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Chronic administration of morphine decreases level of dynorphin a in the rat nucleus accumbens
Neuropharmacology Vol.32,No.7,pp.703709,1993 Printed in Great Britain. All rights reserved
0028-3908/93 WlO+O.OO Copyright 0 1993 Pergamon Press Ltd
CHRONIC ADMINISTRATION OF MORPHINE DECREASES LEVEL OF DYNORPHIN A IN THE RAT NUCLEUS ACCUMBENS R. Yu. YUKHANANOV,’Q. Z. ZHAI,~ S. PERSSON,~ C. POSTS and F. NYBERC~* ‘Department of Pharmacology, Russian Academy of Medical Sciences, Baltiskaya St. 8, Moscow, Russia and 2Department of Pharmaceutical Biosciences, Division of Pharmacology, Uppsala University, P.O. Box 591, Uppsala, Sweden (Accepted 28 December 1992) Summary-The effect of chronically administered morphine on the levels of dynorphin A in distinct regions of the brain (including medial frontal cortex, olfactory tubercule, nucleus accumbens, dorsal and medial striatum), was determined in male Sprague-Dawley rats. The drug was delivered through a subcutaneously implanted Azlet miniosmotic pump over a period of 5 days. The concentration of peptide was probed by radioimmunoassay, following pre-separation of tissue extracts by reversed phase separation on a SepPak C-18 cartridge. The result showed that the level of dynorphin A remained unaltered in all regions studied immediately before (tolerance) and 20 hr after (withdrawal) the pump was removed. A significant decrease in the level of dynorphin was found in the n. accumbens 48 hr (abstinence) after removal of the pump. It is suggested that previously observed changes in the reward system during abstinence may be connected with dynorphinergic neurones in the limbic system. Key words-morphine
dependence, dynorphin A, miniosmotic pump, hyperalgesia, n. accumbens.
Extensive studies have been carried out in order to elucidate the role of opioid peptides in the consequences of opiate addiction. Many attempts to find any change in the level of e.g. Met-enkephalin, Leuenkephalin and dynorphins in the brain of morphinetreated animals have failed (Childers, Simantov and Snyder, 1977; Fratta, Yang, Hong and Costa, 1977; Ho, Way, Chan, Cheng, Kwok and Wen, 1982; Shani, Azov and Weisman, 1979; Wesche, Hollt and Herz, 1977). However, in a recent study a reduction in the level of dynorphin A(l-17) in the spinal cord of the rat 24 hr after cessation of chronic administration of morphine was found (Nylander, Sakaruda, Le GrevCs and Terenius, 1991). It was recently found that dynorphin A(l-13) was reduced in the spinal cord but increased in the pituitary and hypothalamus, 18 hr after removal of morphine pellets in morphinetolerant rats (Rattan, Kao, Tejawani and Bhargava, 1992). Studies have also revealed an increase in dynorphin-related peptides after 7 days of treatment with opiate (Trujillo and Akil, 1990). It is well known that the development of dependence on opiate has a complex dynamic and is also affected by the paradigm of administration of opioids (Shani et al., 1979). In this study, it was decided to determine the levels of dynorphin at all the different stages of the opiate dependence, namely tolerance, withdrawal and abstinence. It is clear that these *Address correspondence to: Dr Fred Nyberg, Division of Pharmacology, Sweden.
BMC, Box 591, S-751 24 Uppsala,
stages have qualitative differences but the time-table of their appearance strongly depends on the route of administration of the narcotic and therefore, in this study, their appearance was checked by testing the behaviour of the animals. Morphine was administrated through miniosmotic pumps, implanted subcutaneously on the back of male rats (Persson, Post, Alari, Nyberg and Terenius, 1989). The level of dynorphin A(l-17) was measured in areas of the brain corresponding to the mesolimbic, mesostriatal and mesocortical dopamine system, which are known to mediate the euphoric effects of many narcotics. METHODS Animal experiment
Male Sprague-Dawley rats (Alab, Sweden), weighing 220-240 g at the beginning of the experiment, were used. The animals were housed in a room with controlled humidity (60%) and temperature (24°C) and with a fixed lighting schedule (light from 9-21 hr). The water and food were given ad libitum. The rats were adapted to the environment at least 1 week before the start of the experiment. One day before implantation of the pump, the animals were placed in individual cages with transparent walls, where they were kept until the end of the experiment. The rats were divided randomly into three groups, each consisting of 6 experimental and 6 control animals. Miniosmotic pumps (Alzet, 2MLl) were filled with morphine acetate (70 mg/ml) or saline and were implanted subcutaneously on the back under 703
704
R. Yl_l. ~UKHANANOV
diethyl ether anaesthesia. Five days (12&124 hr) later, the pumps were removed. Animals in the first group (tolerance or T-group) were decapitated after 120 hr, just before removal of the pumps. The second group of animals (withdrawal or W-group) were decapitated 20 hr after the pumps were removed (144 hr after implantation). Rats in the third group (abstinence or A-group) were decapitated 48 hr following removal of the pump. Every day the rats were weighed and their water consumption was determined. The nociceptive sensitivity
