesiology, University of Wisconsin–Madison, Madison, WI 53792. To whom requests for reprints should be addressed.
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tion with intron-directed probes does not directly detect the rate of transcription, but gives information regarding the activity of transcription, because the level of heteronuclear RNA (hnRNA) is normally very low, and even small changes in the rate of transcription can dramatically alter its concentration in a short period of time [7,16]. In this study, we have used intron directed in situ hybridization histochemistry to determine the time course of the PPE gene transcription in the basal ganglia following a single morphine injection into the rat. The level of PPE hnRNA was determined using probes directed against intronic sequences, and the levels of PPE mRNA was determined using a probe directed against an exonic sequence of the PPE gene. As an indicator of rapid changes in gene expression activity, the level of c- fos mRNA was also determined. It has been shown that acute effects of morphine depend on the sex steroid milieu of the organism [25,42], and that estrogen may alter the PPE gene transcription in brain areas such as the ventromedial nucleus of hypothalamus [13,29]. Although there is little evidence for estrogen or androgen receptors in neurons of the striatum, it has been shown that castration and treatment with gonadal steroids can affect the dopaminergic activity in mesostriatal pathways [32]. Castration of male rats may alter dopamine concentration in the n. accumbens [22], increase dopamine D2 receptors in the striatum [38], and change the ratio between the isoforms of D2 receptors [12]. Thus, to exclude a potentially complex interaction of morphine and sex steroids, we have used gonadectomized male and female rats in these studies. MATERIALS AND METHODS Animals and Surgery Female and male Sprague–Dawley (250–280 g, Harlan– Sprague–Dawley Inc., Indianapolis, IN) rats were used. Animals were housed in translucent shoebox-type plastic cages in temperature and humidity-controlled rooms, which were on a 12– 12 light–dark schedule. Food and water were available ad lib. Beginning 1 week prior to morphine injections, the animals were handled daily for 5–10 min. Gonadectomy was performed under ether anesthesia 8 days prior to morphine injection. All animal protocols were approved by the IACUC at Loyola University, Chicago. Gonadectomized rat of both sexes were divided into three groups: animals in one group (untreated) were kept in their home cage prior to sacrifice, another group (vehicle) received a SC physiological saline injection (1.0 ml/kg), and third group (morphine) received a SC morphine injection (10 mg/kg). The animals were decapitated at different times (0.5, 2, 4, and 24 h) following injection. The brains were immediately removed from the skull, frozen in isopentane ( 0307C), and stored at 0707C prior to sectioning. In Situ Hybridization Histochemistry In situ hybridization histochemistry (ISHH) was performed according to protocols described elsewhere [14]. Briefly, the brains were cut at 20 mM sections with a cryostat (Leitz 1600) and sections were mounted onto Superfrost plus slides (Fisher Scientific). Sections were post fixed in 4% formaldehyde for 5 min, rinsed with 2 1 SSC (0.3 M NaCl, 0.03 M Na citrate, pH 7.0) and acetylated in 0.1 M triethanolamine containing 0.25% acetic anhydride, dehydrated in alcohol, and delipidated in chloroform. Hybridization solution (0.6 M NaCl, 10 mM Tris-HCl, 50% formamide, 10% dextran sulfate, 0.01% tRNA, 0.05% yeast RNA, 0.05% Denhardt’s solution, 0.1% sodium thiosulfate, 100
mM DTT, and 0.1% SDS, 0.01% denaturated DNA) containing 20 1 10 6 cpm/ml radiolabeled probes was incubated with sections at 447C for oligoprobes and 607C for riboprobes. Some control sections were hybridized with an excess of unlabeled probe (2001 ) or pretreated with RNAse A (50 mg/ml, 30 min at 377C) prior to hybridization. Following 24-h incubation with oligo probes, slides were washed with 50% formamide in 21 SSC at 407C. When using cRNA probes, nonhybridized riboprobe was digested with RNase A (20 mg/ml, 377C, 30 min). Following dehydration through alcohols, sections were coated with photographic emulsion (Kodak, NTB-3) and stored in the dark at 47C for 3 weeks for c- fos mRNA, 4 weeks for PPE mRNA, and 15 weeks for PPE hnRNA. Radiolabeled oligoprobes were used to detect PPE mRNAs and hnRNA, and a radiolabeled riboprobe was used to detect cfos mRNA. The oligonucleotide probe for PPE mRNA was characterized elsewhere and complimentary to the nucleotides 1472– 1520 [20] and the oligonucleotide probes for PPE hnRNA were complimentary to the nucleotides 650–697 (IntronA) or 1110– 1158 (Intron B) of the PPE gene sequence [31]. Antisense 35SATP (1000 Ci/mmol) labeled oligonucleotides were prepared by polyadenylation using TdT enzyme. Specific activity of the oligoprobes were approximately 2–3 1 10 7 cpm/pmol. The c- fos riboprobe was prepared by in vitro transcription from subcloned fragment of rat cDNA in the pGEM3Zf( 0 ) plasmid using SP6 RNA polymerase. The plasmid was linearized with RSAI and transcribed using SP6 DNA-dependent RNA polymerase in the presence of 35S-UTP (1000 Ci/mmol). The resulting c- fos cRNA probe was complimentary to nucleotide 1838–2116 of rat c- fos mRNA [14]. Completeness of transcription was determined by polyacrylamide (5% acrylamide, 7.5% urea) gel electrophoresis. Only probes more than 90% full length were used for ISHH. Specific activity of the labeled probe was approximately 3 1 10 8 cpm/ mg. Image Analysis The analysis of emulsion coated sections was performed following counter staining with cresyl violet. Sections were atlas matched [27] and the number of labeled cells and the number of grains per cells were counted using NIH image software (v 1.57). Criteria to determine a labeled cell included a grain density of greater than 31 background and the presence of a visible stained nucleus. Statistical Analysis The data were express as percent of the value observed in untreated animals to minimize variance. Transformation did not alter the normal distribution of the data. Differences in the level of c- fos and PPE mRNA and PPE hnRNA following treatment (untreated, vehicle injection, morphine injection), sex (male, female) and time (0.5, 2, 4, and 24 h) and interactions between groups were determined using three-way ANOVA followed by post hoc Sheffe’s test. RESULTS Characterization of the Probes for PPE Localization. Localization of the PPE mRNA and hnRNA were examined in untreated nongonadectomized male rats. The distributions of the PPE mRNA and hnRNA were compared on consecutive sections through the telencephalon and diencephalon (bregma from 3.7 until 04.8 [27], Fig. 1). Intense PPE mRNA
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FIG. 1. The distribution of PPE RNA in the rat brain. The left panel shows the hybridization signal using an oligonucleotide probe against PPE Intron A. The right panel shows the hybridization signal using an oligonucleotide probe against Intron B. The middle panel shows the distribution of the hybridization signal using an exonic probe for PPE mRNA. This series of photographs show an identical distribution of Intron A and B directed probes detecting hnRNA (primary transcript), which are very similar to the distribution of PPE mRNA.
