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Modulation of preproenkephalin mRNA levels in brain regions and spinal cord of rats treated chronically with morphine
Peptides, Vol. 16, No. 3, pp. 415-419, 1995 Copyright 6 1995 Ekvier Science Ltd Printed in the USA. All rights reserved 0196978149.5 $9.50 + .OU
Pergamon
0196-9%1(94)00199-5
Modulation of Preproenkephalin mRNA Levels in Brain Regions and Spinal Cord of Rats Treated Chronically With Morphine KRISHNAMURTHY
P. GUDEHITHLU
AND
HEMENDRA
N. BHARGAVA’
Department of Pharmaceutics and Pharmacodynamics (M/C 865), The University of Illinois at Chicago, Health Sciences Center, 833 South Wood Street, Chicago, IL 60612 Received 15 August
1994
GUDEHITHLU, K. P. AND H. N. BHARGAVA. Modularion of preproenkephalin mRNA levels in brain regions and spinal cord of rats treated chronically with morphine. PEPTIDES 16(3) 4 15 -4 19, 1995.-The effect of morphine tolerance/dependence and abstinence on the preproenkephalin (PPE) gene expression was determined in brain regions and spinal cord of the rat. Male Sprague-Dawley rats were rendered tolerant and physically dependent on morphine by SC implantation of six pellets, each containing 75 mg of morphine base, during a 7-day period. Placebo pellet-implanted rats served as controls. In tolerant rats, the pellets were left in place at the time of sacrifice whereas in abstinent rats, the pellets were removed 16 h prior to sacrificing. The levels of PPE mRNA were determined in brain regions (striatum, cortex, pons-medulla, hypothalamus, amygdala, and midbrain) and spinal cord. The levels of PPE mRNA increased significantly in the cortex (62%) and the spinal cord (352%) of morphinetolerant rats when compared to placebo pellet-implanted control rats. In other brain regions, the levels of PPE mRNA in placebo and morphine-tolerant rats did not differ. On the other hand, in morphine-abstinent rats, the levels of PPE mRNA increased in the striatum (62%) and hypothalamus (34%) but were decreased in pons-medulla (68%). midbrain (51%), and spinal cord (36%) in comparison to the placebo controls. The results clearly demonstrate differential changes in enkephalin gene expression in brain regions and spinal cord of the abstinent and nonabstinent morphine-tolerant/dependent rats. Morphine Rat
tolerance
Morphine
abstinence
PPE mRNA
Brain regions
for reprints
should be addressed
to Dr. Hemendra
Differential
change
levels of methionine-enkephalin in the spinal cord, amygdala, pons-medulla, midbrain, cortex, and striatum, and naloxone-induced withdrawal resulted in decreases in the level of methionine-enkephalin in the amygdala but increases in the hypothalamus and striatum (12). The mechanism by which modification in the levels of enkephalin are produced in morphine-tolerant and abstinent rodents is not clear. It could involve synthesis, release, and degradation of enkephalins. Neurons in the brain and spinal cord can regulate their contents of the opioid peptides and their respective encoding mRNA. PPE mRNA levels in these neurons are regulated by excitatory inputs, blockade, or removal of inhibitory inputs (10,16). Attempts have been made to determine the levels of PPE mRNA in brain tissue of rats treated chronically with morphine. In one study, chronic treatment with morphine did not alter the distribution of enkephalin mRNA in the hypothalamus. However, naloxone-precipitated withdrawal resulted in a massive and extremely rapid accumulation of enkephalin mRNA in cells of the parvocellular nucleus within 2 h, and the level remained high at 24 h (13). In another study, levels of PPE mRNA were reported to be decreased in the striatum of both nonabstinent and abstinent
IN spite of considerable efforts, the mechanisms in the various aspects of opioid addiction. namely tolerance, physical dependence, abstinence syndrome, and self-administration behavior, are far from being clear. Diverse molecules with known or unknown mechanisms appear to modify these processes (2). Since the discovery of opioid peptides in mammalian tissues, attempts have been made to determine their role in opioid tolerance/dependence and abstinence processes. The levels of enkephalins, dynorphins, and /?-endorphin in various tissues of rats treated chronically with morphine have been determined (12,15). Earlier studies failed to find any alterations in the brain enkephalin levels (6,9,18) in rats treated with morphine for 10 days. On the other hand, implantation of 27 morphine pellets during a 36-day period reduced the levels of enkephalins and ,B-endorphin in the brain and pituitary of the rat (14). The level of methionine-enkephalin was found to be decreased in the hippocampus and putamen of monkeys treated with morphine for 10 days (8). Studies in this laboratory have demonstrated that in the rat, morphine tolerance was associated with decreases in the levels of methionine-enkephalin in the pons-medulla, amygdala, and hippocampus. On the other hand, abrupt abstinence from morphine decreased the