The nociceptive sensitivity was measured by the paw pressure test. The animals were tested before implantation (-24 hr) and at 6, 12, 24 hr and subsequently at every 24 hr after implantation of the pump. The paw pressure (PP) test was performed using a commercially available unit (Ugo Basile, Comerio, Italy). The paw pressure was applied through a ballet shaped plastic tip pressure device to the plantar surface of the hind paw, with continuously increasing pressure at a rate of 480mN/sec (48 PP) or 320 mN/sec (32 PP). The pressure at which a rapid removal of the hind paw was observed was assigned as a response threshold. The cut off pressures were 6 and 3 N, respectively. Analgesia and hyperalgesia were calculated according to the following expressions: Analgesia = (X - phone latency)/ (cut off - phone latency) x 100% Hyperalgesia = (cut off - X)/cut off x lOO%, X-latency before and after pump implantation, phone latency-the reaction at 0 hr, just before implantation of the pump. Locomotor activity
Locomotor activity (LA) was measured in the open field after removal of the pump. The animals were placed in a quadrant box for 5 min (1 x 1 m) with a lamp placed over the center. The number of movements along each wall was recorded as an index of locomotor activity. Tissue extraction
After decapitation, the brain was immediately removed and placed on ice. The factory tubercule was cut out by scissors as described elsewhere (Heyna, Hogenboam, Portoghese, Mulder and Padt, Schoffelmeer, 1990) and the brain was dissected on ice, using a rat brain matrix (Activational systems Inc., Forterra Drive Warren, Michigan, U.S.A.) in coronal slices (1 mm). All further dissections were performed according to a stereotaxic atlas (Paxinos and Watson, 1986) and the distance are given from the interaural line (mm). The medial frontal cortex was taken from the slices (13.8-11.8), nucleus accumbens from the next two slices (11.8-9.8) and striatum
et al.
(Caudatus putamen, 10.7-7.7) was dissected into the medioventral and dorsolateral parts. The extraction of brain tissue was performed as described earlier (Christensson-Nylander and Terenius, 1985) with some modifications (i.e. preseparation of extracts was performed using SepPak cartridges instead of SPSephadex mini-columns). The extracts were thus run through reversed-phase Sep-Pak Cl8 cartridges, prewashed with 10ml of 100% methanol (MeOH) and lOm1 water. After adding the acidic extract, the cartridge was washed with 4 ml of water and 4 ml of 20% MeOH, before elution with 4ml of 85% methanol [all solutions contained 0.04% trifluoroacetic acid (TFA)]. The eluates were collected and after evaporation (Savant Vat, Savant, Hicksville, New York) the samples were dissolved in MeOH/O. 1M HCl (1: 1, v/v) before radioimmunoassay. Chromatography
Extracts of the brain tissue were chromatographed on a Sephadex G-SO column (2.5 x 40 cm) with 1M acetic acid containing 10 mM NaCl at a flow rate of 30 ml/hr and fractions of 2 ml were collected, evaporated (Savant Vat) and dissolved in MeOH/O. 1M HCl (1: 1, v/v) before analysis by radioimmunoassay. A new chromatographic system. SMARTTM system (Pharmacia, Uppsala, Sweden), equipped with a mRP-column C2/Cl8 3.2/3, was used for further analysis of the immunoreactive material. The column was developed with a linear gradient of acetonitrile (o-60%), containing 0.04% TFA. The flow rate was 0.24 ml/min and fractions of 0.2 ml were collected. Radioimmunoassay (RIA)
The antiserum (denoted 84) was raised in rabbits against the peptide-thyroglobulin conjugate and the iodinated peptide was used as a tracer. The characteristics of the antiserum has been described elsewhere (Christensson-Nylander and Terenius, 1985). The radioimmunoassay (RIA) for dynorphin A( l-l 7) was based on the non-immune pre-separation method and conducted as described previously (ChristenssonNylander, Nybert, Ragnarsson and Terenius, 1985; Nyberg, Christensson-Nylander and Terenius, 1987) with the exception that the bound peptide was separated from unbound using a sheep anti-rabbit antibody (Pharmacia Decanting Suspension, 200 ml of a 4 times diluted solution). The crossreactivity of the with dynorphin A( l-8), dynorphin antiserum A(I-13), dynorphin B, a-neoendorphin, Leu-enkephalin and Leu-enkaphahn-Arg6 was less than 0.1% and with dynorphin A(7-17) 100%. The detection limit of the RIA was 334 fmol/tube. Peptide and chemicals
The synthetic was purchased
dynorphin A used in this study from Bachem AG. Bubendorf,
Chronic morphine decreases dynorphin Switzerland. The chromatography materials were from Pharmacia, Uppsala, Sweden. All other solvents and chemicals were of analytical grade from commercial sources. Statistical analysis The statistical analysis of data was performed by parametric or non-parametric one-way analysis of variance (ANOVA), followed by Scheffe or Kruskal-Wallis tests, respectively. All calculations were carried out with the “Logstat” program.