labeling was observed in the caudate-putamen (CP), olfactory tubercle (OT), nucleus accumbens (NA) (Fig. 1, middle panels a, b, and c). Moderate to high labeling was also observed in hypothalamic areas such as anterior hypothalamic nucleus, ventromedial nucleus, and perifornical region (Fig. 1, middle, panels d, e, and f). Moderate labeling have been also found in the amygdala (Fig. 1, middle, panels e and f), nucleus arcuatus, and mammilary bodies (data not shown). From emulsion coated sections labeled cells were also detected in the paraventricular nucleus of the hypothalamus, in piriform, and neocortex (layers II–VI) and in the granule cells of the dentate gyrus of the hippocampus (data not shown).
The distribution of the hybridization signal for both intronic probes were identical (Fig. 1, left and right panels). The maximal signal was observed in the caudate-putamen (CP) and olfactory tubercle (OT), and less intense hybridization was seen in NA (Fig. 1, right and left a, b, c, and d). Labeled cells were observed in the perifornical region, in the anterior hypothalamic area, and amygdala. No labeled cells were seen in the cortex and hippocampus. Specificity. The specificity of c- fos–directed probe and exonic probe for PPE mRNA has been determined elsewhere [14,20]. Following hybridization with intron-directed probes, silver grains were concentrated over stained nuclei, whereas the
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YUKHANANOV AND HANDA creased number of cells was observed beginning at 0.5 h and lasted up to 24 h following morphine injection. Saline injection had no effect on the level PPE hnRNA in NAC. Similarly, in the OT, morphine injection did not change the number of grains per labeled cell, but there was a decrease in the percent of PPE hnRNA expressing cells from 2 h until 4 h after injection (Fig. 5A and B, p õ 0.05). There was also a trend toward a decrease in the percent of labeled cells at 0.5 h and 24 h following morphine injection (Fig. 5B). Saline injection had no effect on the level of PPE hnRNA in the OT. There were no changes in number of grains per cell or percent of labeled cells in the dorsal striatum following morphine injection (Fig. 6A and B). PPE mRNA level. The levels of PPE mRNA were not altered following morphine or vehicle injection in the NAC, OT, and dorsal striatum either the number of grain per cell or the percent of PPE mRNA expressing cells (data not shown). c-fos mRNA level. The levels of c- fos mRNA were increased in the dorsal striatum (Fig. 7). There were no changes in the
FIG. 2. The distribution of PPE mRNA (panel A) and hnRNA (panel B) over cells of the NA as demonstrated by in situ hybridization. This photomicrograph shows the hybridization of oligonucleotide probes to exonic (panel A) or intronic (panel B) sequences of the PPE gene over stained nuclei. Both panels are bright-field microphotographs ( 1720). Tissue sections were coated with NTB3 emulsion and exposed for 4 weeks for exonic probes, 15 weeks for intronic probes and then counterstained with cresyl violet.
exonic probe for PPE mRNA produced a more diffuse silver grain distribution (Fig. 2A and B). RNase pretreatment of the sections completely abolished the signal for mRNA and hnRNA. Addition of a 2001 excess of the unlabeled IntronA and IntronB probes eliminated specific labeling; remaining labeling was similar to that following RNase treatment and was considered background. Effect of Morphine Injection Three-way ANOVA showed that there were no sex differences in the relative levels of PPE hnRNA and mRNA or c- fos mRNA following vehicle or morphine treatment; therefore, the graphs represent the combined results from male and female rats. PPE hnRNA level. Morphine injection had no effect on the average PPE hnRNA grain level per cell in nucleus accumbens core (NAC, Fig. 3A), but decreased the number of PPE hnRNA expressing cells (Fig. 3B, Fig. 4A and B, p õ 0.01). The de-
FIG. 3. Effect of morphine or vehicle injection on the levels of PPE hnRNA in the nucleus accumbens core (NAC) as determined by in situ hybridization. The number of grains per cell (A) were counted and expressed as percent of the matched sections of the untreated untreated rats. The percent of labeled cells (B) were determined as the portion of all counted cells (150–200). Tissue sections were coated with NTB3 emulsion, exposed 16 weeks and then counterstained with cresyl violet. Each bar represents the means { SEM of six to eight animals. **p õ 0.01, *p õ 0.05 compared to untreated animals.
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353 percent of PPE hnRNA-labeled cells was less than we observed for PPE mRNA. This discrepancy in the distribution of mRNA and hnRNA may be explained by differences in the rate of transcription of the PPE gene between brain areas and a longer halflife for PPE mRNA vs. hnRNA, coupled with the lower sensitivity of the assay for hnRNA. It appears that PPE transcription is maximal in the CP and OT and minimal in the cortex and hippocampus. It should also be noted that our initial mapping experiments were accomplished in nongonadectomized male rats, whereas further studies were performed in gonadectomized male and female rats. We determined that there was no sex difference the levels of PE RNAs, and the distribution of the RNAs did not appear to change between gonadectomized and nongonadectomized rats. However, this latter point should be addressed in more detail with a separate study. We did not find any difference in the intensity of the signal when comparing Intron A or Intron B directed probes. Both probes give the uniform distribution of the optical density. This
FIG. 4. The expression of PPE hnRNA in the NAC 2 h following vehicle (A) or morphine injection (B) as demonstrated by in situ hybridization. Panels are dark-field microphotographs at 1130. Tissue sections were coated with NTB3 emulsion and exposed for 15 weeks and counterstained with cresyl violet.