’ Requests
Spinal cord
N. Bhargava.
415
416
GUDEHITHLU
morphine-tolerant/dependent rats (17). Thus, the two brain regions appeared to show differential processing of enkephalins in rats treated chronically with morphine. Little is known about the effect of exogenous opioid drugs on the molecular changes in the functioning of endogenous brain and spinal cord opioid peptide systems. Because our previous studies showed that methionine-enkephalin levels are decreased in various brain regions and spinal cord of morphine-tolerant and abstinent rats but naloxone-induced withdrawal resulted in increases in hypothalamic and striatal methionine-enkephalin levels (12), the present studies were undertaken to determine the levels of PPE mRNA in several brain regions and spinal cord of morphine-tolerant and abstinent rats. METHOD
Animals Male Sprague-Dawley rats weighing 250-275 g (Sasco King Animal Co, Oregon, WI) were housed three to a cage in a room with controlled ambient temperature (23 + l”C), humidity (50 + lo%), and a 12-h dark-light cycle (0600 to 1800 h). The animals were housed under these conditions for at least 4 days prior to being used. Food and water were made available continuously. Drugs Morphine and placebo pellets were supplied by the National Institute on Drug Abuse (Rockville, MD) through the courtesy of Drs. Rao S. Rapaka and Paul S. Hillery. Induction Morphine
ofTolerance to and Physical Dependence on
Rats were rendered tolerant to and physically dependent on morphine by SC implantation of six morphine pellets as described previously (45). Each pellet contained 75 mg of morphine free base. Rats that served as controls were implanted with six placebo pellets. The pellets were implanted under light ether anesthesia. One pellet was implanted on the first day, two on the third day, and three on the fifth day. When assessed on day 8, this procedure has been shown to produce a high degree of tolerance to the analgesic and hyperthermic actions of morphine and also a high degree of physical dependence (3). Determination Cord
AND BHARGAVA
reagent as described above. After the second centrifugation, an equal volume of isopropanol was added to the aqueous phase and the sample was placed on dry ice for 60 min to precipitate RNA. Samples were centrifuged at 10,000 X g for 15 min and the resulting pellet was dissolved in a solution containing guanidinium thiocyanate, sodium citrate, sarcosyl, and 2-mercaptoethanol, and the isopropanol step was repeated. The RNA pellet was resuspended in 75% ethanol, placed on dry ice for 20 mitt, centrifuged, vacuum-dried, and dissolved in 30 ,nl of diethylpyrocabonate-treated water. After heating at 65°C for 10 mitt, a l~1 aliquot was used to estimate the concentration of RNA by measuring absorbance at 260 nm in a UV-spectrophotometer. Total RNA was separated by denaturing electrophoresis on a 0.66 M formaldehyde, 1.1% agarose gel and was blotted for hybridization to hybond N’ membrane (Amersham). All the control and treatment samples were processed on the same gels. To prepare radioactive probe for PPE mRNA, the plasmid PS064 @omega, Madison, WI) containing the Sacl-SmaI 945bp insert from cDNA clone for PPE, a gift from Dr. S.L. Sabol (19) was nick-translated using a kit from Amersham International (Arlington Heights, IL). The specific radioactivity of the probe was approximately l.O- 1.5 x lo7 cprn/pg DNA. The blot was hybridized overnight at 42°C with the [32P]a-dCTP-radiolabeled probe in 40% formamide, 0.75 M NaCl, 75 rm?4 sodium citrate, 7 mM Tris-HCl, pH 7.5, 0.02% ficoll, 0.02% polyvinylpyrrolidone, 25 &ml herring sperm DNA, 10% dextran sulfate, and 0.5% SDS. The blot was washed at 50°C in 2 x SSC (SSC = NaCl, 0.15 M; sodium citrate, 0.015 M, adjusted to pH 7.0 with HClO.05 M), 1% sodium dodecyl sulfate (SDS) for 30 min and in 0.2 x SSC, 1% SDS for 30 min. After washing, the blot was exposed to X-ray film. The quality and integrity of RNA loaded in each well was assessed by ethidinium bromide staining of the gel before transferring to a nylon membrane. The blot was exposed to Fuji medical X-ray film for 1- 10 days. The autoradiogram was analyzed using a GS 300 scanning densitometer (Hoefer Scientific Instruments, San Francisco, CA). Signal density for the PPE mRNA on the X-ray film was normalized by calculating the ratio of the PPE mRNA to the signal for 18s rRNA (from the negative film of the ethidinium bromide-stained gel) on the same blot. The data were subjected to analysis of variance (ANOVA) followed by Scheffe’s s-test. A value of p < 0.05 was considered to be significant.
of PPE r&WA in Brain Regions and Spinal
PPE mRNA levels were determined in brain regions and spinal cord of morphine-tolerant and abstinent rats. In tolerant rats (n = 6), the pellets were left in place whereas in abstinent rats (n = 6), the pellets were removed 16 h prior to sacrificing. The placebo-pelleted controls were n = 6 for the tolerant group and n = 6 for the abstinent group. The brain and spinal cord were isolated. The brain was dissected into seven regions, namely striatum, cortex, pons-medulla, hypothalamus, hippocampus, amygdala, and midbrain. Total RNA from the brain regions or spinal cord was isolated by the method of Chomczynski and Sacchi (7). The tissue was homogenized with a glass-Teflon homogenizer in a solution containing 4 M guanidinium thiocyanate, 25 r&f sodium citrate (pH 7.0), 0.5% sarcosyl, and 0.1 M 2-mercaptoethanol. Then 0.1 vol. of 2 h4 sodium acetate (pH 4.0) and 1 vol. of water-saturated phenol/chloroform mixture were added to the homogenate, with thorough mixing after the addition of each reagent. The samples were cooled on ice for 20 min and then centrifuged at 10,000 X g for 15 min. The aqueous phase was treated again with phenol
RESULTS
Northern blot analysis of total RNA indicated the previously reported size of PPE message (1.4 kb) for all conditions and brain regions examined. Figure 1 depicts a representative autoradiogram of a Northern blot showing the PPE mRNA in striatum of chronically treated rats with morphine along with their respective placebo controls. The density signals for 18s rRNA from the negetive film of ethidinium bromide-stained gels are also presented for comparison. Of all the tissues examined, the striatum had the highest level of PPE mRNA, moderate levels were seen in the hypothalamus, cortex, amygdala, and midbrain, and the lowest were in the spinal cord. Under the present experimental conditions, the PPE mRNA signals for hippocampus were not detected. In morphine-tolerant rats, highly significant increases in the levels of PPE mRNA were observed in the cortex (62%, p < 0.05) and spinal cord (352%, p < 0.001) in comparison to placebo-pelleted controls. Nonsignificant decreases were observed in the striatum (3%), pons-medulla (5%), amygdala (17%), and midbrain (16%), and a nonsignificant increase was observed in the hypothalamus (12%) when
CHRONIC
MORPHINE
ON
PREPROENKEPHALIN
GENE
EXPRESSION
417
4
0 PLACEBO-TREATED m MORPHINE-TOLERANT
1.40 kb PPE mRNA
28s
FIG. 2. Effect
18S FIG. 1. Northern blots of PPE. mRNA from striatum of tolerant and abstinent rats chronically treated with morphine and their respective placebo controls. Total RNA was extracted from the tissues and 20 pg of total RNA for each sample el’ectrophoresed and transferred to nylon
of morphine tolerance on the levels of PPE mRNA in brain regions and spinal cord of the rat. Male Sprague-Dawley rats were implanted with either six morphine or six placebo pellets during a ‘I-day period. The animals were sacrificed on the eighth day. Total RNA was isolated and the prepared Northern blot was hybridized with 32P-labeled PPE probe. Data were expressed as mean t- SEM for three to six observations. *p < 0.05 vs. the placebo controls.