40
RESULTS
The rate of increase in weight of morphine-treated animals was the same as in controls, only during the first 48 hr [Fig. l(a)]. Four days after implantation of the pump, the weights of animals infused with opiate were significantly smaller than those of controls, although they continued to increase. During the first 24 hr after the pumps were removed (withdrawal) an abrupt loss in weight was observed in morphinetreated rats [Fig. l(a)]. The consumption of water was quite stable in all groups of rats during implantation of the pump (up to 120 hr) and then at 24 hr after
a) --o-
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Fig. 1. The weight (a) and water consumption (b) of the rats during and after implantation of the nimiosmotic pumps with morphine (0) or saline (0). The pumps were implanted at 0 hr and removed at 120hr. Each point represents mean f SEM (n = 6-16). The weight is expressed as the difference between weight at the given time and the weight at the moment of implantation (0 hr). The water consumption was measured every 24 hr. Data are expressed as ml/kg/day. Data were analysed by **P < 0.01as compared to controls at the same ANOVA, followed by Kruskal-Wallis test. *P c 0.05; time.
R. Yu. YUKHANANOVet
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-24
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Fig. 2. The sensitivity to pain during administration of morphine was measured by the paw pressure test (48 PP) and analgesia is expressed as a percentage (see Methods section). The miniosmotic pumps with morphine (0) or saline (0) were implanted subcutaneously at 0 hr. Data are presented as means + SEM (n = 15-17). Data were analysed by ANOVA, followed by Kruskal-Wallis. **P < 0.01 as compared to control at the same time.
removal of the pump it decreased acutely in morphine-treated rats, restoring to the control level 2 days following removal of the pump [abstinence, Fig. l(b)]. At 24 hr after implantation of the pump, a significantly decreased sensitivity to pain was observed in morphine-treated rats using the 48 paw pressure test (Fig. 2). The analgesia disappeared and sensitivity to pain returned to the control levels 48 hr after implantation of the pump. Hyperalgesia was observed using the 32 paw pressure test after removal of the pump. The sensitivity to pain was significantly increased at 18 hr following removal of the pump [Fig. 3(a)]. The locomotor activity slowly decreased after removing the pump but remained the same in both control and morphinetreated rats [Fig. 3(b)]. The levels of dynorphin A in the three control groups (controls to the T-group, W-group and Agroup) did not differ significantly from each other (the data not shown) and therefore all data from these groups were combined into one control group (control). The concentration of dynorphin in this control group was greatest in n. accumbens (Table 1) and smallest in medial frontal cortex. The level of peptide in the medial striatum was more than two times higher than that in the dorsal counterpart (P < 0.001, Scheffe-test). In morphine-infused rats, no changes in levels of dynorphin A were observed in any regions, except for the n. accumbens in the abstinent group, where a significant decrease (33%) was found [F(3,23) = 5.17, P < 0.011. When comparing the abstinent group with its corresponding control group,
the decrease in the level of peptide was even more significant. The level of dynorphin A in tolerance (T-group) and withdrawal (W-group) groups (Table 1) did not differ from control in any regions. In recovery studies of the extraction procedure, it was found that following Sep-Pak separation of the synthetic peptide, about 90% of the immunoreactivity was eluted with 85% methanol. The recovery of peptide after extraction and separation was 65570%. In order to characterize the immunoreactivity, the extract of the whole brain (without cerebellum) was chromatographed by gel-filtration on a Sephadex G-50 column. Almost all immunoreactivity ( > 90%) eluted in one peak, which coincided with the elution volume of synthetic dynorphin A. The dynorphin A immunoreactivity, collected from the gel-filtration run, was further chromatographed using the SMART system. The immunoreactivity was eluted at the same concentration of acetonitrile (25.8%) as the synthetic peptide (data not shown). DISCUSSION
It is evident from this study that the distribution of dynorphin A immunoreactivity varied in different regions of the brain. It was thus found that the level of dynorphin A was low in medial frontal cortex and high in the n. accumbens (Table 1). This finding is in good agreement with previously described distribution pattern of dynorphin A in the brain of the rat (Fallon and Leslie, 1986; Zamir, Palkovits and Brownstein, 1983). In the present experiments, the level of dynorphin A was significantly higher in the medial striatum than in the dorsal striatum. It is
Chronic morphine decreases dynorphin
101
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.
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.
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.
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Fig. 3. The sensitivity b pain (a) and locomotor activity (b) were de&rmined after cessation of administration of morphine (after removal of pump). The miniosmotic pumps with morphine (0) or saline (O), were implanted su~u~neously 120 hr before removal. The abscissa gives the time after removal of pump. Sensitivity to pain (hyperalgesia) was measured by the paw pressure (32 PP) test. The locomotor activity was determined in the quadrant open field and expressed as a number of runs along the walls. Data are expressed as means f SEM (n = 6-12). Data were analysed by ANOVA, followed by Kruskal-Wallis test. *P < 0.05 as compared to control at the same time.
known that the caudopallidal region is not homogeneous and that there is a different regulation of the level of dynorphin A by dopamine (DA) in its medial and dorsal parts (Li, Sam, McGinty, Jang, Douglass, Calevetta and Hong, 1988). The medial parts of
the striatum, olfactory tubercule and n. accumbens belong to the mesolimbic system and receive dopaminergic terminals from the ventral tegmental area (AlO), while the dorsal part receives a dopaminergic input, mainly from substantia nigra and probably
Table 1. The con~ntrationof dynorphinA in variousregionsof the brain of the rat after chronic administration of morphine
- _.. . .