percent of labeled cells, but only in the number of grains per cells at 2 h after morphine injection. There were also increases in the percent of labeled cells (Fisher’s protected least significance difference test, p õ 0.05) in both morphine and vehicle-treated rats, which probably reflects the effect of injection-induced stress (Fig. 7B). In the NAC and OT there were no changes in the level c- fos mRNA either with respect to the signal intensity per cell or the total cell number following vehicle or morphine injections (data not shown). DISCUSSION Specificity of ISHH and Localization of PPE hnRNA The distribution of PPE mRNA seen in our studies was very similar to that reported earlier [15]: the greatest levels of mRNA were detected in the CP, NA, and OT, and there was an absence of PPE mRNA in the globus pallidus. In the diencephalon, intense labeling was detected in the ventromedial nucleus, in the mammilary bodies, in the perifornical area, in the anterior hypothalamic area, and amygdala. The localization of PPE hnRNA closely followed the localization of PPE mRNA. The regions that contain the highest level of PPE mRNA—CP, OT, and NA— also had detectable levels of hnRNA. In the CP, the percent of cells labeled for PPE mRNA or hnRNA was almost identical. By film autoradiography there was no PPE hnRNA signal from the hypothalamic area; however, with emulsion coated sections we observed a few labeled cells in the perifornical area, ventromedial nucleus, and dorsal part of anterior hypothalamus. However, the
FIG. 5. Effect of morphine and vehicle injection on the levels of PPE hnRNA in olfactory tubercle (OT) as determined by in situ hybridization. The number of grains (A) were counted over labeled cells and expressed as percent of the matched sections of the untreated rats. The percent of labeled cells (B) were determined as the portion of all counted cells (150–200) and expressed as percent of the untreated rats. Tissue sections were coated with NTB3 emulsion, exposed 16 weeks, and then counter stained with cresyl violet. Each bar represents the means { SEM of six to eight animals. *p õ 0.05 compared to corresponding untreated animals.
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YUKHANANOV AND HANDA A [9]. Our probe complimentary to Intron A detects both normal and alternative spliced mRNA. Thus, using this probe we cannot verify the presence or absence of alternative transcripts in the brain. Morphine Injection The major finding of this study is that a single morphine injection can decrease PPE expression in the NAC and OT. The lowered level of PPE hnRNA in NAC was observed up to 24 h following a single morphine injection. In OT the decreased level of hnRNA was observed after 2 h and disappeared by 24 h. In OT, the higher overall level of mRNA and hnRNA may mask an earlier (0.5 h) decrease of PPE hnRNA levels. Because we detected decreases in the percent of labeled cells but not in the number of grain per cells both in NAC and OT, it is possible that morphine can turn off PPE transcription in some cells without affecting others. Analysis of individual cells revealed that there were no alterations in PPE mRNA or hnRNA levels in the dorsal striatum after morphine or vehicle injections. In contrast, we found an increase in c- fos mRNA levels in the dorsal striatum within 2 h following morphine injection. These results disagree with previous studies showing that morphine increases the level of c- fos mRNA, with greater elevation in the ventral striatum than in the
FIG. 6. Effect of morphine and vehicle injections on the levels of PPE hnRNA in dorsal striatum as determined by in situ hybridization. Tissue sections were coated with NTB3 emulsion, exposed 16 weeks, and then counterstained with cresyl violet. For further details, see Fig. 5.
fact suggests that both probes detect the transcript of the same gene. Thus, in further studies, we hybridized with both probes together to increase the signal. There are areas (e.g., cortex and hippocampus) where we never observed PPE hnRNA labeling in spite of the presence of PPE mRNA. However, there were never observations of PPE hnRNA without the presence of PPE mRNA. The signal from intron-directed probes, corresponding to primary transcript, was concentrated over stained nuclei, whereas that from the exonic probe (PPE mRNA) was distributed over the cytoplasm. RNase pretreatment and the addition of excess of unlabeled oligonucleotides abolished the signal. Thus, the similar distribution of the antisense oligo probe to exonic sequence and both antisense oligo probes to intronic sequences; the identical distribution of both intronic oligoprobes; the presence of hybridization signal following incubation with probes against intronic sequences only in those brain areas were PPE mRNA has been reported; the absence of signal following RNase treatment and coincubation with an excess of unlabeled probes, together provide evidence to conclude that the signal from the intronic oligonucleotide probes corresponds to the primary transcript of the PPE gene. An alternative splicing of the PPE gene has been reported for testis, where instead of a 1400 bp mRNA, there is a 1750 bp mRNA containing approximately 350 bp of 3 * end of the intron
FIG. 7. Effect of morphine and vehicle injection on the levels of c- fos mRNA in dorsal striatum determined by in situ hybridization. # p õ 0.05 (Fisher’s test compared to corresponding untreated animals). For further details, see Fig. 5.
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MORPHINE AND PROENKEPHALIN mRNA dorsal striatum [4,21]. This difference may be explained by different paradigms of morphine administration. In the present study, we used a single injection of morphine, whereas Liu et al. [21] administered morphine continuously. In our studies, the percent of c- fos mRNA-expressing cells was increased in NAC within 0.5 h following morphine or vehicle injections, perhaps reflecting an injection-induced stress. Morphine injection increased the level of c- fos mRNA in the dorsal striatum, whereas PPE hnRNA decreased in NAC and OT. The elevation of c- fos mRNA disappears within 2 h after injection, whereas PPE hnRNA remains low even 24 h following injection. Thus, the difference in temporal and spatial patterns of c- fos mRNA and PPE hnRNA alterations exclude the possibility that changes in PPE transcription following morphine injections are mediated by c- fos gene products. One explanation for the absence of PPE mRNA alterations following a single morphine injection may be that either the level of PPE hnRNA does not reflect real changes in transcription, or that small alterations in transcription cannot change the level of mature PPE mRNA over the short term. The latter explanation seems more reasonable, because chronic morphine administration or 6-OHDA lesion altered the levels of PPE mRNA after 5– 7 days of treatment [28,37]. Morphine appears to have long-lasting effects on PPE transcription because levels of PPE hnRNA are decreased in OT by 4 h following morphine injection and in NAC the level of PPE hnRNA was decreased as long as 24 h after morphine injection. At this time there is no detectable level of morphine in the brain, and morphine-induced analgesia is completely gone. It is unknown which regulatory factors might control this long-lasting inhibition of transcription. Additional knowledge of these factors could be extremely important for explaining morphine dependence. In the present study, we have neither determined if the effects of morphine on PPE RNA are truly opiate receptor mediated, nor have we determined the opiate receptor subtype that might mediate morphine’s effect on PPE hnRNA. These important questions should be addressed in future studies. Furthermore, we do not know whether the effect of morphine on PPE hnRNA is direct or transsynaptic. It is possible that the differential effects of morphine on PPE transcription in the NAC and OT could be through a dopaminergic mechanism. Stimulation of dopamine (DA) release by morphine from mesolimbic and mesocortical projections into in NA, OT, and medial frontal cortex, but not from the nigrostriatal projection into the dorsal striatum has been shown [1,5,41]. Indirect evidence for this is the colocalization of D2 receptor and PPE mRNA in the same spiny striatal neurons in the ventral part of striatum, including the NAC, usually designated as the limbic striatum [10]. It has been shown that inhibition of D2 receptor and subsequent increase of adenylate cyclase activity stimulates PPE mRNA levels [28] and a single morphine injection decreases the forskolin-induced elevation of PPE mRNA levels in the striatum [18]. Chronic morphine administration was found to decrease levels of the transcription factor cAMP response element-binding protein (CREB) in the NAc, but not in other brain regions [40]. Thus, activation of D2 dopamine, and the resulting inhibition of adenylate cyclase may decrease PPE transcription following a single morphine injection. Alternatively, morphine’s effect on the PPE transcription may not be mediated by D2 receptor activation but by attenuating the tonic activity of glutamatergic pathways from cortex that increase the level of the PPE mRNA in the striatum [3]. In conclusion, the observed decrease in PPE hnRNA levels in the limbic striatum following acute morphine indicates the ability of morphine to alter the levels of PPE gene transcription. Further
355 investigation of the regulatory mechanism of PPE transcription in the limbic striatum may help determine the mechanisms of long-term neurobiological changes following chronic administration of opioids. ACKNOWLEDGEMENT
This work was supported by NSF IBN 9408890 and USPHS AA 08696. This work was presented in part at the 26th INRC Meeting in Scotland, July 9–13, 1995.
