Abstract

Increasing evidence suggests that in addition to the mesoaccumbens dopamine (DA) system other neurotransmitter and brain systems are also involved in opiate addiction. Recent evidence points to a major involvement of brain norepinephrine (NE) in the behavioral and central effects of opiates and, more specifically, indicates that NE in the prefrontal cortex may have a critical role in rewarding effects of opiates. Moreover, a body of data points to regions within the medial prefrontal cortex (mpFC) acting as final common pathway of drug relapse behavior. The present experiments were aimed at investigating the possibility of a selective involvement of the prefrontal cortical NE in the rewarding and reinstating effects of morphine. In a first set of experiments, we found that morphine enhances NE and DA release in the mpFC and DA release in the nucleus accumbens, as measured by intra-cerebral microdialysis. Selective depletion of medial prefrontal cortical noradrenergic afferents abolished the morphine-induced increase in DA release in the nucleus accumbens. In a second series of experiments, we demonstrated that the same lesion impaired both conditioned place preference (CPP) induced by morphine and reinstatement of an extinguished CPP. The present results indicate that an intact prefrontal cortical NE transmission is necessary for morphine-induced rewarding effects, reinstatement, and mesoaccumbens dopamine release.

Introduction

Increased dopamine (DA) transmission in the nucleus accumbens (NAc) is known to mediate the rewarding/reinforcing effects of addictive drugs (Di Chiara and Imperato, 1988; Wise and Rompre, 1989; Pontieri et al., 1995; Koob et al., 1998; Robbins and Everitt, 1999).

However, recent evidence points to a major involvement of brain norepinephrine (NE) in the behavioral and central effects of opiates. α2-Adrenergic receptors have been shown to have a role in morphine-induced conditioned place preference (CPP) (Zarrindast et al., 2002). Mice lacking α1b-adrenergic receptors are hyposensitive to the stimulating effects on locomotor activity and non-responsive to the rewarding effect of morphine measured in the CPP test (Drouin et al., 2002). Prazosin, an α1-adrenergic antagonist injected locally into the prefrontal cortex, reduces accumbal DA-related locomotor hyperactivity induced by morphine (Drouin et al., 2001). These data suggest that cortical NE might have a critical role in the rewarding effects of opiates.

Relapse is a major characteristic of drug addiction (see Nestler, 2002, for review). The behavior of addicts after a long period of abstinence is characterized by a high rate of relapse to the drug (Jaffe, 1990). The reinstatement model, based on the CPP, is widely used to study mechanisms underlying relapse to drug seeking (Mueller and Stewart, 2000; Wang et al., 2000; Sanchez and Sorg, 2001; Lu et al., 2002; Mueller et al., 2002). Drug-induced reinstatement seems to be a result of the rewarding properties of abused drugs involving the mesolimbic dopaminergic system (see Shaham et al., 2003, for review). Opiates increase extracellular dopamine in the NAc (Di Chiara et al., 1993) and drug priming reinstates drug-seeking behavior by activating the mesolimbic dopaminergic incentive motivation system (Robinson and Berridge, 1993). Moreover, lesions of the NAc shell and the ventral tegmental area (VTA) abolish the morphine-primed reactivation of an extinguished CPP (Wang et al., 2002). Finally, activation by local morphine infusion of DA neurons in the VTA reinstates heroin-seeking behavior (Stewart, 1984; Di Chiara and North, 1992). Taken together, these data suggest that mesolimbic dopamine release is a mechanism underlying the rewarding properties of opiates as well as the reinstatement of compulsive opiate-seeking behavior.

Nevertheless, some evidence indicates that several other neurotransmitter and brain systems are involved in opiate addiction. In particular, the NE system might be a possible candidate. Noradrenergic transmission is involved in both opiate withdrawal (Maldonado, 1997; Aston-Jones et al., 1999; Delfs et al., 2000) and reinstatement (Shaham et al., 2000Wang et al., 2001).

Although it is known that neural mechanisms underlying reinstatement induced by drug re-exposure, drug cues and stressors are to some degree different (Shaham et al., 2003), recent evidence indicates regions within the medial prefrontal cortex (mpFC) as a ‘final common pathway’ of drug relapse behavior (Neisewander et al., 2000; McFarland and Kalivas, 2001; See, 2002; Capriles et al., 2003).

We have recently shown that an intact prefrontal cortical NE transmission is critical for psychostimulant-induced mesoaccumbens dopamine release (Ventura et al., 2003a). Since morphine is thought to produce rewarding effects and reinstatement to drug-seeking behavior by increasing DA release in the NAc, we hypothesize that an intact prefrontal cortical NE transmission is a necessary condition for morphine-induced reward and mesoaccumbens DA release, as well as for morphine-induced reinstatement of an extinguished CPP.