T-!Zrolm w---r
W-IZrouo
A-nrotm
4.7 k 0.6 (16) 16.1 * 1.6 (17) 6.16? 3.1 (14) 31.4* I.6 (18) 13.3 & I.0 (161
6.3 + 1.2 (6) 18.8 f 2.7 (6) 64.7 + 5.4 (5) 35.1 + 2.9 (6) 14.7 * 1.1 161
4.6 +_0.6 (6) 16.7 + 2.1 (6) 59.7 f 3.9 (5) 27.1 f 3.6 (6) 13.2 + 2.8 (5)
4.0 It 0.5 (6) 12.2 & 3.2 (4) 41.0 + 3.3’ (5) 32.1 & 6.5 (4) 17.3 + 1.9 (61
chtro1
Medial frontal cortex Tuberculum olfactorium N. accumbens Medial striatum Dorsal strialum
I
/
v
a
The concentration of dyoorphin was measured by RIA in acidic extracts after Sep-Pak separation. Each column represents means fSEM (n) fmol/mg of tissue. *P < 0.01 (Sch&e test) as compared to control. The T-group (tolerance) was decapitated 120 hr after impIan~tion of the pump; W-group (withdmwa1) was decapitated 20 hr following removal of the pump (144 hr after implan~tion). A-group (abstinence) was decapitated 48 hr after ternoval of the pump (I 68 hr after implantation)
708
R. Yu. YUKHA#AXUVet al,
also regulate the activity in a dyno~hiner~c striato-nigral pathway (Lindvali and Bj&khmd, 1974; Ungerstedt, 1971; Christensson-Nylander, HerreraMarschitz, Staines, Hiikfelt, Terenius, Ungerstedt, Cuello, Oertel and Goldstein, 1986). The mesolimbic system has been suggested to regulate the emotional components of food and water consumption (Cooper, Jackson and Kirham, 1985; Wise, 1983) and the relatively large concentration of dynorphin A in this region provides additional evidence for its role in the physiological regulation of feeding and drinking. The returning of the latency of reaction to pain to control level, 48 hr following implantation of the pump (Fig. 2) indicated the development of tolerance to the analgesic effect of morphine. In spite of the development of tolerance to morphine, no changes in the levels of dynorphin was found. Previously, some alterations in proopiomelanocortinand proenkephalin-derived peptides, after the development of tolerance to morphine were reported (Bronstein, Przewlocki and Akil, 1990; Gudehithlu, Tejwani and Bhargava, 1991; Martinez, Vargas, Fuente, Garzia de1 Rio and Milanes, 1990; Sweep, van Ree and Wiegand, 1988). Moreover, a decrease in the level of prodyno~hin mRNA in the striatum has been observed (Romualdi, Lesa and Ferri, 1990) but these changes do not seem to have any profound effects on the steady-state level of dynorphin in the striatum, where the levels of the peptide remained stable (Table 1) or even seem to increase (Romualdi et al., 1990; Trujillo and Akil, 1990). In this study, the withdrawal state was characterized by a loss of weight, reduced consumption of water and hyperalgesia. These signs reached their maximum at 18-26 hr after removal of the miniosmotic pump. Although “strong” signs of abstinence, like diarrhoea, irritability, teeth chattering, etc. (Wei, Loh and Shen, 1969) were not seen, the observed items clearly indicated when the rat reached this stage. One possible explanation for the lack of pronounced overt signs of withdrawal may be the route of administration of morphine. Xn this study, miniosmotic pumps were used from which the drug was released in relatively small doses, while in most studies implantation of pellets or intermittent injections were used. In this study, the withdrawal almost disappeared 48 hr after removing the pump, although the weights of morphine-infused animals were still significantly smaller than those of controls but the rate of the increase in weight was the same as in all rats. The only alteration in the concentration of dynorphin in the present experiment occurred in the n. accumbens only during abstinence, after disappearance of the signs of withdrawal (Table 1). The level of peptide was significantly decreased. It is interesting that the level of dynorphin was changed only in the n. accumbens, an area of brain, known to be involved in the control of the reward process (Wise, 1989). It has been shown that dynorphin may reinforce the
consummator reward and this action of the peptide is mediated through the limbic system, possibly through the n. accumbens (Cooper et al., 1985; Smith and Lee, 1988; Wise, 1983). This region is found to be involved in the reward action of many narcotics (Koob, Vaccarini, Amalric and Bloom, 1987; Koob, Wall and Bloom, 1984; Wise 1989) but dynorphin itself was shown to induce aversive behaviour, following injection in the n. accumbens (Bals-Kubick, Shippenberg and Herz, 1990). It is therefore difficult to understand what the observed decrease in the level of dynorphin reflects. However, it is inviting to propose that it may be connected with the alleviation of the withdrawal signs, like reducing consumption of water and food. In conclusion, this study does not indicate any changes in the level of dynorphin A in the medial frontal cortex, dorsal striatum and mesolimbic system, after the development of tolerance and withdrawal. In the abstinence state, however, the level of dynorphin was significantly lowered but only in the n. accumbens. From this and previously published results (Nylander et al., 1991) it is suggested that dynorphin does not participate in the development of tolerance to opiates, but rather in the mechanism overcoming withdrawal and abstinence. Acknowledgements-This study was supported by the National Institute of Drug Abuse (NIDA) and the Swedish Medical Research Council (grant 9459 and 10357). The authors are indebted to Dr I. Nylander, Karolinska Institute for helpful discussions.