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YUKHANANOV AND HANDA in rat striatum: A model for acute and chronic opioid actions in brain. Mol. Brain Res. 32:313–320; 1995. Kosterlitz, H. W.; Huges, J. Some thoughts on the significance of enkephalin, the endogenous ligand. Life Sci. 17:91–96; 1975. Lightman, S. L.; Young, W. S. III. Changes in hypothalamic preproenkephalin A mRNA following stress and opiate withdrawal. Nature 328:643–645; 1987. Liu, J.; Nickolenko, J.; Sharp, F. R. Morphine induces c- fos and jun-B in striatum and nucleus accumbens via D1 and N-methyl-Daspartate receptors. Proc. Natl. Acad. Sci. USA 91:8537–8541; 1994. Mitchell, J. B.; Stewart, J. Effects of castration, steroids replacement, and sexual experience on mesolimbic dopamine and sexual behaviors in the male rat. Brain Res. 491:116–127; 1989. Mocchetti, I.; Ritter, A.; Costa, E. Down-regulation of proopiomelanocortin synthesis and beta-endorphin utilization in hypothalamus of morphine-tolerant rats. J. Mol. Neurosci. 1:33–38; 1989. Morris, B. J.; Hunt, S. P. Proenkephalin mRNA levels in rat striatum are increased and decreased respectively, by selective D2 and D1 dopamine receptor antagonists. Neurosci. Lett. 125:201–204; 1991. Nomikos, G.; Spyraki, C.; Kazandjan, A.; Sfikakis, A. Estrogen treatment to ovariectomized rats modifies morphine induced behavior. Pharmacol. Biochem. Behav. 27:611–617; 1987. Nylander, I.; Sakurada, T.; Le Greve, P.; Terenius, L. Spinal cord level of dynorphin peptides, substance P and CGRP after subchronic morphine administration in the rat. Neuropharmacology 30:1219– 1223; 1991. Paxinos, G.; Watson, C. The rat brain in stereotaxic coordinates. Sydney: Academic Press; 1986. Pollack, A. E.; Wooten, G. F. Differential regulation of striatal preproenkephalin mRNA by D1 and D2 dopamine receptors. Mol. Brain Res. 12:111–119; 1992. Romano, G. J.; Mobbs, C. V.; Lauber, A.; Howels, R. D.; Pfaff, D. W. Differential regulation of proenkephalin gene expression by estrogen in the ventromedial hypothalamus of male and female rats: Implication for the molecular basis of sexually differentiated behavior. Brain Res. 536:63–68; 1990. Romualdi, P.; Lesa, G.; Ferri, S. Chronic opiate agonists down regulate prodynorphin gene expression in rat brain. Brain Res. 563:132–136; 1991.
31. Rosen, H.; Douglass, J.; Herbert, E. Isolation and characterization of the rat proenkephalin gene. J. Biol. Chem. 259:14309–14313; 1984. 32. Ryan–Jastrow, T.; Gnegy, M. E. Castration blocks chronic sulpiride-induced desensitization of striatal D1 receptor-stimulated adenylate cyclase activity in male rats. J. Pharmacol. Exp. Ther. 248:626–631; 1989. 33. Sawynok, J.; Pinsky, G.; LaBella, F. S. Minireview on the specificity of naloxone as an opiate antagonist. Life Sci. 25:1621–1632; 1979. 34. Shani, J.; Azov, R.; Weisman, B. A. Enkephalin level in rat brain after various regimen of morphine administration. Neurosci. Lett. 12:319–322; 1979. 35. Tang, F.; Costa, E.; Schwarz, J. P. Increases in proenkephalin mRNA and enkephalin content of the rat striatum after daily injection of haloperidol for 2 to 3 weeks. Proc. Natl. Acad. Sci. USA 80:3841– 3844; 1983. 36. Trujillo, K. A.; Akil, H. Pharmacological regulation of striatal prodynorphin peptides. Prog. Clin. Biol. Res. 328:223–226; 1990. 37. Uhl, G. R.; Ryan, J. P.; Schwartz, J. P. Morphine alters preproenkephalin gene expression. Brain Res. 459:391–397; 1988. 38. Watanabe, H.; Matsumoto, K.; Imamura, L.; Suzuki, Y. Castration increases striatal D-2 dopamine receptors in mid-life rats. Jpn. J. Pharmacol. 50:79–81; 1989. 39. Wesche, D.; Ho¨llt, V.; Herz, A. Radioimmunoassay of enkephalins. Naunyn Schmiedeberg’s Arch. Pharmacol. 301:79–82; 1977. 40. Widnell, K. L.; Self, D. W.; Lane, S. B.; Russell, D. S.; Vaidya, V. A.; Miserendino, M. J.; Rubin, C. S.; Duman, R. S.; 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. 276:306–315; 1996. 41. Wood, P. L.; Rao, T. S. Morphine stimulation of mesolimbic and mesocortical but not nigrostriatal; Dopamine release in the rat as reflected by changes in 3-methoxytyramine levels. Neuropharmacology 30:399–401; 1991. 42. Yukhananov, R. Y.; Handa, R. J. Estrogen prolongs morphine analgesia in the rat. Soc. Neurosci. Abstr. 20:135; 1994. 43. Yukhananov, R. Y.; Zhai, Z. Q.; Persson, S.; Post, C.; Nyberg, F. Chronic morphine decreases dynorphin (1–17) level in the rat n. accumbens. Neuropharmacology 32:703–709; 1993.
