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Keywords:

  • caudate–putamen;
  • dopaminergic D1 receptors;
  • drugs of abuse;
  • nucleus accumbens;
  • protein kinase A activity

Abstract

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Animals
  5. Induction of cocaine sensitization
  6. Induction of morphine sensitization
  7. Immunoblotting
  8. Experimental protocols
  9. Experiment 1: effect of cocaine sensitization on DARPP-32 phosphorylation pattern
  10. Experiment 2: effect of morphine sensitization on DARPP-32 phosphorylation pattern
  11. Experiment 3: time course of the effect of acute morphine exposure on DARPP-32 phosphorylation pattern in control and morphine-sensitized rats
  12. Tissue processing
  13. Drugs
  14. Statistical analysis
  15. Results
  16. Effect of cocaine sensitization on DARPP-32 phosphorylation pattern
  17. Effect of morphine sensitization on DARPP-32 phosphorylation pattern
  18. Time-course of the effect of acute morphine exposure on DARPP-32 phosphorylation in control and morphine-sensitized rats
  19. Discussion
  20. Acknowledgement
  21. References

This study reports some of the modifications in dopaminergic signalling that accompany cocaine and morphine behavioural sensitization. Cocaine-sensitized rats showed increased phosphorylation of dopamine- and cyclic AMP-regulated phosphoprotein Mr 32 kDa (DARPP-32) at threonine-75 (Thr75) and decreased DARPP-32 phosphorylation at Thr34, in the caudate–putamen (CPu) and nucleus accumbens (NAc) 7 days after sensitization assessment. Conversely, in morphine-sensitized rats, no apparent modifications in DARPP-32 phosphorylation pattern were observed. Morphine-sensitized rats have increased binding and coupling of µ-opioid receptors and increased dopaminergic transmission in striatal areas and, upon morphine challenge, exhibit dopamine D1 receptor-dependent stereotypies. Thus, the DARPP-32 phosphorylation pattern was studied in morphine-sensitized rats at different times after morphine challenge. Morphine challenge increased levels of phospho-Thr75 DARPP-32 and decreased levels of phospho-Thr34 DARPP-32 in a time-dependent manner in the CPu and NAc. In order to assess whether these modifications were related to modified cyclic AMP-dependent protein kinase (PKA) activity, the phosphorylation levels of two other PKA substrates were examined, the GluR1 and NR1 subunits of α-amino-3-hydroxy-5-methylisoxazole-4-propionate and NMDA receptors respectively. The phosphorylation levels of GluR1 and NR1 subunits decreased in parallel with those of phospho-Thr-34 DARPP-32, supporting the hypothesis that morphine challenge elicited a decrease in PKA activity in morphine-sensitized rats.

Abbreviations used
Cdk5

cyclin-dependent kinases

CPu

caudate–putamen

DA

dopamine

DARPP

DA and cyclic AMP-regulated phosphoprotein

NAc

nucleus accumbens

PKA

cyclic AMP-dependent protein kinase

Repeated exposure to drugs of abuse causes enduring cellular changes (Hope et al. 1992; Self and Nestler 1995) that may mediate long-lasting behavioural modifications, such as sensitization. Sensitization, or reverse-tolerance, is a condition in which the repeated administration of a drug dose elicits increasing behavioural and neurochemical effects. Once established, the behavioural modifications can be retrieved even after prolonged periods of drug abstinence by environmental cues, stress or additional drug exposure (Nestler 2001). Despite the different mechanisms of action, repeated administration of many drugs of abuse share some common effects, such as perturbation of the mesolimbic dopaminergic system (Robinson and Becker 1986; Kalivas and Stewart 1991). Dopamine (DA) signalling in the striatum is mediated through adenylyl cyclase activation by D1-like receptors or inhibition by D2-like receptors (Stoof and Kebabian 1981). DA and cyclic AMP-regulated phosphoprotein (Mr 32 kDa) (DARPP-32) (Walaas et al. 1983) is a major target protein for the cyclic AMP–cyclic AMP-dependent protein kinase (PKA) pathway. This protein is highly concentrated in the striatum and in the olfactory tubercle (Ouimet et al. 1984) where it plays a central role in the integration of synaptic inputs to these brain regions. The function of DARPP-32 is determined by its phosphorylation state. DA, by means of D1 receptor-mediated activation of PKA, phosphorylates DARPP-32 at threonine-34 (Thr34) and thereby converts DARPP-32 into a potent inhibitor of protein phosphatase-1 (Hemmings et al. 1984). Phosphorylation at threonine-75 (Thr75) by cyclin-dependent kinase 5 (Cdk5) converts DARPP-32 into an inhibitor of PKA (Bibb et al. 1999) and antagonizes the PKA–Thr34-DARPP-32–protein phosphatase-1 cascade. That is, DARPP-32 can act either as a phosphatase inhibitor or as a kinase inhibitor, depending on its relative state of phosphorylation at the PKA and Cdk5 sites. The acute administration of cocaine activates PKA, increases DARPP-32 phosphorylation at Thr34, and causes a relative decrease in Thr75 phosphorylation (Nishi et al. 2000). Conversely, repeated cocaine treatment induces an increased expression of Cdk5, and a steady, significant increase in the Thr75 phosphorylated species of DARPP-32 (Bibb et al. 2001).