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0028-3908/93 WlO+O.OO Copyright 0 1993 Pergamon Press Ltd
CHRONIC ADMINISTRATION OF MORPHINE DECREASES LEVEL OF DYNORPHIN A IN THE RAT NUCLEUS ACCUMBENS R. Yu. YUKHANANOV,’Q. Z. ZHAI,~ S. PERSSON,~ C. POSTS and F. NYBERC~* ‘Department of Pharmacology, Russian Academy of Medical Sciences, Baltiskaya St. 8, Moscow, Russia and 2Department of Pharmaceutical Biosciences, Division of Pharmacology, Uppsala University, P.O. Box 591, Uppsala, Sweden (Accepted 28 December 1992) Summary-The effect of chronically administered morphine on the levels of dynorphin A in distinct regions of the brain (including medial frontal cortex, olfactory tubercule, nucleus accumbens, dorsal and medial striatum), was determined in male Sprague-Dawley rats. The drug was delivered through a subcutaneously implanted Azlet miniosmotic pump over a period of 5 days. The concentration of peptide was probed by radioimmunoassay, following pre-separation of tissue extracts by reversed phase separation on a SepPak C-18 cartridge. The result showed that the level of dynorphin A remained unaltered in all regions studied immediately before (tolerance) and 20 hr after (withdrawal) the pump was removed. A significant decrease in the level of dynorphin was found in the n. accumbens 48 hr (abstinence) after removal of the pump. It is suggested that previously observed changes in the reward system during abstinence may be connected with dynorphinergic neurones in the limbic system. Key words-morphine
dependence, dynorphin A, miniosmotic pump, hyperalgesia, n. accumbens.
Extensive studies have been carried out in order to elucidate the role of opioid peptides in the consequences of opiate addiction. Many attempts to find any change in the level of e.g. Met-enkephalin, Leuenkephalin and dynorphins in the brain of morphinetreated animals have failed (Childers, Simantov and Snyder, 1977; Fratta, Yang, Hong and Costa, 1977; Ho, Way, Chan, Cheng, Kwok and Wen, 1982; Shani, Azov and Weisman, 1979; Wesche, Hollt and Herz, 1977). However, in a recent study a reduction in the level of dynorphin A(l-17) in the spinal cord of the rat 24 hr after cessation of chronic administration of morphine was found (Nylander, Sakaruda, Le GrevCs and Terenius, 1991). It was recently found that dynorphin A(l-13) was reduced in the spinal cord but increased in the pituitary and hypothalamus, 18 hr after removal of morphine pellets in morphinetolerant rats (Rattan, Kao, Tejawani and Bhargava, 1992). Studies have also revealed an increase in dynorphin-related peptides after 7 days of treatment with opiate (Trujillo and Akil, 1990). It is well known that the development of dependence on opiate has a complex dynamic and is also affected by the paradigm of administration of opioids (Shani et al., 1979). In this study, it was decided to determine the levels of dynorphin at all the different stages of the opiate dependence, namely tolerance, withdrawal and abstinence. It is clear that these *Address correspondence to: Dr Fred Nyberg, Division of Pharmacology, Sweden.
BMC, Box 591, S-751 24 Uppsala,
stages have qualitative differences but the time-table of their appearance strongly depends on the route of administration of the narcotic and therefore, in this study, their appearance was checked by testing the behaviour of the animals. Morphine was administrated through miniosmotic pumps, implanted subcutaneously on the back of male rats (Persson, Post, Alari, Nyberg and Terenius, 1989). The level of dynorphin A(l-17) was measured in areas of the brain corresponding to the mesolimbic, mesostriatal and mesocortical dopamine system, which are known to mediate the euphoric effects of many narcotics. METHODS Animal experiment
Male Sprague-Dawley rats (Alab, Sweden), weighing 220-240 g at the beginning of the experiment, were used. The animals were housed in a room with controlled humidity (60%) and temperature (24°C) and with a fixed lighting schedule (light from 9-21 hr). The water and food were given ad libitum. The rats were adapted to the environment at least 1 week before the start of the experiment. One day before implantation of the pump, the animals were placed in individual cages with transparent walls, where they were kept until the end of the experiment. The rats were divided randomly into three groups, each consisting of 6 experimental and 6 control animals. Miniosmotic pumps (Alzet, 2MLl) were filled with morphine acetate (70 mg/ml) or saline and were implanted subcutaneously on the back under 703
704
R. Yl_l. ~UKHANANOV
diethyl ether anaesthesia. Five days (12&124 hr) later, the pumps were removed. Animals in the first group (tolerance or T-group) were decapitated after 120 hr, just before removal of the pumps. The second group of animals (withdrawal or W-group) were decapitated 20 hr after the pumps were removed (144 hr after implantation). Rats in the third group (abstinence or A-group) were decapitated 48 hr following removal of the pump. Every day the rats were weighed and their water consumption was determined. The nociceptive sensitivity