membranes. The blot was hybridized with a 32P-radiolabeledPPE probe and autoradiograms scanned with a densitometer. The 18.9 and 28s rRNA bands from the negative
film of the ethidinium
bromide-stained
gel are shown for comparison. compared to placebo pellet-implanted rat tissues (Fig. 2). Much more dramatic changes were found in the levels of PPE mRNA in brain regions and spinal cord of morphine-tolerant/dependent rats from which pellets had been removed (i.e., the abstinent rats). Highly significant increases in the levels of PPE mRNA were observed in the striatum (62%, p < 0.05) and hypothalamus (34%, p < 0.05), whereas significant decreases were found in the pons-medulla (68%, p
found to be decreased in the striatum (49%) and cortex (11.5%) but were increased in the hippocampus and unchanged in the hypothalamus of morphine-tolerant rats (1). In this study, the effect of withdrawal was not determined. Obviously, there are differences in the results of various studies on the changes in PPE mRNA levels of the central tissues of morphine-treated rats and they may be due to the method used to induce tolerance and physical dependence, and abstinence. Lightman and Young (13) injected rats subcutaneously with 8 mg/kg twice daily on day 1 and with 16 mg/kg in the morning of day 2 and then implanted osmotic minipumps (Alzet 2 ml 1) that delivered 60 mg/kg per day for 12 days. Two, 5,9, and 25 h after removal of the pumps, naloxone (3 mg/kg) was injected subcutaneously. Uhl et al., (17) 5
0 PLACEBO-TREATED .
MORPHINE-ABSTINENT
4 DISCUSSION The present studies clearly demonstrate that the levels of PPE mRNA, which is a measure of gene expression for the opioid peptide enkephalin, vary from region to region in the brain. The highest density of PPE mRNA was found in the striatum. On the other hand, levels in the spinal cord were very low. In morphinetolerant rats, significant increases in the levels of PPE mRNA were found in the cortex and the spinal cord. On the other hand, in the abstinent rats, the levels were increased in the striatum and hypothalamus and were decreased significantly in the pons-medulla, midbrain, and spinal cord. Limited information is available on the changes in PPE mRNA levels in animals treated chronically with morphine. In one study, marked increases in PPE mRNA were found in the parvocellular paraventricular nucleus of the hypothalamus of morphine-abstinent rats (13). No other brain regions or spinal cord were examined. Uhl et al. (17) determined the levels of PPE mRNA in the striatum and found them to be decreased in both morphine-tolerant and abstinent rats. In another study, the levels of PPE m.RNA were determined in two brain regions and were
5; ’ 5’;: Ly& 3. El
ZB c.vl 2 22 PI.5 = 1
FIG. 3. Effect of morphine abstinence on the levels of PPE mRNA in brain regions and spinal cord of the rat. Male Sprague-Dawley rats were implanted with either six morphine or six placebo pellets during a 7day period. The animals were sacrificed 16 h after the pellet removal. Total RNA was isolated and the prepared Northern blot was hybridized with 32P-labeled PPE probe. Data were expressed as mean 2 SEM for three to six observations. *p < 0.05 vs. the placebo controls.
418
GUDEHITHLU
AND BHARGAVA
TABLE 1
TABLE 2
COMPARISON OF THE CHANGES IN THE LEVELS OF PPE mRNA IN BRAIN REGIONS AND SPINAL CORD OF MORPHINE-TOLERANT RATS AS REPORTED IN PREVIOUS AND PRESENT STUDIES
COMPARISON OF THE CHANGES IN THE LEVELS OF PPE mRNA IN BRAIN REGIONS AND SPINAL CORD OF MORPHINE-ABSTINENT RATS AS REPORTED IN PREVIOUS AND PRESENT STUDIES
Uhl et al. (14)
Tissue Striatum
1
Lightman and Young (11)
Basheer and Tern@ (1)
Present Study
1 t 1 0
0 Not Detected t 0
ND ND ND ND
0 0 0 t
Hippocampus Cortex Hypothalamus Pons-medulla
ND ND ND ND
ND ND ND ND ND
Midbrain
ND
ND
Amygdala Spinal cord
ND ND
ND ND
ND = not determined;
t = increased;
1 = decreased;
0 = no change.