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Effect of Morphine on Proenkephalin Gene Expression in the Rat Brain RUSTAM Y. YUKHANANOV 1 AND ROBERT J. HANDA 2 Department of Cell Biology, Neurobiology and Anatomy, Loyola University Medical Center, 2160 S. First Ave., Maywood, IL 60153, USA [Received 20 June 1996; Revised 22 October 1996; Accepted 2 December 1996] ABSTRACT: Previous studies indicate that an acute injection of morphine does not effect the level of opioid peptides and their mRNA in the brain. However, due to the presence of a large pool of mRNA and possible opposing changes in turnover rate it is often difficult to visualize the transitory and relatively small alterations in gene transcription by examining mRNA level. Therefore, in situ hybridization with probes directed against intronic sequences to measure the primary transcript of proenkephalin (PPE) mRNA (heteronucleic RNA, hnRNA) in the rat brain following morphine administration was used in this study. The distribution of the hybridization signal of probes against both the A and B intron of the PPE gene were identical and coincide with the distribution PPE mRNA. Thus, to increase the sensitivity of this assay both probes were concurrently hybridized. Female and male Sprague–Dawley rats were gonadectomized and injected with morphine (10 mg/kg, SC). We detected no changes in PPE mRNA levels in the striatum, olfactory tubercle (OT) and n. accumbens core (NAC) at any time following morphine administration. However, from 0.5 h until 24 h following morphine injection, the levels of PPE hnRNA in NAC and OT but not in the dorsal striatum were significantly decreased. The level of c-fos mRNA was increased only in the dorsal striatum following morphine injections. These data show that morphine administration can acutely change opioid peptide gene transcription. The observed decrease of PPE hnRNA levels for 24 h following a single morphine injection may indicate its importance for the development of acute and chronic dependence. However, the significance of these alterations in PPE gene transcription in term of the acute effect of morphine is not clear, because the steady-state level of mRNA was not changed. Q 1997 Elsevier Science Inc.
lowing chronic morphine treatment, which results in a deficiency of inhibitory enkephalinergic control following narcotics withdrawal. Because the tonic activity of enkephalinergic neurons are low and injections of opioid antagonists, for example, naloxone, produce sparse or no effect on intact animals [33], this deficit may be hidden, but following stress or other activating stimuli, the deficits in enkephalinergic pathways may provoke narcotic intake by addicts. Earlier studies of opioid peptide levels in different brain areas failed to detect any changes following the development of morphine tolerance and dependence [6,8,17,34,39]. The use of more specific antisera and the focus on specific brain areas revealed altered proopiomelanocortin synthesis and processing in the pituitary and hypothalamus following morphine administration [2,11,23]. It has also been shown that the implantation of a morphine pellet decreased the levels of PPE mRNA in the striatum [37]. However, the levels of opioid peptides were unchanged in the rat rendered tolerant to morphine and during a naloxoneprecipitated withdrawal reaction [23]. The results of studies examining the effect of morphine on the levels of prodynorphin and prodynorphin-derived peptides is also contradictory. Chronic morphine administration reduces the level of prodynorphin mRNA in the striatum [30], but may increase [36] or decrease [43] the concentration of dynorphin immunoreactivity in striatum and may increase it in the spinal cord [26]. These data do not provide sufficient information to formulate a coherent conclusion concerning the role of mu, kappa, and delta opioid receptors on opioid peptide expression in the brain following chronic morphine administration. However, there were consistently no changes in the opioid peptide and PPE mRNA levels following a single morphine injection [2,6,8,11,17,23,34,39]. Similarly, changes in PPE mRNA and enkephalin immunoreactivity following treatment with dopamine antagonists or 6-OHDA lesions required at least 7 days of treatment [24,28,35]. These data can be explained by a large pool of mRNA buffering small changes in the level of transcription. There are presently two acceptable methods to detect the rate of transcription. Nuclear run-on assay, and hybridization with probes directed against intronic sequences of a gene. Run-on assay directly reflects the level of transcription but cannot be used for anatomical mapping of transcription alterations. Hybridiza-
KEY WORDS: Morphine, Proenkephalin, in situ hybridization, n. Accumbens, c-fos, Striatum, Intronic probe, Heteronuclear RNA.
INTRODUCTION The role of opioid peptides in the regulation of emotion and mood makes it feasible that deficits in enkephalinergic pathways promote the long-term craving in the morphine addict. Opioid peptide participation in the development of morphine dependence, as proposed by Kosterlitz and Huges [19], is based on the hypothetical feedback inhibition of enkephalinergic pathways fol1 2
Present address: Department of Anesthesiology, University of Wisconsin–Madison, Madison, WI 53792. To whom requests for reprints should be addressed.