The nociceptive sensitivity was measured by the paw pressure test. The animals were tested before implantation (-24 hr) and at 6, 12, 24 hr and subsequently at every 24 hr after implantation of the pump. The paw pressure (PP) test was performed using a commercially available unit (Ugo Basile, Comerio, Italy). The paw pressure was applied through a ballet shaped plastic tip pressure device to the plantar surface of the hind paw, with continuously increasing pressure at a rate of 480mN/sec (48 PP) or 320 mN/sec (32 PP). The pressure at which a rapid removal of the hind paw was observed was assigned as a response threshold. The cut off pressures were 6 and 3 N, respectively. Analgesia and hyperalgesia were calculated according to the following expressions: Analgesia = (X - phone latency)/ (cut off - phone latency) x 100% Hyperalgesia = (cut off - X)/cut off x lOO%, X-latency before and after pump implantation, phone latency-the reaction at 0 hr, just before implantation of the pump. Locomotor activity
Locomotor activity (LA) was measured in the open field after removal of the pump. The animals were placed in a quadrant box for 5 min (1 x 1 m) with a lamp placed over the center. The number of movements along each wall was recorded as an index of locomotor activity. Tissue extraction
After decapitation, the brain was immediately removed and placed on ice. The factory tubercule was cut out by scissors as described elsewhere (Heyna, Hogenboam, Portoghese, Mulder and Padt, Schoffelmeer, 1990) and the brain was dissected on ice, using a rat brain matrix (Activational systems Inc., Forterra Drive Warren, Michigan, U.S.A.) in coronal slices (1 mm). All further dissections were performed according to a stereotaxic atlas (Paxinos and Watson, 1986) and the distance are given from the interaural line (mm). The medial frontal cortex was taken from the slices (13.8-11.8), nucleus accumbens from the next two slices (11.8-9.8) and striatum
et al.
(Caudatus putamen, 10.7-7.7) was dissected into the medioventral and dorsolateral parts. The extraction of brain tissue was performed as described earlier (Christensson-Nylander and Terenius, 1985) with some modifications (i.e. preseparation of extracts was performed using SepPak cartridges instead of SPSephadex mini-columns). The extracts were thus run through reversed-phase Sep-Pak Cl8 cartridges, prewashed with 10ml of 100% methanol (MeOH) and lOm1 water. After adding the acidic extract, the cartridge was washed with 4 ml of water and 4 ml of 20% MeOH, before elution with 4ml of 85% methanol [all solutions contained 0.04% trifluoroacetic acid (TFA)]. The eluates were collected and after evaporation (Savant Vat, Savant, Hicksville, New York) the samples were dissolved in MeOH/O. 1M HCl (1: 1, v/v) before radioimmunoassay. Chromatography
Extracts of the brain tissue were chromatographed on a Sephadex G-SO column (2.5 x 40 cm) with 1M acetic acid containing 10 mM NaCl at a flow rate of 30 ml/hr and fractions of 2 ml were collected, evaporated (Savant Vat) and dissolved in MeOH/O. 1M HCl (1: 1, v/v) before analysis by radioimmunoassay. A new chromatographic system. SMARTTM system (Pharmacia, Uppsala, Sweden), equipped with a mRP-column C2/Cl8 3.2/3, was used for further analysis of the immunoreactive material. The column was developed with a linear gradient of acetonitrile (o-60%), containing 0.04% TFA. The flow rate was 0.24 ml/min and fractions of 0.2 ml were collected. Radioimmunoassay (RIA)
The antiserum (denoted 84) was raised in rabbits against the peptide-thyroglobulin conjugate and the iodinated peptide was used as a tracer. The characteristics of the antiserum has been described elsewhere (Christensson-Nylander and Terenius, 1985). The radioimmunoassay (RIA) for dynorphin A( l-l 7) was based on the non-immune pre-separation method and conducted as described previously (ChristenssonNylander, Nybert, Ragnarsson and Terenius, 1985; Nyberg, Christensson-Nylander and Terenius, 1987) with the exception that the bound peptide was separated from unbound using a sheep anti-rabbit antibody (Pharmacia Decanting Suspension, 200 ml of a 4 times diluted solution). The crossreactivity of the with dynorphin A( l-8), dynorphin antiserum A(I-13), dynorphin B, a-neoendorphin, Leu-enkephalin and Leu-enkaphahn-Arg6 was less than 0.1% and with dynorphin A(7-17) 100%. The detection limit of the RIA was 334 fmol/tube. Peptide and chemicals
The synthetic was purchased
dynorphin A used in this study from Bachem AG. Bubendorf,
Chronic morphine decreases dynorphin Switzerland. The chromatography materials were from Pharmacia, Uppsala, Sweden. All other solvents and chemicals were of analytical grade from commercial sources. Statistical analysis The statistical analysis of data was performed by parametric or non-parametric one-way analysis of variance (ANOVA), followed by Scheffe or Kruskal-Wallis tests, respectively. All calculations were carried out with the “Logstat” program.