The first aim of the present study was to investigate whether the modifications induced by repeated treatment with cocaine on the DARPP-32 phosphorylation state were long-lasting and possibly related to cocaine-induced behavioural sensitization. Cocaine and morphine sensitization are commonly considered to be similar processes that might contribute to the increased risk of relapse after withdrawal (Nestler 2001). Repeated treatment with these drugs of abuse has been reported to induce, in the caudate–putamen (CPu) and nucleus accumbens (NAc), accumulation of ΔFosB, a complex transcription factor that has a remarkably long half-life (Hope et al. 1994; Nye and Nestler 1996), and that may play a role in adaptive neuronal responses. However, to our knowledge, no data are available on the phosphorylation state of DARPP-32 in morphine-sensitized rats. Thus, the second aim of the present study was to investigate whether rats repeatedly treated with morphine would present, as observed after cocaine, basal modifications in the DARPP-32 phosphorylation pattern, and, if this were the case, whether such modifications were temporally related to morphine-induced behavioural sensitization. In order to assess whether the observed modifications in DARPP-32 phosphorylation pattern were related to a possible modification in PKA activity, we also measured the degree of phosphorylation of two other PKA substrates, that is the glutamate receptor subunits GluR-1 and NR-1 (Tingley et al. 1993; Snyder et al. 2000).

Animals

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Animals
  5. Induction of cocaine sensitization
  6. Induction of morphine sensitization
  7. Immunoblotting
  8. Experimental protocols
  9. Experiment 1: effect of cocaine sensitization on DARPP-32 phosphorylation pattern
  10. Experiment 2: effect of morphine sensitization on DARPP-32 phosphorylation pattern
  11. Experiment 3: time course of the effect of acute morphine exposure on DARPP-32 phosphorylation pattern in control and morphine-sensitized rats
  12. Tissue processing
  13. Drugs
  14. Statistical analysis
  15. Results
  16. Effect of cocaine sensitization on DARPP-32 phosphorylation pattern
  17. Effect of morphine sensitization on DARPP-32 phosphorylation pattern
  18. Time-course of the effect of acute morphine exposure on DARPP-32 phosphorylation in control and morphine-sensitized rats
  19. Discussion
  20. Acknowledgement
  21. References

Experiments were carried out on male Sprague–Dawley rats (Charles River, Calco, Italy) weighing 125–150 g at their arrival in the vivarium. Animals were housed five per cage (59 × 38.5 × 20 cm) for the entire duration of the experiments; they were moved to a different cage or apparatus only for the time required for behavioural manipulation. They were kept in an environment maintained at a constant temperature and humidity, with free access to food and water. A 12-h inverted light–dark cycle (07.00 hours lights off, 19.00 hours on) was used. Experiments were carried out between 09.00 and 17.00 hours under a red light and controlled-noise conditions in a testing room separated from and adjacent to the main animal room, under the same conditions of temperature and humidity. Rats were allowed at least 1 week of habituation to the animal colony and when experimental procedures began they weighed 200–225 g.

The procedures used in this study were in strict accordance with the European legislation on the use and care of laboratory animals (EEC Council Directive 86/609), with the guidelines of the National Institutes of Health on the use and care of laboratory animals, and were approved by the University of Siena Ethics Committee.

Induction of cocaine sensitization

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Animals
  5. Induction of cocaine sensitization
  6. Induction of morphine sensitization
  7. Immunoblotting
  8. Experimental protocols
  9. Experiment 1: effect of cocaine sensitization on DARPP-32 phosphorylation pattern
  10. Experiment 2: effect of morphine sensitization on DARPP-32 phosphorylation pattern
  11. Experiment 3: time course of the effect of acute morphine exposure on DARPP-32 phosphorylation pattern in control and morphine-sensitized rats
  12. Tissue processing
  13. Drugs
  14. Statistical analysis
  15. Results
  16. Effect of cocaine sensitization on DARPP-32 phosphorylation pattern
  17. Effect of morphine sensitization on DARPP-32 phosphorylation pattern
  18. Time-course of the effect of acute morphine exposure on DARPP-32 phosphorylation in control and morphine-sensitized rats
  19. Discussion
  20. Acknowledgement
  21. References

Sensitization was induced by administering cocaine (40 mg/kg, i.p. every other day) for a total of 14 days. All cocaine (or saline) treatments were carried out in locomotor activity cages (M/P40 Fc Electronic Motility Meter; Motor Products, Stockholm, Sweden). Rats were placed individually in the cage for 25 min after cocaine (or saline) administration to strengthen environmental contingencies and to favour the development of sensitization. At the end of the 14-day protocol, animals were left undisturbed for 10 days before behavioural sensitization was assessed. Sprague–Dawley rats present a high degree of variability in their sensitivity to cocaine. In our experimental conditions, repeated administration of a low dose of cocaine (10 mg/kg) induced a sensitized response to cocaine challenge in only 70% of animals, which showed stereotypies of moderate intensity. The present protocol (cocaine 40 mg/kg on alternate days for 14 days) was selected as it consistently yields a sensitized response to a 10 mg/kg cocaine challenge in 90–95% of treated rats, with intense stereotypies that are absent in control animals (Gambarana et al. 1998).