implanted 11 pellets, each containing 75 mg of morphine base, during a S-day period. Both abstinent (24-48 h after pellet removal) and nonabstinent rats (with pellet left intact) were used. Finally, Basheer and Tempel (1) made rats tolerant to morphine by implanting two 75 mg pellet on day 1 and four pellets on day 3. The animals were sacrificed on day 5 with pellets left in place. The results of the previous studies and the present studies on the changes in the levels of PPE mRNA in tissues of morphine tolerant and abstinent rats are summarized in Tables 1 and 2. It is clear from the studies of Basheer and Tempel(1) that the changes in PPE mRNA levels in different regions of the brain do not show similar direction in rodents treated chronically with morphine. The present studies demonstrate that, in morphine-tolerant rats, the levels of PPE mRNA are increased in the cortex and spinal cord. Both of these regions are important in the elicitation of the analgesic response to morphine. On the other hand, differential changes in the levels of PPE mRNA in brain regions and spinal cord of morphine-abstinent rats were noted. The levels were increased in the striatum and hypothalamus but were decreased in the pons-medulla, midbrain, and spinal cord. Some of these changes can be related to the symptoms of the abstinence syndrome, like hyperactivity, stereotyped jumping, and hypothermic response (2). The changes in the hypothalamus are consistent with the studies of Lightman and Young (13), and the changes may be related to the endocrine changes observed during withdrawal (10,13). On the other hand, changes in striatal levels of PPE mRNA may be responsible for motor function. It is interesting to note that naloxone-induced withdrawal has been shown to increase the levels of methionine-enkephalin in the hypothalamus and striatum but not in abrupt abstinence from morphine (12). In abrupt abstinence, the levels in the striatum were decreased and were unchanged in the hypothalamus. This observation suggests that the processing of enkephalins in abrupt vs. the antagonist-induced abstinence is different. Our previous stud-
Tissue Striatum
Hippocampus Cortex Hypothalamus Pons-medulla Midbrain Amygdala Spinal cord
Uhl et al. (14) 1
ND ND ND ND ND ND ND
ND = not determined;
Lightman and Young (11)
Basheer and Ten@ (1)
ND ND ND t ND ND ND ND
ND ND ND ND ND ND ND ND
t = increased;
1 = decreased;
Present Study
t Not Detected 0 t 1 1 0 1 0 = no change.
in methionine-enkephalin levels in the central tissues indicate that in morphine-tolerant rats, the levels were decreased in the pons-medulla, amygdala, and hippocampus (12). During abstinence, the direction of change in the levels of methionine-enkephalin and PPE mRNA were similar in the ponsmedulla, midbrain, and spinal cord but were dissimilar in the amygdala, cortex, striatum, and hypothalamus. These directions of changes or the brain areas affected are not the same as seen for the levels of PPE mRNA, and these differences are due to the fact that the site of synthesis of PPE mRNA and the site of expression of methionine-enkephalin peptide are different. Furthermore, the rate of synthesis of PPE mRNA, Met-enkephalin peptide, and the rate of release of this peptide may also be under different regulations and we cannot exclude a selective impact of morphine on any one of the above processes. Monitoring the expression of an opioid peptide gene during morphine administration and withdrawal has revealed a specific temporal pattern of changes in mRNA and peptide levels. Elucidation of rate of PPE mRNA synthesis by nuclear run-on assay may enhance our understanding of interactions between the opiate tolerance and dependence and the expression of PPE mRNA. In summary, the present studies have examined the changes in the levels of PPE mRNA in several brain regions and spinal cord of rats tolerant to morphine as well as those undergoing protracted abstinence from morphine. The results indicate that the changes in PPE mRNA levels in brain regions and spinal cord of morphine-tolerant or abstinent rats do not always parallel the changes in the levels of methionine-enkephalin and thus suggest the involvement of other enkephalins and possibly other processes, such as their release and degradation. ies on the changes
ACKNOWLEDGEMENTS These studies
were supported by a Research Scientist Development Award K02-DA-00130 to Hemendra N. Bhargava from the National Institute on Drug Abuse, National Institutes of Health.
REFERENCES I. Basheer, G,, and Neurosci. 2. Bhargava, physical behavior.
R.; Tempel, A. Morphine-induced reciprocal alterations in opioid peptide mRNA levels in discrete brain regions. J. Res. 36551-557; 1993. H. N. Diversity of agents which modify opioid tolerance, dependence, abstinence syndrome and self-administrative Pharmacol. Rev. 46:293-324; 1994.
3. Bhargava, H. N.; Matwyshyn, G. A.; Gerk, P. M.; et al. Effect of naltrexone pellet implantation on morphine tolerance and physical dependence in the rat. Gen. Pharmacol. 25: 149- 155; 1994. 4. Bhargava, H. N. Rapid induction and quantitation of morphine dependence in the raf by pellet implantation. Psychopharmacology (Berlin) 52:55-62; 1977.
CHRONIC MORPHINE ON PREPROENKEPHALIN GENE EXPRESSION
5. Bhargava, H. N. Quantitation of morphine tolerance induced by pellet implantation in the rat. J. Pharm. Pharmacol. 30:133- 135; 1978. 6. Childers, S. R.; Simantov, IL; Snyder, S. H. Enkephalin: Radioimmunoassay and radioreceptar assay in morphine dependent rats. Eur. J. Pharmacol. 46:289-293; 1977. 7. Chomczynski, P.; Sacchi, N. Single-step method of RNA isolation by acid guauidinium thiocyanate-phenol-chloroform extraction. Anal. B&hem. 162:156- 1.59; 1987. 8. Elsworth, J. D.; Redmond, I). E., Jr.; Roth, R. H. Effect of morphine treatment and withdrawal on endogenous methionine and leucineenkephalin levels in primatc brain, B&hem. Pharmacol. 35:34153417; 1986. 9. Fratta, W.; Yang, H. Y. T.; Hong, J.; Costa, E. Stability of metenkephalin content in brain structures of morphine-dependent or foot shock stressed rats. Nature ‘268:452-453; 1977. 10. George, R. Hypothalamus Anterior pituitary gland. In: Clouet, D. H., ed. Narcotic drugs, biochemical pharmacology. New York: Plenum Press; 1971:283-299. 11. Gudehithlu, K. P.; Duchemin, A. M.; Tejwani, G. A.; Neff, N. H.; Hadjiconstantinou, M. Preproenkephalin mRNA and methionineenkephalin increase in mouse striatum after 1-methyl&phenyl1,2,3,6-tetrahydropyridine treatment. J. Neurochem. 56: 1043- 1048; 1991. 12. Gudehithlu, K. P.; Tejwaui, G. A.; Bhargava, H. N. P-Endorphin and methionine-enkephalin levels in discrete brain regions, spinal
13.