40
RESULTS
The rate of increase in weight of morphine-treated animals was the same as in controls, only during the first 48 hr [Fig. l(a)]. Four days after implantation of the pump, the weights of animals infused with opiate were significantly smaller than those of controls, although they continued to increase. During the first 24 hr after the pumps were removed (withdrawal) an abrupt loss in weight was observed in morphinetreated rats [Fig. l(a)]. The consumption of water was quite stable in all groups of rats during implantation of the pump (up to 120 hr) and then at 24 hr after
a) --o-
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Fig. 1. The weight (a) and water consumption (b) of the rats during and after implantation of the nimiosmotic pumps with morphine (0) or saline (0). The pumps were implanted at 0 hr and removed at 120hr. Each point represents mean f SEM (n = 6-16). The weight is expressed as the difference between weight at the given time and the weight at the moment of implantation (0 hr). The water consumption was measured every 24 hr. Data are expressed as ml/kg/day. Data were analysed by **P < 0.01as compared to controls at the same ANOVA, followed by Kruskal-Wallis test. *P c 0.05; time.
R. Yu. YUKHANANOVet
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-24
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Fig. 2. The sensitivity to pain during administration of morphine was measured by the paw pressure test (48 PP) and analgesia is expressed as a percentage (see Methods section). The miniosmotic pumps with morphine (0) or saline (0) were implanted subcutaneously at 0 hr. Data are presented as means + SEM (n = 15-17). Data were analysed by ANOVA, followed by Kruskal-Wallis. **P < 0.01 as compared to control at the same time.
removal of the pump it decreased acutely in morphine-treated rats, restoring to the control level 2 days following removal of the pump [abstinence, Fig. l(b)]. At 24 hr after implantation of the pump, a significantly decreased sensitivity to pain was observed in morphine-treated rats using the 48 paw pressure test (Fig. 2). The analgesia disappeared and sensitivity to pain returned to the control levels 48 hr after implantation of the pump. Hyperalgesia was observed using the 32 paw pressure test after removal of the pump. The sensitivity to pain was significantly increased at 18 hr following removal of the pump [Fig. 3(a)]. The locomotor activity slowly decreased after removing the pump but remained the same in both control and morphinetreated rats [Fig. 3(b)]. The levels of dynorphin A in the three control groups (controls to the T-group, W-group and Agroup) did not differ significantly from each other (the data not shown) and therefore all data from these groups were combined into one control group (control). The concentration of dynorphin in this control group was greatest in n. accumbens (Table 1) and smallest in medial frontal cortex. The level of peptide in the medial striatum was more than two times higher than that in the dorsal counterpart (P < 0.001, Scheffe-test). In morphine-infused rats, no changes in levels of dynorphin A were observed in any regions, except for the n. accumbens in the abstinent group, where a significant decrease (33%) was found [F(3,23) = 5.17, P < 0.011. When comparing the abstinent group with its corresponding control group,
the decrease in the level of peptide was even more significant. The level of dynorphin A in tolerance (T-group) and withdrawal (W-group) groups (Table 1) did not differ from control in any regions. In recovery studies of the extraction procedure, it was found that following Sep-Pak separation of the synthetic peptide, about 90% of the immunoreactivity was eluted with 85% methanol. The recovery of peptide after extraction and separation was 65570%. In order to characterize the immunoreactivity, the extract of the whole brain (without cerebellum) was chromatographed by gel-filtration on a Sephadex G-50 column. Almost all immunoreactivity ( > 90%) eluted in one peak, which coincided with the elution volume of synthetic dynorphin A. The dynorphin A immunoreactivity, collected from the gel-filtration run, was further chromatographed using the SMART system. The immunoreactivity was eluted at the same concentration of acetonitrile (25.8%) as the synthetic peptide (data not shown). DISCUSSION
It is evident from this study that the distribution of dynorphin A immunoreactivity varied in different regions of the brain. It was thus found that the level of dynorphin A was low in medial frontal cortex and high in the n. accumbens (Table 1). This finding is in good agreement with previously described distribution pattern of dynorphin A in the brain of the rat (Fallon and Leslie, 1986; Zamir, Palkovits and Brownstein, 1983). In the present experiments, the level of dynorphin A was significantly higher in the medial striatum than in the dorsal striatum. It is
Chronic morphine decreases dynorphin
101
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.
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.
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.
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Fig. 3. The sensitivity b pain (a) and locomotor activity (b) were de&rmined after cessation of administration of morphine (after removal of pump). The miniosmotic pumps with morphine (0) or saline (O), were implanted su~u~neously 120 hr before removal. The abscissa gives the time after removal of pump. Sensitivity to pain (hyperalgesia) was measured by the paw pressure (32 PP) test. The locomotor activity was determined in the quadrant open field and expressed as a number of runs along the walls. Data are expressed as means f SEM (n = 6-12). Data were analysed by ANOVA, followed by Kruskal-Wallis test. *P < 0.05 as compared to control at the same time.
known that the caudopallidal region is not homogeneous and that there is a different regulation of the level of dynorphin A by dopamine (DA) in its medial and dorsal parts (Li, Sam, McGinty, Jang, Douglass, Calevetta and Hong, 1988). The medial parts of
the striatum, olfactory tubercule and n. accumbens belong to the mesolimbic system and receive dopaminergic terminals from the ventral tegmental area (AlO), while the dorsal part receives a dopaminergic input, mainly from substantia nigra and probably
Table 1. The con~ntrationof dynorphinA in variousregionsof the brain of the rat after chronic administration of morphine
- _.. . .