In order to assess sensitization, rats were placed individually in locomotor activity cages and were injected with cocaine (10 mg/kg, i.p.). Locomotor activity was defined as the number of beam interruptions during the observation period. Stereotypy ratings were scored by a skilled experimenter who was unaware of the experimental group, according to the rating scale devised by Creese and Iversen (1975) and modified as follows: 0, asleep or motionless; 1, active; 2, active with intermittent bursts of sniffing; 3, discontinuous stereotyped sniffing, head-weaving and rearing; 4, frequent stereotyped sniffing, head-weaving, turning, cage crossing and rearing; 5, continuous stereotyped sniffing, head-weaving, turning and rearing, frequently confined in a corner; 6, continuous, intense stereotypies that disrupt gross motility.

Observation sessions lasted a total of 45 min; the first 5 min after injection were excluded; total locomotor activity counts and the cumulative score reflecting stereotypy activity during the final 40 min were recorded for each rat.

Induction of morphine sensitization

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Animals
  5. Induction of cocaine sensitization
  6. Induction of morphine sensitization
  7. Immunoblotting
  8. Experimental protocols
  9. Experiment 1: effect of cocaine sensitization on DARPP-32 phosphorylation pattern
  10. Experiment 2: effect of morphine sensitization on DARPP-32 phosphorylation pattern
  11. Experiment 3: time course of the effect of acute morphine exposure on DARPP-32 phosphorylation pattern in control and morphine-sensitized rats
  12. Tissue processing
  13. Drugs
  14. Statistical analysis
  15. Results
  16. Effect of cocaine sensitization on DARPP-32 phosphorylation pattern
  17. Effect of morphine sensitization on DARPP-32 phosphorylation pattern
  18. Time-course of the effect of acute morphine exposure on DARPP-32 phosphorylation in control and morphine-sensitized rats
  19. Discussion
  20. Acknowledgement
  21. References

Sensitization was induced by administering morphine (10 mg per kg per day, s.c.) for 7 days. All treatments were carried out in cages with a grid floor. At the end of the 7-day protocol rats were left undisturbed for a week before behavioural sensitization was assessed.

Rats were placed individually in locomotor activity cages (M/P40 Fc Electronic Motility Meter), given a 15-min habituation period, and then injected with morphine (5 mg/kg, s.c.). Locomotor activity was defined as the number of interruptions of a beam during the observation period. Stereotypy ratings were scored from 0 to 6 by experimenters unaware of the treatment according to a rating scale derived from that used for cocaine: 0, asleep or motionless; 1, active; 2, active with intermittent bursts of stereotypies; 3, discontinuous stereotyped sniffing, licking and biting; 4, frequent stereotyped sniffing, licking and biting; 5, continuous stereotyped licking and biting; 6, continuous, intense oral stereotypies that disrupt gross motility.

Observation sessions lasted a total of 95 min; the first 5 min after injection were excluded; total locomotor activity counts and the cumulative score reflecting stereotypy activity during the final 90 min were recorded for each rat.

Immunoblotting

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Animals
  5. Induction of cocaine sensitization
  6. Induction of morphine sensitization
  7. Immunoblotting
  8. Experimental protocols
  9. Experiment 1: effect of cocaine sensitization on DARPP-32 phosphorylation pattern
  10. Experiment 2: effect of morphine sensitization on DARPP-32 phosphorylation pattern
  11. Experiment 3: time course of the effect of acute morphine exposure on DARPP-32 phosphorylation pattern in control and morphine-sensitized rats
  12. Tissue processing
  13. Drugs
  14. Statistical analysis
  15. Results
  16. Effect of cocaine sensitization on DARPP-32 phosphorylation pattern
  17. Effect of morphine sensitization on DARPP-32 phosphorylation pattern
  18. Time-course of the effect of acute morphine exposure on DARPP-32 phosphorylation in control and morphine-sensitized rats
  19. Discussion
  20. Acknowledgement
  21. References