14.
15.
16. 17. 18.
19.
cord, pituitary gland and serum of morphine tolerant-dependent and abstinent rats. Brain Res. 553:284-290, 1991. Lightman, S. L.; Young, W. S., III. Changes in hypothalamic preproenkephalin A mRNA following stress and opiate withdrawal. Nature 328643645; 1987. Przewlocki, R.; Hollt, V.; Duka, Th.; et al. Long-term morphine treatment decreases endorphin levels in rat brain and pituitary. Brain Res 174:357-361; 1979. Rattan, A. K.; Koo, K. L.; Tejwani, G. A.; Bhargava, H. N. The effect of morphine tolerance-dependence and abstinence on dynorphin (1 - 13) levels in discrete brain regions, spinal cord, pituitary gland and peripheral tissues of the rat. Brain Res. 584:207-212; 1992. Schwartz, 1. P.; Costa, E. Hybridization approaches to the study of neuropeptides. Annu. Rev. Neurosci. 9:277-304; 1986. Uhl, G. R.; Ryan, J. P.; Schwartz, J. P. Morphine alters preproenkephalin gene expression. Brain Res. 459:391-397; 1988. Wesche, D.; Hollt, V.; Herz, A. Radioimmunoassays of enkephalins. Regional distribution in rat brain after morphine treatment and hypophysectomy. Naunyn-Schmiedebergs Arch. Exp. Pathol. Pharmakol. 301:79-82; 1977. Yoshikawa, K.; Williams, C.; Sabol, S. L. Rat brain preproenkephalin mRNA: cDNA cloning, primary structure and distribution in the central nervous system. J. Biol. Chem. 259:14301-14308; 1984.
Pergamon
0196-9%1(94)00199-5
Modulation of Preproenkephalin mRNA Levels in Brain Regions and Spinal Cord of Rats Treated Chronically With Morphine KRISHNAMURTHY
P. GUDEHITHLU
AND
HEMENDRA
N. BHARGAVA’
Department of Pharmaceutics and Pharmacodynamics (M/C 865), The University of Illinois at Chicago, Health Sciences Center, 833 South Wood Street, Chicago, IL 60612 Received 15 August
1994
GUDEHITHLU, K. P. AND H. N. BHARGAVA. Modularion of preproenkephalin mRNA levels in brain regions and spinal cord of rats treated chronically with morphine. PEPTIDES 16(3) 4 15 -4 19, 1995.-The effect of morphine tolerance/dependence and abstinence on the preproenkephalin (PPE) gene expression was determined in brain regions and spinal cord of the rat. Male Sprague-Dawley rats were rendered tolerant and physically dependent on morphine by SC implantation of six pellets, each containing 75 mg of morphine base, during a 7-day period. Placebo pellet-implanted rats served as controls. In tolerant rats, the pellets were left in place at the time of sacrifice whereas in abstinent rats, the pellets were removed 16 h prior to sacrificing. The levels of PPE mRNA were determined in brain regions (striatum, cortex, pons-medulla, hypothalamus, amygdala, and midbrain) and spinal cord. The levels of PPE mRNA increased significantly in the cortex (62%) and the spinal cord (352%) of morphinetolerant rats when compared to placebo pellet-implanted control rats. In other brain regions, the levels of PPE mRNA in placebo and morphine-tolerant rats did not differ. On the other hand, in morphine-abstinent rats, the levels of PPE mRNA increased in the striatum (62%) and hypothalamus (34%) but were decreased in pons-medulla (68%). midbrain (51%), and spinal cord (36%) in comparison to the placebo controls. The results clearly demonstrate differential changes in enkephalin gene expression in brain regions and spinal cord of the abstinent and nonabstinent morphine-tolerant/dependent rats. Morphine Rat
tolerance
Morphine
abstinence
PPE mRNA
Brain regions
for reprints
should be addressed
to Dr. Hemendra
Differential
change
levels of methionine-enkephalin in the spinal cord, amygdala, pons-medulla, midbrain, cortex, and striatum, and naloxone-induced withdrawal resulted in decreases in the level of methionine-enkephalin in the amygdala but increases in the hypothalamus and striatum (12). The mechanism by which modification in the levels of enkephalin are produced in morphine-tolerant and abstinent rodents is not clear. It could involve synthesis, release, and degradation of enkephalins. Neurons in the brain and spinal cord can regulate their contents of the opioid peptides and their respective encoding mRNA. PPE mRNA levels in these neurons are regulated by excitatory inputs, blockade, or removal of inhibitory inputs (10,16). Attempts have been made to determine the levels of PPE mRNA in brain tissue of rats treated chronically with morphine. In one study, chronic treatment with morphine did not alter the distribution of enkephalin mRNA in the hypothalamus. However, naloxone-precipitated withdrawal resulted in a massive and extremely rapid accumulation of enkephalin mRNA in cells of the parvocellular nucleus within 2 h, and the level remained high at 24 h (13). In another study, levels of PPE mRNA were reported to be decreased in the striatum of both nonabstinent and abstinent
IN spite of considerable efforts, the mechanisms in the various aspects of opioid addiction. namely tolerance, physical dependence, abstinence syndrome, and self-administration behavior, are far from being clear. Diverse molecules with known or unknown mechanisms appear to modify these processes (2). Since the discovery of opioid peptides in mammalian tissues, attempts have been made to determine their role in opioid tolerance/dependence and abstinence processes. The levels of enkephalins, dynorphins, and /?-endorphin in various tissues of rats treated chronically with morphine have been determined (12,15). Earlier studies failed to find any alterations in the brain enkephalin levels (6,9,18) in rats treated with morphine for 10 days. On the other hand, implantation of 27 morphine pellets during a 36-day period reduced the levels of enkephalins and ,B-endorphin in the brain and pituitary of the rat (14). The level of methionine-enkephalin was found to be decreased in the hippocampus and putamen of monkeys treated with morphine for 10 days (8). Studies in this laboratory have demonstrated that in the rat, morphine tolerance was associated with decreases in the levels of methionine-enkephalin in the pons-medulla, amygdala, and hippocampus. On the other hand, abrupt abstinence from morphine decreased the