We investigated the effects of mpFC-selctive NE depletion on the behavioral effects of morphine, and on mesoaccumbens DA release in mice of the C57BL/6J inbred strain. This background is commonly used in molecular approaches and is known to be highly sensitive to the behavioral effects of opiates (Oliverio et al., 1983). The effects of morphine on NE and DA release in the mpFC of naïve or NE-depleted animals were also assessed.

Materials and Methods

Animals

Male mice of the inbred C57BL/6JIco (C57) strain (Charles River, Como, Italy), 8–9 weeks old at the time of the experiments, were housed as previously described (Ventura et al., 2001). All experiments were conducted in accordance with Italian national law (DL no. 116, 1992) governing the use of animals for research.

Drugs

Chloral hydrate, 6-hydroxydopamine (6-OHDA) and GBR 12909 (GBR) were purchased from Sigma (Sigma Aldrich, Milan, Italy); morphine hydrochloride from Carlo Erba (Carlo Erba, Milan, Italy). Morphine (5, 10, 20 or 30 mg/kg), chloral hydrate (450 mg/kg) and GBR (15 mg/Kg) were dissolved in saline (0.9% NaCl) and injected intraperitoneally (i.p.) in a volume of 10 ml/kg. 6-OHDA was dissolved in saline containing sodium metabisulphite (0.1 M).

Microdialysis

Animals were anesthetized with chloral hydrate, mounted in a stereotaxic frame (David Kopf Instruments, Tujunga, CA) equipped with a mouse adapter and implanted unilaterally with a guide cannula (stainless steel, shaft o.d. 0.38 mm, Metalant AB, Stockholm, Sweden,) in the mpFC or in the NAc. The length of the guide cannula was 1 mm for mpFC and 4.5 mm for NAc. The guide cannula was fixed with epoxy glue, and dental cement was added for further stabilization. The coordinates from bregma (measured according to the atlas of Franklin and Paxinos, 1998) were: +2.52 AP; 0.6 L, for mpFC and +1.60 AP; 0.6 L, for NAc (mostly including the shell subdivision; Franklin and Paxinos, 1998; Ventura et al., 2004a,b). The probe (dialysis membrane length 2 mm for mpFC and 1 mm for NAc; o.d. 0.24 mm, MAB 4 cuprophane microdialysis probe, Metalant AB) was introduced 24 h after implantation of the guide cannula. This procedure was chosen following preliminary experiments as well as previous reports that showed a full recovery of mice for behavioral as well as neurochemical testing (Foster Olive, 2000; Ventura et al., 2003a, 2004a,b). Animals were returned to their home cages and the outlet and inlet probe tubing were protected by locally applied parafilm. The animals were lightly anesthetized to facilitate manual insertion of the microdialysis probe into the guide cannula. The membranes were tested for in vitro recovery of DA and NE [relative recovery (%): DA = 10.7 ± 0.82; NE = 12.2 ± 0.75; n = 20] on the day before use in order to verify recovery.

The microdialysis probe was connected to a CMA/100 pump (Carnegie Medicine, Stockholm, Sweden) through PE-20 tubing and an ultra-low torque dual-channel liquid swivel (Model 375/D/22QM, Instech Laboratories, Inc., Plymouth Meeting, PA) to allow free movement. Artificial CSF (147 mM NaCl, 2.2 mM CaCl2 and 4 mM KCl) (Pontieri et al., 1995) was pumped through the dialysis probe at a constant flow rate of 2 μl/min. Experiments were carried out 22–24 h after probe placement. Each animal was placed in a circular cage provided with microdialysis equipment (Instech Laboratories, Inc.) and with home cage bedding on the floor. Dialysis perfusion was started 1 h later. Following the start of dialysis perfusion mice were left undisturbed for ∼2 h before the collection of baseline samples. Dialysate was collected every 20 min for 180 min. Only data from mice with a correctly placed cannula have been reported. Placements were judged by methylene blue staining. The location of microdialysis probes in the mpFC and the NAc is shown in Figure 1. Twenty microliters of the dialysate samples were analyzed by high-performance liquid chromatography (HPLC). The remaining 20 μl were kept for possible subsequent analysis. Concentrations (pg/20 μl) were not corrected for probe recovery. The mean concentration of the three samples collected immediately before treatment (< 10% variation) was taken as basal concentration.

Figure 1.

Location of microdialysis probes in the medial prefrontal cortex and the nucleus accumbens. Silhouettes of probe tracks were drawn onto representative sections of the mouse brain and the range of implantation sites. The circle represents the cortical area punched for tissue analysis. For details see method section. The numbers indicate millimeters rostral to bregma according to Franklin and Paxinos (1998).

Figure 1.