Frozen tissue samples from rat brain were prepared by solubilization in 1% sodium dodecyl sulfate and 50 mm NaF. Aliquots containing 30–50 µg total protein were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis (12% polyacrylamide gel for DARPP-32, 7.5% polyacrylamide gel for α-amino-3-hydroxy-5-methylisoxazole-4-propionate and NMDA receptor subunits) and transferred to nitrocellulose membranes. Small aliquots of the homogenate were retained for protein determination by the Lowry protein assay method using bovine serum albumin as a standard (Lowry et al. 1951). Immunoblotting was carried out with phosphorylation state-specific antibodies against phospho-Thr34 DARPP-32 and phospho-Thr75 DARPP-32 (Cell Signalling Technology, Beverly, MA, USA), phospho-Ser845 GluR1 and phospho-Ser897 NR1 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), or with non- phosphorylation state-specific antibodies against total DARPP-32 (Cell Signalling Technology), total GluR1 and NR1 (Santa Cruz Biotechnology) and Cdk5 (Santa Cruz Biotechnology). Antibody binding was detected using a chemiluminescence detection system (Pierce Biotechnology Inc., Rockford, IL, USA) and quantified with the Versa Doc 1000 Imaging System (Bio-Rad Laboratories, Hercules, CA, USA). Samples containing the same amount of total protein from control and treated rats were run on the same immunoblots and analysed together. For each experiment, values obtained from treated rats were calculated as the percentage of control values.

Levels of total DARPP-32 and total GluR1 and NR1 were not modified compared with levels in control groups by any of the treatments in either the NAc or CPu (data not shown).

A group of rats were subjected to the cocaine sensitization protocol (n = 12; COCA); a second group of rats received saline (1 mL/kg i.p.) every other day for 14 days (n = 8, CTR 1). Both groups were challenged with cocaine (10 mg/kg i.p.) after a 10-day washout period to assess the development of behavioural sensitization. Rats were killed after a further 7-day washout, sufficient to exclude effects related to the challenge dose of cocaine.

A group of rats were subjected to the morphine sensitization protocol (n = 12, MF 1); a second group of rats received saline (1 mL/kg s.c.) every other day for 7 days (n = 8, CTR 2). Both groups were challenged with morphine (5 mg/kg s.c.) after a 10-day washout period to assess the development of behavioural sensitization. Rats were killed after a further 7-day washout, sufficient to exclude effects related to the challenge dose of morphine.

As there was no apparent modification in the phosphorylation pattern of DARPP-32 in morphine-sensitized rats, we studied the possible effect of acute morphine administration in control and morphine-sensitized rats. A group of rats underwent the morphine sensitization procedure (n = 36, MF 2); a second group of rats received saline (n = 36, CTR 3). After a 10-day washout period, sensitization was assessed and after a further 7-day period each group was subdivided into six subgroups of six animals each. In each group, a subgroup received saline (1 mL/kg s.c.) and was immediately killed. Rats in the other subgroups received a morphine challenge (5 mg/kg s.c.) and were then killed at different time points after acute morphine administration (30 min, and 1, 2, 4 and 6 h).

In all three experiments, only rats showing a clear-cut sensitized response were used for subsequent immunoblotting experiments. Brains were quickly removed, and the NAc and CPu were dissected out from 1-mm coronal slices on an ice-cooled plate. Tissues were immediately frozen on dry ice and stored at −80°C until utilized for immunoblotting.

Statistical analysis

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Animals
  5. Induction of cocaine sensitization
  6. Induction of morphine sensitization
  7. Immunoblotting
  8. Experimental protocols
  9. Experiment 1: effect of cocaine sensitization on DARPP-32 phosphorylation pattern
  10. Experiment 2: effect of morphine sensitization on DARPP-32 phosphorylation pattern
  11. Experiment 3: time course of the effect of acute morphine exposure on DARPP-32 phosphorylation pattern in control and morphine-sensitized rats
  12. Tissue processing
  13. Drugs
  14. Statistical analysis
  15. Results
  16. Effect of cocaine sensitization on DARPP-32 phosphorylation pattern
  17. Effect of morphine sensitization on DARPP-32 phosphorylation pattern
  18. Time-course of the effect of acute morphine exposure on DARPP-32 phosphorylation in control and morphine-sensitized rats
  19. Discussion
  20. Acknowledgement
  21. References

Statistical analyses were performed using commercially available software (GraphPad Prism 3.0; GraphPad Software Inc., San Diego, CA, USA). All data are expressed as mean ± SEM. Statistical comparisons between two experimental groups were made by parametric unpaired t-test. When more than two groups were compared, a non-parametric one-way anova (Kruskal–Wallis test) was used. When the Kruskal–Wallis test demonstrated that differences between groups were significant (p < 0.05), the data were subjected to post-hoc analysis using the Mann–Whitney test.