’ Requests
Spinal cord
N. Bhargava.
415
416
GUDEHITHLU
morphine-tolerant/dependent rats (17). Thus, the two brain regions appeared to show differential processing of enkephalins in rats treated chronically with morphine. Little is known about the effect of exogenous opioid drugs on the molecular changes in the functioning of endogenous brain and spinal cord opioid peptide systems. Because our previous studies showed that methionine-enkephalin levels are decreased in various brain regions and spinal cord of morphine-tolerant and abstinent rats but naloxone-induced withdrawal resulted in increases in hypothalamic and striatal methionine-enkephalin levels (12), the present studies were undertaken to determine the levels of PPE mRNA in several brain regions and spinal cord of morphine-tolerant and abstinent rats. METHOD
Animals Male Sprague-Dawley rats weighing 250-275 g (Sasco King Animal Co, Oregon, WI) were housed three to a cage in a room with controlled ambient temperature (23 + l”C), humidity (50 + lo%), and a 12-h dark-light cycle (0600 to 1800 h). The animals were housed under these conditions for at least 4 days prior to being used. Food and water were made available continuously. Drugs Morphine and placebo pellets were supplied by the National Institute on Drug Abuse (Rockville, MD) through the courtesy of Drs. Rao S. Rapaka and Paul S. Hillery. Induction Morphine
ofTolerance to and Physical Dependence on
Rats were rendered tolerant to and physically dependent on morphine by SC implantation of six morphine pellets as described previously (45). Each pellet contained 75 mg of morphine free base. Rats that served as controls were implanted with six placebo pellets. The pellets were implanted under light ether anesthesia. One pellet was implanted on the first day, two on the third day, and three on the fifth day. When assessed on day 8, this procedure has been shown to produce a high degree of tolerance to the analgesic and hyperthermic actions of morphine and also a high degree of physical dependence (3). Determination Cord
AND BHARGAVA
reagent as described above. After the second centrifugation, an equal volume of isopropanol was added to the aqueous phase and the sample was placed on dry ice for 60 min to precipitate RNA. Samples were centrifuged at 10,000 X g for 15 min and the resulting pellet was dissolved in a solution containing guanidinium thiocyanate, sodium citrate, sarcosyl, and 2-mercaptoethanol, and the isopropanol step was repeated. The RNA pellet was resuspended in 75% ethanol, placed on dry ice for 20 mitt, centrifuged, vacuum-dried, and dissolved in 30 ,nl of diethylpyrocabonate-treated water. After heating at 65°C for 10 mitt, a l~1 aliquot was used to estimate the concentration of RNA by measuring absorbance at 260 nm in a UV-spectrophotometer. Total RNA was separated by denaturing electrophoresis on a 0.66 M formaldehyde, 1.1% agarose gel and was blotted for hybridization to hybond N’ membrane (Amersham). All the control and treatment samples were processed on the same gels. To prepare radioactive probe for PPE mRNA, the plasmid PS064 @omega, Madison, WI) containing the Sacl-SmaI 945bp insert from cDNA clone for PPE, a gift from Dr. S.L. Sabol (19) was nick-translated using a kit from Amersham International (Arlington Heights, IL). The specific radioactivity of the probe was approximately l.O- 1.5 x lo7 cprn/pg DNA. The blot was hybridized overnight at 42°C with the [32P]a-dCTP-radiolabeled probe in 40% formamide, 0.75 M NaCl, 75 rm?4 sodium citrate, 7 mM Tris-HCl, pH 7.5, 0.02% ficoll, 0.02% polyvinylpyrrolidone, 25 &ml herring sperm DNA, 10% dextran sulfate, and 0.5% SDS. The blot was washed at 50°C in 2 x SSC (SSC = NaCl, 0.15 M; sodium citrate, 0.015 M, adjusted to pH 7.0 with HClO.05 M), 1% sodium dodecyl sulfate (SDS) for 30 min and in 0.2 x SSC, 1% SDS for 30 min. After washing, the blot was exposed to X-ray film. The quality and integrity of RNA loaded in each well was assessed by ethidinium bromide staining of the gel before transferring to a nylon membrane. The blot was exposed to Fuji medical X-ray film for 1- 10 days. The autoradiogram was analyzed using a GS 300 scanning densitometer (Hoefer Scientific Instruments, San Francisco, CA). Signal density for the PPE mRNA on the X-ray film was normalized by calculating the ratio of the PPE mRNA to the signal for 18s rRNA (from the negative film of the ethidinium bromide-stained gel) on the same blot. The data were subjected to analysis of variance (ANOVA) followed by Scheffe’s s-test. A value of p < 0.05 was considered to be significant.