Location of microdialysis probes in the medial prefrontal cortex and the nucleus accumbens. Silhouettes of probe tracks were drawn onto representative sections of the mouse brain and the range of implantation sites. The circle represents the cortical area punched for tissue analysis. For details see method section. The numbers indicate millimeters rostral to bregma according to Franklin and Paxinos (1998).

The HPLC system consisted of an Alliance (Waters Corporation, Milford, MA) system and a coulometric detector (ESA Model 5200A Coulochem II) provided with a conditioning cell (M 5021) and an analytical cell (M 5011). The conditioning cell was set at 400 mV, electrode 1 at 200 mV, and electrode 2 at −250 mV. A Nova-Pack C18 column (3.9 × 150 mm, Waters) maintained at 33°C was used. The flow rate was 1.1 ml/min. The mobile phase was as previously described (Westerink et al., 1998). The assay detection limit was 0.1 pg.

NE Depletion in the mpFC

Anesthesia and surgical set are described in the preceding paragraph. Animals were injected with GBR (15 mg/kg) 30 min before 6-OHDA micro-injection in order to protect dopaminergic neurons. Bilateral injection of 6-OHDA (1.5 μg/0.1 μl/2 min for each side) was made into the mpFC [coordinates: +2.52 AP; ± 0.6 L; −2.0 V with respect to bregma (Franklin and Paxinos, 1998)], through a stainless steel cannula (0.15 mm o.d., UNIMED, Switzerland), connected to a 1 μl syringe by a polyethylene tube and driven by a CMA/100 pump. The cannula was left in place for an additional 2 min after the end of the infusion. Sham animals (Sham) were subjected to the same treatment but received intracerebral vehicle. Animals were used for microdialysis or behavioral experiments 7 days after surgery. In order to rule out that this procedure induced trauma or rough damage in the mpFC affecting microdialyis data, histological analysis was performed. Visualization of dialysis probe location in the mpFC after 6-OHDA local infusion was evidentiated by Niss'l staining (Fig. 2). Animals were deeply anesthetized with chloral hydrate and transcardially perfused with saline followed by ice-cold 10% neutral buffered formalin. Brains were dissected, further fixed, and cryoprotected in 30% sucrose.

Figure 2.

Photomicrographs of coronal sections (2.68 mm and 2.22 mm anterior to bregma) showing the microdialysis probe trace in the medial prefrontal cortex. The arrows indicate tissue next to the probe that was not damaged by previous neurotoxic lesion procedure (for details see the Materials and Methods and Results sections).

Figure 2.

Photomicrographs of coronal sections (2.68 mm and 2.22 mm anterior to bregma) showing the microdialysis probe trace in the medial prefrontal cortex. The arrows indicate tissue next to the probe that was not damaged by previous neurotoxic lesion procedure (for details see the Materials and Methods and Results sections).

NE and DA tissue levels in the mpFC were assessed as previously described (Ventura et al., 2002, 2003a) to evaluate the extent of depletion.

The brain was fixed vertically on the freeze plate of a freezing microtome. Punches of both hemispheres were obtained from brain slices (coronal sections) no thicker than 300 μm. Stainless steel tubes of 2.3 mm (mpFC) i.d. were used. The coordinates were measured according to the atlas of Franklin and Paxinos (1998) (coronal sections as mm from Bregma), as follows: mpFC two slices from 2.96 to 2.34. Punching localization is shown in Figure 1. The punches were stored in liquid nitrogen until the day of analysis. DA and NE were determined simultaneously by a reverse-phase HPLC procedure coupled with coulochem electrochemical detection. On the day of the analysis frozen samples were weighed and homogenized in HC1O4 0.1 N containing sodium metabisulphite 6 mM and EDTA 1mM. The homogenates were centrifuged at 10 000 g for 20 min at 4°C. Aliquots of the supernatant were transferred to the HPLC system. The HPLC system is described in the preceding paragraph; the potentials were set at +450 and +100 mV at the analytical and the conditioning cell, respectively. The Nova-Pack Phenyl column (3.9 × 150 mm) and the Sentry Guard Nova-Pack pre-column (3.9 × 20 mm) were purchased from Waters Assoc. The flow rate was 1.0 ml/min. The mobile phase consisted of 3% methanol in 0.1 M sodium phosphate buffer, pH 3, Na2EDTA 0.1 mM, and 1-octane sulfonic acid sodium salt (Aldrich) 0.5 mM.