Effect of cocaine sensitization on DARPP-32 phosphorylation pattern

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Animals
  5. Induction of cocaine sensitization
  6. Induction of morphine sensitization
  7. Immunoblotting
  8. Experimental protocols
  9. Experiment 1: effect of cocaine sensitization on DARPP-32 phosphorylation pattern
  10. Experiment 2: effect of morphine sensitization on DARPP-32 phosphorylation pattern
  11. Experiment 3: time course of the effect of acute morphine exposure on DARPP-32 phosphorylation pattern in control and morphine-sensitized rats
  12. Tissue processing
  13. Drugs
  14. Statistical analysis
  15. Results
  16. Effect of cocaine sensitization on DARPP-32 phosphorylation pattern
  17. Effect of morphine sensitization on DARPP-32 phosphorylation pattern
  18. Time-course of the effect of acute morphine exposure on DARPP-32 phosphorylation in control and morphine-sensitized rats
  19. Discussion
  20. Acknowledgement
  21. References

Table 1 shows that the stereotypy scores and motility counts induced by the cocaine challenge (10 mg/kg, i.p.) were significantly higher in COCA rats than in CTR 1 animals (Table 1). Seven days after assessment of sensitization, both groups of rats were killed and the pattern of phosphorylation of DARPP-32 was measured in the NAc and CPu. Rats in the COCA group showed higher levels of phospho-Thr75 DARPP-32 and lower levels of phospho-Thr34 DARPP-32 than CTR rats, both in the NAc and in the CPu (Figs 1a and b). Moreover, levels of Cdk5 were higher in rats in the COCA group than in CTR rats (Fig. 1c).

Table 1.  Locomotor activity and stereotypy score of control, cocaine and morphine-sensitized rats
GroupnChallengeStereotypy scoreMotility counts
  1. Values are the mean ± SEM motility counts and stereotypy scores of rats used in experiment 1 (CTR 1 and COCA), experiment 2 (CTR 2 and MF 1) and experiment 3 (CTR 3 and MF 2). Control rats received saline (1 mL/kg) during induction of cocaine or morphine sensitization, and cocaine and morphine were administered as described in Materials and Methods. Rats were challenged with cocaine (10 mg/kg, i.p.) or morphine (5 mg/kg, s.c.) 10 days after the last treatment. ***p < 0.001 versus respective control group (Student t-test).

CTR 18Cocaine0.9 ± 0.3859 ± 230.2
COCA12Cocaine4.2 ± 0.3***3625 ± 535.6***
CTR 28Morphine1.0 ± 0.2835 ± 245.1
MF 112Morphine4.3 ± 0.2***3863 ± 751.8***
CTR 336Morphine0.9 ± 0.2787 ± 199.5
MF 236Morphine5.0 ± 0.3***3567 ± 845.4***
image

Figure 1. Effect of cocaine sensitization on DARPP-32 phosphorylation state and Cdk5 expression level. Levels of phospho-Thr75 DARPP-32 (a) and phospho-Thr34 DARPP-32 (b) and Cdk5 (c) in the CPu and NAc of control and cocaine-sensitized rats. Data are expressed as mean ± SEM percentage of control values. **p < 0.01 versus CTR group (Mann–Whitney test).

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Effect of morphine sensitization on DARPP-32 phosphorylation pattern

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Animals
  5. Induction of cocaine sensitization
  6. Induction of morphine sensitization
  7. Immunoblotting
  8. Experimental protocols
  9. Experiment 1: effect of cocaine sensitization on DARPP-32 phosphorylation pattern
  10. Experiment 2: effect of morphine sensitization on DARPP-32 phosphorylation pattern
  11. Experiment 3: time course of the effect of acute morphine exposure on DARPP-32 phosphorylation pattern in control and morphine-sensitized rats
  12. Tissue processing
  13. Drugs
  14. Statistical analysis
  15. Results
  16. Effect of cocaine sensitization on DARPP-32 phosphorylation pattern
  17. Effect of morphine sensitization on DARPP-32 phosphorylation pattern
  18. Time-course of the effect of acute morphine exposure on DARPP-32 phosphorylation in control and morphine-sensitized rats
  19. Discussion
  20. Acknowledgement
  21. References

Stereotypy scores and motility counts were significantly higher in morphine-sensitized rats than in control animals (MF 1 and CTR 2 in Table 1). The pattern of phosphorylation of DARPP-32 was measured in the NAc and CPu of CTR and MF rats killed 7 days after the assessment of sensitization. No significant differences in the levels of phospho-Thr34 or phospho-Thr75 DARPP-32 were demonstrated between CTR and MF rats by the Mann–Whitney test in the two areas examined (phospho-Thr75, CPu: CTR 100% and MF 96.5 ± 4%; NAc: CTR 100% and MF 108.7 ± 4%; phospho-Thr34, CPu: CTR 100% and MF 101.2 ± 8%; NAc: CTR 100% and MF 105.3 ± 6%). Moreover, Cdk5 levels did not differ in the NAc and CPu between the two groups (CPu: CTR 100% and MF 107.3 ± 5%; NAc: CTR 100% and MF 105.8 ± 4%).