of PPE r&WA in Brain Regions and Spinal
PPE mRNA levels were determined in brain regions and spinal cord of morphine-tolerant and abstinent rats. In tolerant rats (n = 6), the pellets were left in place whereas in abstinent rats (n = 6), the pellets were removed 16 h prior to sacrificing. The placebo-pelleted controls were n = 6 for the tolerant group and n = 6 for the abstinent group. The brain and spinal cord were isolated. The brain was dissected into seven regions, namely striatum, cortex, pons-medulla, hypothalamus, hippocampus, amygdala, and midbrain. Total RNA from the brain regions or spinal cord was isolated by the method of Chomczynski and Sacchi (7). The tissue was homogenized with a glass-Teflon homogenizer in a solution containing 4 M guanidinium thiocyanate, 25 r&f sodium citrate (pH 7.0), 0.5% sarcosyl, and 0.1 M 2-mercaptoethanol. Then 0.1 vol. of 2 h4 sodium acetate (pH 4.0) and 1 vol. of water-saturated phenol/chloroform mixture were added to the homogenate, with thorough mixing after the addition of each reagent. The samples were cooled on ice for 20 min and then centrifuged at 10,000 X g for 15 min. The aqueous phase was treated again with phenol
RESULTS
Northern blot analysis of total RNA indicated the previously reported size of PPE message (1.4 kb) for all conditions and brain regions examined. Figure 1 depicts a representative autoradiogram of a Northern blot showing the PPE mRNA in striatum of chronically treated rats with morphine along with their respective placebo controls. The density signals for 18s rRNA from the negetive film of ethidinium bromide-stained gels are also presented for comparison. Of all the tissues examined, the striatum had the highest level of PPE mRNA, moderate levels were seen in the hypothalamus, cortex, amygdala, and midbrain, and the lowest were in the spinal cord. Under the present experimental conditions, the PPE mRNA signals for hippocampus were not detected. In morphine-tolerant rats, highly significant increases in the levels of PPE mRNA were observed in the cortex (62%, p < 0.05) and spinal cord (352%, p < 0.001) in comparison to placebo-pelleted controls. Nonsignificant decreases were observed in the striatum (3%), pons-medulla (5%), amygdala (17%), and midbrain (16%), and a nonsignificant increase was observed in the hypothalamus (12%) when
CHRONIC
MORPHINE
ON
PREPROENKEPHALIN
GENE
EXPRESSION
417
4
0 PLACEBO-TREATED m MORPHINE-TOLERANT
1.40 kb PPE mRNA
28s
FIG. 2. Effect
18S FIG. 1. Northern blots of PPE. mRNA from striatum of tolerant and abstinent rats chronically treated with morphine and their respective placebo controls. Total RNA was extracted from the tissues and 20 pg of total RNA for each sample el’ectrophoresed and transferred to nylon
of morphine tolerance on the levels of PPE mRNA in brain regions and spinal cord of the rat. Male Sprague-Dawley rats were implanted with either six morphine or six placebo pellets during a ‘I-day period. The animals were sacrificed on the eighth day. Total RNA was isolated and the prepared Northern blot was hybridized with 32P-labeled PPE probe. Data were expressed as mean t- SEM for three to six observations. *p < 0.05 vs. the placebo controls.
membranes. The blot was hybridized with a 32P-radiolabeledPPE probe and autoradiograms scanned with a densitometer. The 18.9 and 28s rRNA bands from the negative
film of the ethidinium
bromide-stained
gel are shown for comparison. compared to placebo pellet-implanted rat tissues (Fig. 2). Much more dramatic changes were found in the levels of PPE mRNA in brain regions and spinal cord of morphine-tolerant/dependent rats from which pellets had been removed (i.e., the abstinent rats). Highly significant increases in the levels of PPE mRNA were observed in the striatum (62%, p < 0.05) and hypothalamus (34%, p < 0.05), whereas significant decreases were found in the pons-medulla (68%, p
found to be decreased in the striatum (49%) and cortex (11.5%) but were increased in the hippocampus and unchanged in the hypothalamus of morphine-tolerant rats (1). In this study, the effect of withdrawal was not determined. Obviously, there are differences in the results of various studies on the changes in PPE mRNA levels of the central tissues of morphine-treated rats and they may be due to the method used to induce tolerance and physical dependence, and abstinence. Lightman and Young (13) injected rats subcutaneously with 8 mg/kg twice daily on day 1 and with 16 mg/kg in the morning of day 2 and then implanted osmotic minipumps (Alzet 2 ml 1) that delivered 60 mg/kg per day for 12 days. Two, 5,9, and 25 h after removal of the pumps, naloxone (3 mg/kg) was injected subcutaneously. Uhl et al., (17) 5
0 PLACEBO-TREATED .
MORPHINE-ABSTINENT
4 DISCUSSION The present studies clearly demonstrate that the levels of PPE mRNA, which is a measure of gene expression for the opioid peptide enkephalin, vary from region to region in the brain. The highest density of PPE mRNA was found in the striatum. On the other hand, levels in the spinal cord were very low. In morphinetolerant rats, significant increases in the levels of PPE mRNA were found in the cortex and the spinal cord. On the other hand, in the abstinent rats, the levels were increased in the striatum and hypothalamus and were decreased significantly in the pons-medulla, midbrain, and spinal cord. Limited information is available on the changes in PPE mRNA levels in animals treated chronically with morphine. In one study, marked increases in PPE mRNA were found in the parvocellular paraventricular nucleus of the hypothalamus of morphine-abstinent rats (13). No other brain regions or spinal cord were examined. Uhl et al. (17) determined the levels of PPE mRNA in the striatum and found them to be decreased in both morphine-tolerant and abstinent rats. In another study, the levels of PPE m.RNA were determined in two brain regions and were
5; ’ 5’;: Ly& 3. El
ZB c.vl 2 22 PI.5 = 1
FIG. 3. Effect of morphine abstinence on the levels of PPE mRNA in brain regions and spinal cord of the rat. Male Sprague-Dawley rats were implanted with either six morphine or six placebo pellets during a 7day period. The animals were sacrificed 16 h after the pellet removal. Total RNA was isolated and the prepared Northern blot was hybridized with 32P-labeled PPE probe. Data were expressed as mean 2 SEM for three to six observations. *p < 0.05 vs. the placebo controls.