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tion with intron-directed probes does not directly detect the rate of transcription, but gives information regarding the activity of transcription, because the level of heteronuclear RNA (hnRNA) is normally very low, and even small changes in the rate of transcription can dramatically alter its concentration in a short period of time [7,16]. In this study, we have used intron directed in situ hybridization histochemistry to determine the time course of the PPE gene transcription in the basal ganglia following a single morphine injection into the rat. The level of PPE hnRNA was determined using probes directed against intronic sequences, and the levels of PPE mRNA was determined using a probe directed against an exonic sequence of the PPE gene. As an indicator of rapid changes in gene expression activity, the level of c- fos mRNA was also determined. It has been shown that acute effects of morphine depend on the sex steroid milieu of the organism [25,42], and that estrogen may alter the PPE gene transcription in brain areas such as the ventromedial nucleus of hypothalamus [13,29]. Although there is little evidence for estrogen or androgen receptors in neurons of the striatum, it has been shown that castration and treatment with gonadal steroids can affect the dopaminergic activity in mesostriatal pathways [32]. Castration of male rats may alter dopamine concentration in the n. accumbens [22], increase dopamine D2 receptors in the striatum [38], and change the ratio between the isoforms of D2 receptors [12]. Thus, to exclude a potentially complex interaction of morphine and sex steroids, we have used gonadectomized male and female rats in these studies. MATERIALS AND METHODS Animals and Surgery Female and male Sprague–Dawley (250–280 g, Harlan– Sprague–Dawley Inc., Indianapolis, IN) rats were used. Animals were housed in translucent shoebox-type plastic cages in temperature and humidity-controlled rooms, which were on a 12– 12 light–dark schedule. Food and water were available ad lib. Beginning 1 week prior to morphine injections, the animals were handled daily for 5–10 min. Gonadectomy was performed under ether anesthesia 8 days prior to morphine injection. All animal protocols were approved by the IACUC at Loyola University, Chicago. Gonadectomized rat of both sexes were divided into three groups: animals in one group (untreated) were kept in their home cage prior to sacrifice, another group (vehicle) received a SC physiological saline injection (1.0 ml/kg), and third group (morphine) received a SC morphine injection (10 mg/kg). The animals were decapitated at different times (0.5, 2, 4, and 24 h) following injection. The brains were immediately removed from the skull, frozen in isopentane ( 0307C), and stored at 0707C prior to sectioning. In Situ Hybridization Histochemistry In situ hybridization histochemistry (ISHH) was performed according to protocols described elsewhere [14]. Briefly, the brains were cut at 20 mM sections with a cryostat (Leitz 1600) and sections were mounted onto Superfrost plus slides (Fisher Scientific). Sections were post fixed in 4% formaldehyde for 5 min, rinsed with 2 1 SSC (0.3 M NaCl, 0.03 M Na citrate, pH 7.0) and acetylated in 0.1 M triethanolamine containing 0.25% acetic anhydride, dehydrated in alcohol, and delipidated in chloroform. Hybridization solution (0.6 M NaCl, 10 mM Tris-HCl, 50% formamide, 10% dextran sulfate, 0.01% tRNA, 0.05% yeast RNA, 0.05% Denhardt’s solution, 0.1% sodium thiosulfate, 100
mM DTT, and 0.1% SDS, 0.01% denaturated DNA) containing 20 1 10 6 cpm/ml radiolabeled probes was incubated with sections at 447C for oligoprobes and 607C for riboprobes. Some control sections were hybridized with an excess of unlabeled probe (2001 ) or pretreated with RNAse A (50 mg/ml, 30 min at 377C) prior to hybridization. Following 24-h incubation with oligo probes, slides were washed with 50% formamide in 21 SSC at 407C. When using cRNA probes, nonhybridized riboprobe was digested with RNase A (20 mg/ml, 377C, 30 min). Following dehydration through alcohols, sections were coated with photographic emulsion (Kodak, NTB-3) and stored in the dark at 47C for 3 weeks for c- fos mRNA, 4 weeks for PPE mRNA, and 15 weeks for PPE hnRNA. Radiolabeled oligoprobes were used to detect PPE mRNAs and hnRNA, and a radiolabeled riboprobe was used to detect cfos mRNA. The oligonucleotide probe for PPE mRNA was characterized elsewhere and complimentary to the nucleotides 1472– 1520 [20] and the oligonucleotide probes for PPE hnRNA were complimentary to the nucleotides 650–697 (IntronA) or 1110– 1158 (Intron B) of the PPE gene sequence [31]. Antisense 35SATP (1000 Ci/mmol) labeled oligonucleotides were prepared by polyadenylation using TdT enzyme. Specific activity of the oligoprobes were approximately 2–3 1 10 7 cpm/pmol. The c- fos riboprobe was prepared by in vitro transcription from subcloned fragment of rat cDNA in the pGEM3Zf( 0 ) plasmid using SP6 RNA polymerase. The plasmid was linearized with RSAI and transcribed using SP6 DNA-dependent RNA polymerase in the presence of 35S-UTP (1000 Ci/mmol). The resulting c- fos cRNA probe was complimentary to nucleotide 1838–2116 of rat c- fos mRNA [14]. Completeness of transcription was determined by polyacrylamide (5% acrylamide, 7.5% urea) gel electrophoresis. Only probes more than 90% full length were used for ISHH. Specific activity of the labeled probe was approximately 3 1 10 8 cpm/ mg. Image Analysis The analysis of emulsion coated sections was performed following counter staining with cresyl violet. Sections were atlas matched [27] and the number of labeled cells and the number of grains per cells were counted using NIH image software (v 1.57). Criteria to determine a labeled cell included a grain density of greater than 31 background and the presence of a visible stained nucleus. Statistical Analysis The data were express as percent of the value observed in untreated animals to minimize variance. Transformation did not alter the normal distribution of the data. Differences in the level of c- fos and PPE mRNA and PPE hnRNA following treatment (untreated, vehicle injection, morphine injection), sex (male, female) and time (0.5, 2, 4, and 24 h) and interactions between groups were determined using three-way ANOVA followed by post hoc Sheffe’s test. RESULTS Characterization of the Probes for PPE Localization. Localization of the PPE mRNA and hnRNA were examined in untreated nongonadectomized male rats. The distributions of the PPE mRNA and hnRNA were compared on consecutive sections through the telencephalon and diencephalon (bregma from 3.7 until 04.8 [27], Fig. 1). Intense PPE mRNA
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FIG. 1. The distribution of PPE RNA in the rat brain. The left panel shows the hybridization signal using an oligonucleotide probe against PPE Intron A. The right panel shows the hybridization signal using an oligonucleotide probe against Intron B. The middle panel shows the distribution of the hybridization signal using an exonic probe for PPE mRNA. This series of photographs show an identical distribution of Intron A and B directed probes detecting hnRNA (primary transcript), which are very similar to the distribution of PPE mRNA.
labeling was observed in the caudate-putamen (CP), olfactory tubercle (OT), nucleus accumbens (NA) (Fig. 1, middle panels a, b, and c). Moderate to high labeling was also observed in hypothalamic areas such as anterior hypothalamic nucleus, ventromedial nucleus, and perifornical region (Fig. 1, middle, panels d, e, and f). Moderate labeling have been also found in the amygdala (Fig. 1, middle, panels e and f), nucleus arcuatus, and mammilary bodies (data not shown). From emulsion coated sections labeled cells were also detected in the paraventricular nucleus of the hypothalamus, in piriform, and neocortex (layers II–VI) and in the granule cells of the dentate gyrus of the hippocampus (data not shown).