Time-course of the effect of acute morphine exposure on DARPP-32 phosphorylation in control and morphine-sensitized rats

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Animals
  5. Induction of cocaine sensitization
  6. Induction of morphine sensitization
  7. Immunoblotting
  8. Experimental protocols
  9. Experiment 1: effect of cocaine sensitization on DARPP-32 phosphorylation pattern
  10. Experiment 2: effect of morphine sensitization on DARPP-32 phosphorylation pattern
  11. Experiment 3: time course of the effect of acute morphine exposure on DARPP-32 phosphorylation pattern in control and morphine-sensitized rats
  12. Tissue processing
  13. Drugs
  14. Statistical analysis
  15. Results
  16. Effect of cocaine sensitization on DARPP-32 phosphorylation pattern
  17. Effect of morphine sensitization on DARPP-32 phosphorylation pattern
  18. Time-course of the effect of acute morphine exposure on DARPP-32 phosphorylation in control and morphine-sensitized rats
  19. Discussion
  20. Acknowledgement
  21. References

Upon morphine challenge, stereotypy scores and motility counts were significantly higher in morphine-sensitized rats than in control animals (MF 2 and CTR 3 in Table 1). Table 1 shows cumulative scores and locomotor activity counts in the 90 min of observation. When sensitized rats were killed at different intervals after morphine challenge, they were scored for stereotypies and locomotor activity until the time of death. Morphine-sensitized rats showed a peak of intensity in stereotypies between 30 (score 5.0 ± 0.4) and 60 min (score 5.5 ± 0.2); at 90 min stereotypy scores were similar to those of the CTR group (1.9 ± 0.4 vs. 1.2 ± 0.3 respectively). Locomotor activity was increased compared with that in the CTR group until 90 min; thereafter it was similar in the two groups (data not shown). The levels of phosphorylation of DARPP-32 and GluR1 and NR1 subunits were measured in the NAc and CPu of CTR and MF rats. Analysis of phospho-Thr75 DARPP-32 and phospho-Thr34 DARPP-32 levels in the CPu and NAc by Kruskal–Wallis test showed a significant difference between groups. Mann–Whitney test indicated that in CTR + MF rats acute morphine administration did not modify DARPP-32 phosphorylation pattern in the CPu and NAc at any time after morphine challenge; moreover, no difference was found in the levels of phosphorylation of Ser845 GluR1 and Ser897 NR1 subunits (Figs 2 and 3). Mann–Whitney test revealed that in the MF + MF group 2 h after morphine challenge the levels of phospho-Thr34 DARPP-32 were significantly decreased and the levels of phospho-Thr75 DARPP-32 were significantly increased, both in the CPu and NAc. These modifications in DARPP-32 phosphorylation pattern were associated with a significant decrease in the phosphorylation levels of two other PKA substrates, the GluR1 and NR1 subunits, in both brain areas (Figs 2 and 3). In the MF + MF group, 2 h after morphine challenge, the expression of Cdk5 was unmodified both in the NAc and CPu (CPu: 100% at time 0 and 105.3 ± 11% at 2 h; NAc: 100% at time 0 and 114.0 ± 13% at 2 h).

image

Figure 2. Time course of modifications after morphine challenge in the phosphorylation levels of DARPP-32, GluR1 and NR1 in the CPu of control and morphine-sensitized rats. Levels of phospho-Thr75 DARPP-32 (a), phospho-Thr34 DARPP-32 (b), phospho-Ser845 GluR1 (c) and phospho-Ser897 NR1 (d) were examined using phosphorylation state-specific antibodies. Levels in the CTR + MF group are expressed as mean ± SEM percentage of the levels obtained in control rats that did not receive acute morphine administration. Levels in the MF + MF group are expressed as mean ± SEM percentage of the levels obtained in morphine-sensitized rats that did not receive acute morphine administration. Analysis of the data with the Kruskal–Wallis test indicated a significant difference between groups (KW = 19.82, p < 0.05 for phospho-Thr75; KW = 21.68, p < 0.05 for phospho-Thr34; KW = 25.28, p < 0.01 for phospho-Ser845; KW = 21.48, p < 0.05 for phospho-Ser897). In the MF group, post-hoc analysis demonstrated a significant difference in the levels of phospho-Thr75 and phospho-Thr34 DARPP-32, and phospho-Ser845 GluR1 and phospho-Ser897 NR1 in the rats killed 2 h after morphine challenge compared with levels in rats that did not receive a morphine challenge (**p < 0.01 for both phospho-Thr75 and phospho-Thr34; *p < 0.05 for both phospho-Ser845 and phospho-Ser897). (e) Representative immunoblots showing levels of phospho-Thr75 and phospho-Thr34 DARPP-32 in the CPu of rats in the CTR + MF and MF + MF groups killed 2 h after morphine challenge.