418
GUDEHITHLU
AND BHARGAVA
TABLE 1
TABLE 2
COMPARISON OF THE CHANGES IN THE LEVELS OF PPE mRNA IN BRAIN REGIONS AND SPINAL CORD OF MORPHINE-TOLERANT RATS AS REPORTED IN PREVIOUS AND PRESENT STUDIES
COMPARISON OF THE CHANGES IN THE LEVELS OF PPE mRNA IN BRAIN REGIONS AND SPINAL CORD OF MORPHINE-ABSTINENT RATS AS REPORTED IN PREVIOUS AND PRESENT STUDIES
Uhl et al. (14)
Tissue Striatum
1
Lightman and Young (11)
Basheer and Tern@ (1)
Present Study
1 t 1 0
0 Not Detected t 0
ND ND ND ND
0 0 0 t
Hippocampus Cortex Hypothalamus Pons-medulla
ND ND ND ND
ND ND ND ND ND
Midbrain
ND
ND
Amygdala Spinal cord
ND ND
ND ND
ND = not determined;
t = increased;
1 = decreased;
0 = no change.
implanted 11 pellets, each containing 75 mg of morphine base, during a S-day period. Both abstinent (24-48 h after pellet removal) and nonabstinent rats (with pellet left intact) were used. Finally, Basheer and Tempel (1) made rats tolerant to morphine by implanting two 75 mg pellet on day 1 and four pellets on day 3. The animals were sacrificed on day 5 with pellets left in place. The results of the previous studies and the present studies on the changes in the levels of PPE mRNA in tissues of morphine tolerant and abstinent rats are summarized in Tables 1 and 2. It is clear from the studies of Basheer and Tempel(1) that the changes in PPE mRNA levels in different regions of the brain do not show similar direction in rodents treated chronically with morphine. The present studies demonstrate that, in morphine-tolerant rats, the levels of PPE mRNA are increased in the cortex and spinal cord. Both of these regions are important in the elicitation of the analgesic response to morphine. On the other hand, differential changes in the levels of PPE mRNA in brain regions and spinal cord of morphine-abstinent rats were noted. The levels were increased in the striatum and hypothalamus but were decreased in the pons-medulla, midbrain, and spinal cord. Some of these changes can be related to the symptoms of the abstinence syndrome, like hyperactivity, stereotyped jumping, and hypothermic response (2). The changes in the hypothalamus are consistent with the studies of Lightman and Young (13), and the changes may be related to the endocrine changes observed during withdrawal (10,13). On the other hand, changes in striatal levels of PPE mRNA may be responsible for motor function. It is interesting to note that naloxone-induced withdrawal has been shown to increase the levels of methionine-enkephalin in the hypothalamus and striatum but not in abrupt abstinence from morphine (12). In abrupt abstinence, the levels in the striatum were decreased and were unchanged in the hypothalamus. This observation suggests that the processing of enkephalins in abrupt vs. the antagonist-induced abstinence is different. Our previous stud-
Tissue Striatum
Hippocampus Cortex Hypothalamus Pons-medulla Midbrain Amygdala Spinal cord
Uhl et al. (14) 1
ND ND ND ND ND ND ND
ND = not determined;
Lightman and Young (11)
Basheer and Ten@ (1)
ND ND ND t ND ND ND ND
ND ND ND ND ND ND ND ND
t = increased;
1 = decreased;
Present Study
t Not Detected 0 t 1 1 0 1 0 = no change.
in methionine-enkephalin levels in the central tissues indicate that in morphine-tolerant rats, the levels were decreased in the pons-medulla, amygdala, and hippocampus (12). During abstinence, the direction of change in the levels of methionine-enkephalin and PPE mRNA were similar in the ponsmedulla, midbrain, and spinal cord but were dissimilar in the amygdala, cortex, striatum, and hypothalamus. These directions of changes or the brain areas affected are not the same as seen for the levels of PPE mRNA, and these differences are due to the fact that the site of synthesis of PPE mRNA and the site of expression of methionine-enkephalin peptide are different. Furthermore, the rate of synthesis of PPE mRNA, Met-enkephalin peptide, and the rate of release of this peptide may also be under different regulations and we cannot exclude a selective impact of morphine on any one of the above processes. Monitoring the expression of an opioid peptide gene during morphine administration and withdrawal has revealed a specific temporal pattern of changes in mRNA and peptide levels. Elucidation of rate of PPE mRNA synthesis by nuclear run-on assay may enhance our understanding of interactions between the opiate tolerance and dependence and the expression of PPE mRNA. In summary, the present studies have examined the changes in the levels of PPE mRNA in several brain regions and spinal cord of rats tolerant to morphine as well as those undergoing protracted abstinence from morphine. The results indicate that the changes in PPE mRNA levels in brain regions and spinal cord of morphine-tolerant or abstinent rats do not always parallel the changes in the levels of methionine-enkephalin and thus suggest the involvement of other enkephalins and possibly other processes, such as their release and degradation. ies on the changes
ACKNOWLEDGEMENTS These studies
were supported by a Research Scientist Development Award K02-DA-00130 to Hemendra N. Bhargava from the National Institute on Drug Abuse, National Institutes of Health.
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CHRONIC MORPHINE ON PREPROENKEPHALIN GENE EXPRESSION
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