The distribution of the hybridization signal for both intronic probes were identical (Fig. 1, left and right panels). The maximal signal was observed in the caudate-putamen (CP) and olfactory tubercle (OT), and less intense hybridization was seen in NA (Fig. 1, right and left a, b, c, and d). Labeled cells were observed in the perifornical region, in the anterior hypothalamic area, and amygdala. No labeled cells were seen in the cortex and hippocampus. Specificity. The specificity of c- fos–directed probe and exonic probe for PPE mRNA has been determined elsewhere [14,20]. Following hybridization with intron-directed probes, silver grains were concentrated over stained nuclei, whereas the
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YUKHANANOV AND HANDA creased number of cells was observed beginning at 0.5 h and lasted up to 24 h following morphine injection. Saline injection had no effect on the level PPE hnRNA in NAC. Similarly, in the OT, morphine injection did not change the number of grains per labeled cell, but there was a decrease in the percent of PPE hnRNA expressing cells from 2 h until 4 h after injection (Fig. 5A and B, p õ 0.05). There was also a trend toward a decrease in the percent of labeled cells at 0.5 h and 24 h following morphine injection (Fig. 5B). Saline injection had no effect on the level of PPE hnRNA in the OT. There were no changes in number of grains per cell or percent of labeled cells in the dorsal striatum following morphine injection (Fig. 6A and B). PPE mRNA level. The levels of PPE mRNA were not altered following morphine or vehicle injection in the NAC, OT, and dorsal striatum either the number of grain per cell or the percent of PPE mRNA expressing cells (data not shown). c-fos mRNA level. The levels of c- fos mRNA were increased in the dorsal striatum (Fig. 7). There were no changes in the
FIG. 2. The distribution of PPE mRNA (panel A) and hnRNA (panel B) over cells of the NA as demonstrated by in situ hybridization. This photomicrograph shows the hybridization of oligonucleotide probes to exonic (panel A) or intronic (panel B) sequences of the PPE gene over stained nuclei. Both panels are bright-field microphotographs ( 1720). Tissue sections were coated with NTB3 emulsion and exposed for 4 weeks for exonic probes, 15 weeks for intronic probes and then counterstained with cresyl violet.
exonic probe for PPE mRNA produced a more diffuse silver grain distribution (Fig. 2A and B). RNase pretreatment of the sections completely abolished the signal for mRNA and hnRNA. Addition of a 2001 excess of the unlabeled IntronA and IntronB probes eliminated specific labeling; remaining labeling was similar to that following RNase treatment and was considered background. Effect of Morphine Injection Three-way ANOVA showed that there were no sex differences in the relative levels of PPE hnRNA and mRNA or c- fos mRNA following vehicle or morphine treatment; therefore, the graphs represent the combined results from male and female rats. PPE hnRNA level. Morphine injection had no effect on the average PPE hnRNA grain level per cell in nucleus accumbens core (NAC, Fig. 3A), but decreased the number of PPE hnRNA expressing cells (Fig. 3B, Fig. 4A and B, p õ 0.01). The de-
FIG. 3. Effect of morphine or vehicle injection on the levels of PPE hnRNA in the nucleus accumbens core (NAC) as determined by in situ hybridization. The number of grains per cell (A) were counted and expressed as percent of the matched sections of the untreated untreated rats. The percent of labeled cells (B) were determined as the portion of all counted cells (150–200). Tissue sections were coated with NTB3 emulsion, exposed 16 weeks and then counterstained with cresyl violet. Each bar represents the means { SEM of six to eight animals. **p õ 0.01, *p õ 0.05 compared to untreated animals.
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353 percent of PPE hnRNA-labeled cells was less than we observed for PPE mRNA. This discrepancy in the distribution of mRNA and hnRNA may be explained by differences in the rate of transcription of the PPE gene between brain areas and a longer halflife for PPE mRNA vs. hnRNA, coupled with the lower sensitivity of the assay for hnRNA. It appears that PPE transcription is maximal in the CP and OT and minimal in the cortex and hippocampus. It should also be noted that our initial mapping experiments were accomplished in nongonadectomized male rats, whereas further studies were performed in gonadectomized male and female rats. We determined that there was no sex difference the levels of PE RNAs, and the distribution of the RNAs did not appear to change between gonadectomized and nongonadectomized rats. However, this latter point should be addressed in more detail with a separate study. We did not find any difference in the intensity of the signal when comparing Intron A or Intron B directed probes. Both probes give the uniform distribution of the optical density. This
FIG. 4. The expression of PPE hnRNA in the NAC 2 h following vehicle (A) or morphine injection (B) as demonstrated by in situ hybridization. Panels are dark-field microphotographs at 1130. Tissue sections were coated with NTB3 emulsion and exposed for 15 weeks and counterstained with cresyl violet.
percent of labeled cells, but only in the number of grains per cells at 2 h after morphine injection. There were also increases in the percent of labeled cells (Fisher’s protected least significance difference test, p õ 0.05) in both morphine and vehicle-treated rats, which probably reflects the effect of injection-induced stress (Fig. 7B). In the NAC and OT there were no changes in the level c- fos mRNA either with respect to the signal intensity per cell or the total cell number following vehicle or morphine injections (data not shown). DISCUSSION Specificity of ISHH and Localization of PPE hnRNA The distribution of PPE mRNA seen in our studies was very similar to that reported earlier [15]: the greatest levels of mRNA were detected in the CP, NA, and OT, and there was an absence of PPE mRNA in the globus pallidus. In the diencephalon, intense labeling was detected in the ventromedial nucleus, in the mammilary bodies, in the perifornical area, in the anterior hypothalamic area, and amygdala. The localization of PPE hnRNA closely followed the localization of PPE mRNA. The regions that contain the highest level of PPE mRNA—CP, OT, and NA— also had detectable levels of hnRNA. In the CP, the percent of cells labeled for PPE mRNA or hnRNA was almost identical. By film autoradiography there was no PPE hnRNA signal from the hypothalamic area; however, with emulsion coated sections we observed a few labeled cells in the perifornical area, ventromedial nucleus, and dorsal part of anterior hypothalamus. However, the
FIG. 5. Effect of morphine and vehicle injection on the levels of PPE hnRNA in olfactory tubercle (OT) as determined by in situ hybridization. The number of grains (A) were counted over labeled cells and expressed as percent of the matched sections of the untreated rats. The percent of labeled cells (B) were determined as the portion of all counted cells (150–200) and expressed as percent of the untreated rats. Tissue sections were coated with NTB3 emulsion, exposed 16 weeks, and then counter stained with cresyl violet. Each bar represents the means { SEM of six to eight animals. *p õ 0.05 compared to corresponding untreated animals.