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image

Figure 3. Time course of modifications after morphine challenge in the phosphorylation levels of DARPP-32, GluR1 and NR1 in the NAc of control and morphine-sensitized rats. Levels of phospho-Thr75 DARPP-32 (a), phospho-Thr34 DARPP-32 (b), phospho-Ser845 GluR1 (c) and phospho-Ser897 NR1 (d) were examined using phosphorylation state-specific antibodies. Levels in the CTR + MF group are expressed as mean ± SEM percentage of the levels obtained in control rats that did not receive acute morphine administration. Levels in the MF + MF group are expressed as mean ± SEM percentage of the levels obtained in morphine-sensitized rats that did not receive acute morphine administration. Analysis of the data with the Kruskal–Wallis test indicated a significant difference between groups (KW = 23.08, p < 0.05 for phospho-Thr75; KW = 20.06, p < 0.05 for phospho-Thr34; KW = 21.65, p < 0.05 for phospho-Ser845; KW = 26.36, p < 0.01 for phospho-Ser897). In the MF group, post-hoc analysis demonstrated a significant difference in the levels of phospho-Thr75 and phospho-Thr34 DARPP-32, and phospho-Ser845 GluR1 and phospho-Ser897 NR1 in the rats killed 2 h after morphine challenge compared with levels in rats that did not receive a morphine challenge (**p < 0.01 for both phospho-Thr75 and phospho-Thr34; *p < 0.05 for both phospho-Ser845 and phospho-Ser897). (e) Representative immunoblots showing levels of phospho-Thr75 and phospho-Thr34 DARPP-32 in the NAc of rats in the CTR + MF and MF + MF groups killed 2 h after a morphine challenge.

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Discussion

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Animals
  5. Induction of cocaine sensitization
  6. Induction of morphine sensitization
  7. Immunoblotting
  8. Experimental protocols
  9. Experiment 1: effect of cocaine sensitization on DARPP-32 phosphorylation pattern
  10. Experiment 2: effect of morphine sensitization on DARPP-32 phosphorylation pattern
  11. Experiment 3: time course of the effect of acute morphine exposure on DARPP-32 phosphorylation pattern in control and morphine-sensitized rats
  12. Tissue processing
  13. Drugs
  14. Statistical analysis
  15. Results
  16. Effect of cocaine sensitization on DARPP-32 phosphorylation pattern
  17. Effect of morphine sensitization on DARPP-32 phosphorylation pattern
  18. Time-course of the effect of acute morphine exposure on DARPP-32 phosphorylation in control and morphine-sensitized rats
  19. Discussion
  20. Acknowledgement
  21. References

Repeated cocaine administration (20 mg/kg for 8 days, rats killed 12 h after the last treatment) increases the levels of phospho-Thr75 DARPP-32 and decreases those of phospho-Thr34 DARPP-32 in the CPu and NAc of rats killed 24 h after the last treatment (Bibb et al. 2001). This effect has been attributed to a cocaine-induced increase in ΔFosB expression, as ΔFosB induces Cdk5 expression and Thr75 of DARPP-32 is a specific substrate of this kinase (Bibb et al. 2001). In the present study we observed that rats exposed to a cocaine sensitization protocol showed an increase in levels of phospho-Thr75 DARPP-32 and a decrease in levels of phospho-Thr34 DARPP-32 compared with levels in control rats, both in the CPu and NAc. In addition, they had increased levels of Cdk5 in the same two brain areas. Rats infused in the NAc with Cdk5 inhibitors show a higher locomotor response to repeated cocaine administration than rats infused with saline (Bibb et al. 2001). This finding led to the hypothesis that the increased levels of Cdk5 and phospho-Thr75 DARPP-32 after chronic cocaine represent an adaptive response to the development of cocaine behavioural sensitization (Bibb et al. 2001). Our results seem to be consistent with this hypothesis as the increase in Cdk5 and phospho-Thr75 DARPP-32 levels and the reduction in phospho-Thr34 DARPP-32 levels were still present several days after the last cocaine injection, at a time when the behavioural sensitization state was well developed.

Long-term behavioural sensitization to drugs of abuse is a long-lasting phenomenon (Paulson et al. 1991; Pollock and Kornetsky 1996; Nestby et al. 1997) and the neural adaptive mechanisms underlying it are considered to be similar to other forms of long-term plasticity in the CNS (Nestler 2001). However, several traits seem to distinguish cocaine and morphine sensitization. For instance, the concurrent stimulation of DA D1- and D2-like receptors is a condition necessary for the full expression of cocaine sensitization (Capper-Loup et al. 2002), whereas the expression of morphine sensitization seems to be more heavily dependent on DA D1 receptor transmission (Pollock and Kornetsky 1989; Scheggi et al. 2000). However, not all the literature supports these differences, and DA D2 receptor transmission has also been implicated in the expression of morphine behavioural sensitization (Jeziorski and White 1995; Piepponen et al. 1996; Del Rosario et al. 2002). The complex processes of neuronal adaptation in morphine and cocaine sensitization have distinct biochemical substrates, although the expression of the sensitized locomotor activity seems to be dependent in both cases on dopaminergic transmission. Thus, we studied the phosphorylation pattern of DARPP-32 in morphine-sensitized rats too.