T-!Zrolm w---r
W-IZrouo
A-nrotm
4.7 k 0.6 (16) 16.1 * 1.6 (17) 6.16? 3.1 (14) 31.4* I.6 (18) 13.3 & I.0 (161
6.3 + 1.2 (6) 18.8 f 2.7 (6) 64.7 + 5.4 (5) 35.1 + 2.9 (6) 14.7 * 1.1 161
4.6 +_0.6 (6) 16.7 + 2.1 (6) 59.7 f 3.9 (5) 27.1 f 3.6 (6) 13.2 + 2.8 (5)
4.0 It 0.5 (6) 12.2 & 3.2 (4) 41.0 + 3.3’ (5) 32.1 & 6.5 (4) 17.3 + 1.9 (61
chtro1
Medial frontal cortex Tuberculum olfactorium N. accumbens Medial striatum Dorsal strialum
I
/
v
a
The concentration of dyoorphin was measured by RIA in acidic extracts after Sep-Pak separation. Each column represents means fSEM (n) fmol/mg of tissue. *P < 0.01 (Sch&e test) as compared to control. The T-group (tolerance) was decapitated 120 hr after impIan~tion of the pump; W-group (withdmwa1) was decapitated 20 hr following removal of the pump (144 hr after implan~tion). A-group (abstinence) was decapitated 48 hr after ternoval of the pump (I 68 hr after implantation)
708
R. Yu. YUKHA#AXUVet al,
also regulate the activity in a dyno~hiner~c striato-nigral pathway (Lindvali and Bj&khmd, 1974; Ungerstedt, 1971; Christensson-Nylander, HerreraMarschitz, Staines, Hiikfelt, Terenius, Ungerstedt, Cuello, Oertel and Goldstein, 1986). The mesolimbic system has been suggested to regulate the emotional components of food and water consumption (Cooper, Jackson and Kirham, 1985; Wise, 1983) and the relatively large concentration of dynorphin A in this region provides additional evidence for its role in the physiological regulation of feeding and drinking. The returning of the latency of reaction to pain to control level, 48 hr following implantation of the pump (Fig. 2) indicated the development of tolerance to the analgesic effect of morphine. In spite of the development of tolerance to morphine, no changes in the levels of dynorphin was found. Previously, some alterations in proopiomelanocortinand proenkephalin-derived peptides, after the development of tolerance to morphine were reported (Bronstein, Przewlocki and Akil, 1990; Gudehithlu, Tejwani and Bhargava, 1991; Martinez, Vargas, Fuente, Garzia de1 Rio and Milanes, 1990; Sweep, van Ree and Wiegand, 1988). Moreover, a decrease in the level of prodyno~hin mRNA in the striatum has been observed (Romualdi, Lesa and Ferri, 1990) but these changes do not seem to have any profound effects on the steady-state level of dynorphin in the striatum, where the levels of the peptide remained stable (Table 1) or even seem to increase (Romualdi et al., 1990; Trujillo and Akil, 1990). In this study, the withdrawal state was characterized by a loss of weight, reduced consumption of water and hyperalgesia. These signs reached their maximum at 18-26 hr after removal of the miniosmotic pump. Although “strong” signs of abstinence, like diarrhoea, irritability, teeth chattering, etc. (Wei, Loh and Shen, 1969) were not seen, the observed items clearly indicated when the rat reached this stage. One possible explanation for the lack of pronounced overt signs of withdrawal may be the route of administration of morphine. Xn this study, miniosmotic pumps were used from which the drug was released in relatively small doses, while in most studies implantation of pellets or intermittent injections were used. In this study, the withdrawal almost disappeared 48 hr after removing the pump, although the weights of morphine-infused animals were still significantly smaller than those of controls but the rate of the increase in weight was the same as in all rats. The only alteration in the concentration of dynorphin in the present experiment occurred in the n. accumbens only during abstinence, after disappearance of the signs of withdrawal (Table 1). The level of peptide was significantly decreased. It is interesting that the level of dynorphin was changed only in the n. accumbens, an area of brain, known to be involved in the control of the reward process (Wise, 1989). It has been shown that dynorphin may reinforce the
consummator reward and this action of the peptide is mediated through the limbic system, possibly through the n. accumbens (Cooper et al., 1985; Smith and Lee, 1988; Wise, 1983). This region is found to be involved in the reward action of many narcotics (Koob, Vaccarini, Amalric and Bloom, 1987; Koob, Wall and Bloom, 1984; Wise 1989) but dynorphin itself was shown to induce aversive behaviour, following injection in the n. accumbens (Bals-Kubick, Shippenberg and Herz, 1990). It is therefore difficult to understand what the observed decrease in the level of dynorphin reflects. However, it is inviting to propose that it may be connected with the alleviation of the withdrawal signs, like reducing consumption of water and food. In conclusion, this study does not indicate any changes in the level of dynorphin A in the medial frontal cortex, dorsal striatum and mesolimbic system, after the development of tolerance and withdrawal. In the abstinence state, however, the level of dynorphin was significantly lowered but only in the n. accumbens. From this and previously published results (Nylander et al., 1991) it is suggested that dynorphin does not participate in the development of tolerance to opiates, but rather in the mechanism overcoming withdrawal and abstinence. Acknowledgements-This study was supported by the National Institute of Drug Abuse (NIDA) and the Swedish Medical Research Council (grant 9459 and 10357). The authors are indebted to Dr I. Nylander, Karolinska Institute for helpful discussions.
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