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YUKHANANOV AND HANDA A [9]. Our probe complimentary to Intron A detects both normal and alternative spliced mRNA. Thus, using this probe we cannot verify the presence or absence of alternative transcripts in the brain. Morphine Injection The major finding of this study is that a single morphine injection can decrease PPE expression in the NAC and OT. The lowered level of PPE hnRNA in NAC was observed up to 24 h following a single morphine injection. In OT the decreased level of hnRNA was observed after 2 h and disappeared by 24 h. In OT, the higher overall level of mRNA and hnRNA may mask an earlier (0.5 h) decrease of PPE hnRNA levels. Because we detected decreases in the percent of labeled cells but not in the number of grain per cells both in NAC and OT, it is possible that morphine can turn off PPE transcription in some cells without affecting others. Analysis of individual cells revealed that there were no alterations in PPE mRNA or hnRNA levels in the dorsal striatum after morphine or vehicle injections. In contrast, we found an increase in c- fos mRNA levels in the dorsal striatum within 2 h following morphine injection. These results disagree with previous studies showing that morphine increases the level of c- fos mRNA, with greater elevation in the ventral striatum than in the
FIG. 6. Effect of morphine and vehicle injections on the levels of PPE hnRNA in dorsal striatum as determined by in situ hybridization. Tissue sections were coated with NTB3 emulsion, exposed 16 weeks, and then counterstained with cresyl violet. For further details, see Fig. 5.
fact suggests that both probes detect the transcript of the same gene. Thus, in further studies, we hybridized with both probes together to increase the signal. There are areas (e.g., cortex and hippocampus) where we never observed PPE hnRNA labeling in spite of the presence of PPE mRNA. However, there were never observations of PPE hnRNA without the presence of PPE mRNA. The signal from intron-directed probes, corresponding to primary transcript, was concentrated over stained nuclei, whereas that from the exonic probe (PPE mRNA) was distributed over the cytoplasm. RNase pretreatment and the addition of excess of unlabeled oligonucleotides abolished the signal. Thus, the similar distribution of the antisense oligo probe to exonic sequence and both antisense oligo probes to intronic sequences; the identical distribution of both intronic oligoprobes; the presence of hybridization signal following incubation with probes against intronic sequences only in those brain areas were PPE mRNA has been reported; the absence of signal following RNase treatment and coincubation with an excess of unlabeled probes, together provide evidence to conclude that the signal from the intronic oligonucleotide probes corresponds to the primary transcript of the PPE gene. An alternative splicing of the PPE gene has been reported for testis, where instead of a 1400 bp mRNA, there is a 1750 bp mRNA containing approximately 350 bp of 3 * end of the intron
FIG. 7. Effect of morphine and vehicle injection on the levels of c- fos mRNA in dorsal striatum determined by in situ hybridization. # p õ 0.05 (Fisher’s test compared to corresponding untreated animals). For further details, see Fig. 5.
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MORPHINE AND PROENKEPHALIN mRNA dorsal striatum [4,21]. This difference may be explained by different paradigms of morphine administration. In the present study, we used a single injection of morphine, whereas Liu et al. [21] administered morphine continuously. In our studies, the percent of c- fos mRNA-expressing cells was increased in NAC within 0.5 h following morphine or vehicle injections, perhaps reflecting an injection-induced stress. Morphine injection increased the level of c- fos mRNA in the dorsal striatum, whereas PPE hnRNA decreased in NAC and OT. The elevation of c- fos mRNA disappears within 2 h after injection, whereas PPE hnRNA remains low even 24 h following injection. Thus, the difference in temporal and spatial patterns of c- fos mRNA and PPE hnRNA alterations exclude the possibility that changes in PPE transcription following morphine injections are mediated by c- fos gene products. One explanation for the absence of PPE mRNA alterations following a single morphine injection may be that either the level of PPE hnRNA does not reflect real changes in transcription, or that small alterations in transcription cannot change the level of mature PPE mRNA over the short term. The latter explanation seems more reasonable, because chronic morphine administration or 6-OHDA lesion altered the levels of PPE mRNA after 5– 7 days of treatment [28,37]. Morphine appears to have long-lasting effects on PPE transcription because levels of PPE hnRNA are decreased in OT by 4 h following morphine injection and in NAC the level of PPE hnRNA was decreased as long as 24 h after morphine injection. At this time there is no detectable level of morphine in the brain, and morphine-induced analgesia is completely gone. It is unknown which regulatory factors might control this long-lasting inhibition of transcription. Additional knowledge of these factors could be extremely important for explaining morphine dependence. In the present study, we have neither determined if the effects of morphine on PPE RNA are truly opiate receptor mediated, nor have we determined the opiate receptor subtype that might mediate morphine’s effect on PPE hnRNA. These important questions should be addressed in future studies. Furthermore, we do not know whether the effect of morphine on PPE hnRNA is direct or transsynaptic. It is possible that the differential effects of morphine on PPE transcription in the NAC and OT could be through a dopaminergic mechanism. Stimulation of dopamine (DA) release by morphine from mesolimbic and mesocortical projections into in NA, OT, and medial frontal cortex, but not from the nigrostriatal projection into the dorsal striatum has been shown [1,5,41]. Indirect evidence for this is the colocalization of D2 receptor and PPE mRNA in the same spiny striatal neurons in the ventral part of striatum, including the NAC, usually designated as the limbic striatum [10]. It has been shown that inhibition of D2 receptor and subsequent increase of adenylate cyclase activity stimulates PPE mRNA levels [28] and a single morphine injection decreases the forskolin-induced elevation of PPE mRNA levels in the striatum [18]. Chronic morphine administration was found to decrease levels of the transcription factor cAMP response element-binding protein (CREB) in the NAc, but not in other brain regions [40]. Thus, activation of D2 dopamine, and the resulting inhibition of adenylate cyclase may decrease PPE transcription following a single morphine injection. Alternatively, morphine’s effect on the PPE transcription may not be mediated by D2 receptor activation but by attenuating the tonic activity of glutamatergic pathways from cortex that increase the level of the PPE mRNA in the striatum [3]. In conclusion, the observed decrease in PPE hnRNA levels in the limbic striatum following acute morphine indicates the ability of morphine to alter the levels of PPE gene transcription. Further
355 investigation of the regulatory mechanism of PPE transcription in the limbic striatum may help determine the mechanisms of long-term neurobiological changes following chronic administration of opioids. ACKNOWLEDGEMENT
This work was supported by NSF IBN 9408890 and USPHS AA 08696. This work was presented in part at the 26th INRC Meeting in Scotland, July 9–13, 1995.
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