The condition of morphine behavioural sensitization did not apparently modify the pattern of DARPP-32 phosphorylation nor the expression of Cdk5 in the CPu and the NAc. In our experimental conditions, morphine-sensitized rats show a persistent state of functionally inducible increase in DA D1 receptor mediated transmission as (i) they are resistant to unavoidable stress exposure in a manner that is selectively dependent on DA D1 receptor activation (Scheggi et al. 2000) and (ii) they present intense stereotypies selectively antagonized by SCH 23390 administration, upon challenge with a low dose of morphine (Scheggi et al. 2000). Morphine sensitization is associated with an increase in the function and expression of Gs and a decrease in Gi proteins (Van Vliet et al. 1993), which may explain the increase in the basal activity of adenylyl cyclase observed in striatal areas of morphine-sensitized rats (Viganòet al. 2003). On these basis we hypothesized that the administration of a morphine challenge to morphine-sensitized rats would produce a cascade of events leading to PKA activation and, possibly, to a transient increase in DARPP-32 phosphorylation at Thr34. On the contrary, an increase in the levels of phospho-Thr34 was not observed at any time after morphine challenge. Indeed, an increase in phospho-Thr75 DARPP-32 levels and a decrease in phospho-Thr34 DARPP-32 levels, with no change in Cdk5 expression, were consistently observed 2 h after morphine challenge. In order to substantiate a possible decrease in PKA activity, we measured the degree of phosphorylation of two other PKA substrates, glutamate receptor subunits GluR-1 and NR-1 (Tingley et al. 1993; Snyder et al. 2000), in the same animals. The phosphorylation levels of GluR-1 and NR-1 at Ser845 and Ser897 sites respectively were similar in control and morphine-sensitized rats, and in sensitized rats they were both significantly decreased 2 h after a morphine challenge.

In the striatal areas of morphine-sensitized rats, an increased binding of µ-opioid receptors associated with increased coupling of µ-receptors to Gi/O proteins (Viganòet al. 2003) and an increase in dopaminergic transmission (Cadoni and Di Chiara 1999) have been observed. The µ-opioid receptor stimulation induces inhibition of adenylyl cyclase (Childers 1991) and/or an increase in Ca2+ release from intracellular stores (Smart et al. 1994). Thus, it can be hypothesized that an acute morphine challenge should result in the simultaneous stimulation of sensitized µ-opioid and sensitized DA D1 receptors in striatal areas. If this were the case, the ensuing neurochemical and behavioural effects should reflect the algebraic sum of the two opposite signals on adenylyl cyclase activity. Upon acute morphine administration, sensitized rats showed intense stereotypies that reached maximal intensity at 30–60 min and subsided within 90 min, whereas the increase in locomotor activity lasted for 90 min. As these stereotypies are mediated by DA D1 receptor stimulation (Pollock and Kornetsky 1989; Scheggi et al. 2000) and thus by adenylyl cyclase activation, the lack of change in phosphorylation levels of different PKA substrates 30–60 min after challenge may be considered evidence of the simultaneous µ-opioid receptor stimulation. Two hours after morphine administration stereotypies and increased locomotor activity had subsided, suggesting that DA D1 receptor stimulation was over, and the observed decrease in levels of phospho-Thr34 and increase in levels of phospho-Thr75 DARPP-32 were probably related to a residual stimulation of opioid receptors by morphine. The µ-opioid receptor-elicited increase in intraneuronal Ca2+ concentration can, in fact, activate protein phosphatase 2B (PP2B or calcineurin), which dephosphorylates phospho-Thr34 DARPP-32 (King et al. 1984).

In conclusion, the present data confirmed that repeated cocaine treatment induces a tonic increase in Cdk5 and phospho-Thr75 DARPP-32 levels, and a decrease in phospho-Thr34 DARPP-32 levels. Both these effects were long lasting. Morphine sensitization was apparently not accompanied by modifications in the pattern of phosphorylation of DARPP-32 or in Cdk5 expression. Moreover, in control rats morphine challenge had no apparent effects on the phosphorylation pattern of DARPP-32. Thus, the challenge-induced modifications in DARPP-32 phosphorylation in morphine-sensitized rats were probably related to an increased functional responsiveness of µ-opioid and DA D1 receptors. However, in order to ascertain whether the observed neurochemical modifications are necessary for cocaine and/or morphine behavioural sensitization, the temporal persistence of the sensitized behavioural response and of the modifications in DARPP-32 phosphorylation should be compared.

References

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Animals
  5. Induction of cocaine sensitization
  6. Induction of morphine sensitization
  7. Immunoblotting
  8. Experimental protocols
  9. Experiment 1: effect of cocaine sensitization on DARPP-32 phosphorylation pattern
  10. Experiment 2: effect of morphine sensitization on DARPP-32 phosphorylation pattern
  11. Experiment 3: time course of the effect of acute morphine exposure on DARPP-32 phosphorylation pattern in control and morphine-sensitized rats
  12. Tissue processing
  13. Drugs
  14. Statistical analysis
  15. Results
  16. Effect of cocaine sensitization on DARPP-32 phosphorylation pattern
  17. Effect of morphine sensitization on DARPP-32 phosphorylation pattern
  18. Time-course of the effect of acute morphine exposure on DARPP-32 phosphorylation in control and morphine-sensitized rats
  19. Discussion
  20. Acknowledgement
  21. References
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