Behavioral Experiments

Conditioned Place Preference

Behavioral experiments were performed using CPP apparatus (Cabib et al., 1996, 2000). The apparatus comprised two gray Plexiglas chambers (15 × 15 × 20 cm) and a central alley (15 × 5 × 20 cm). Two sliding doors (4 × 20 cm) connected the alley to the chambers. In each chamber two triangular parallelepipeds (5 × 5 × 20 cm) made of black Plexiglas and arranged in different patterns (always covering the same surface of the chamber) were used as conditioned stimuli. The training procedure for place conditioning was described previously (Cabib et al., 1996, 2000; Ventura et al., 2003a). Briefly, on day 1 (pretest), mice were free to explore the entire apparatus for 20 min. During the following 8 days (conditioning phase) mice were confined daily for 40 min alternately in one of the two chambers. For animals in the experimental groups, one of the patterns was consistently paired with saline and the other one with morphine (5, 20 or 30 mg/kg i.P.), during the conditioning phase. Pairings were balanced so that for half of each experimental group, morphine was paired with one of the patterns and for half of them with the other one. For animals in the control group, both chambers were paired with saline. Test for the expression of the CPP was conducted on day 10 using the pretest procedure.

Behavioral data were collected and analyzed by the ‘EthoVision’ (Noldus, The Netherlands) fully automated video tracking system (Spink et al., 2001). Briefly, the experimental system is recorded by a CCD video camera. The signal is then digitized (by a hardware device called a frame grabber) and passed on to the computer's memory. Later, the digital data are analyzed by means of the EthoVision software to obtain ‘time spent’ (s), which is used as raw data for preference scores, in each sector of the apparatus by each subject.

Effects of Selective Prefrontal Cortical NE Depletion on CPP Induced by Morphine

Eight groups of mice were used for the first CPP experiment, conducted as described in the preceding paragraph. Animals were used for behavioral experiments 7 days after surgery. Before conditioning, the mice were randomly assigned to one of the different treatment (saline, morphine 5, 20 or 30 mg/kg) within each group (Sham, NE-depleted).

Effects of Selective Prefrontal Cortical NE Depletion on Morphine-induced Reinstatement of Extinguished CPP

Two groups of mice were used in this experiment. After initial CPP testing [for both groups one of the patterns was consistently paired with saline and the other one with morphine (20 mg/kg, i.p.), during the conditioning phase, as described in the preceding paragraph], mice were given extinction training in which saline was paired four times with each of the outer chambers, once per day, over 8 days (Mueller et al., 2002). After extinction, mice were randomly assigned to one of the two groups (Sham, NE-depleted) and subjected to surgery. Animals were used for the reinstatement test 7 days after surgery. The reinstatement test was conducted as for previous CPP testing, but animals received a priming injection of morphine (5 mg/kg, i.p.) before being placed in the CPP apparatus.

One-way Passive Avoidance

Passive avoidance apparatus and procedure have been described previously (Puglisi-Allegra et al., 1986). Briefly, the apparatus consisted in two chambers (a clear, brightly illuminated box and a dark box) connected by a sliding door and placed on a grid floor. During training, individual mice were placed in the clear box and the latency to enter the dark box (mean step-through latencies) was taken as dependent variable. Once the animal entered the box, the sliding door was closed manually and a 50 Hz scrambled foot-shock of 0.2 mA was delivered though the grid floor for 1 s. On the following day testing was run according to the same procedure as in training without shock delivery.

Two groups of mice (Sham and NE-depleted) were used in this experiment.

Statistics

NE Depletion

The effects of prefrontal NE depletion on tissue levels of DA and NE in the mpFC were analyzed by two-way ANOVA, with the factors: lesion (two levels: Sham, NE-depleted); and experiment (three levels: behavioral experiment, microdialysis in the Nac, microdialysis in the mpFC). Individual between-groups comparisons, where appropriate, were carried out by post-hoc test (Duncan multiple-range test).

Microdialysis

Statistical analyses were carried out on raw data (concentrations: pg/20 μl). The effects of morphine on extracellular NE and DA levels in the mpFC were analyzed by repeated-measures ANOVA with one between factor (treatment, four levels: saline, morphine 5, 10 and 20 mg/kg) and one within factor (time, seven levels: 0, 20, 40, 60, 80, 100, 120). Sphericity assumed modelling with Greenhouse–Geisser adjustment was applied. The effects of prefrontal NE depletion on DA release in the NAc of animals challenged with morphine were analyzed by repeated-measures ANOVA with two between factors (treatment, three levels: saline, morphine 10 and 20 mg/kg; and lesion, two levels: Sham, NE-depleted) and one within factor (time, seven levels: 0, 20, 40, 60, 80, 100, 120). Sphericity-assumed modeling with Greenhouse–Geisser adjustment was applied.The effects of prefrontal NE depletion on NE and DA release in the mpFC of animals challenged with morphine (20 mg/kg) were analyzed by repeated-measures ANOVA with one between factor (lesion, two levels: Sham, NE-depleted) and one within factor (time, seven levels: 0, 20, 40, 60, 80, 100, 120). Sphericity-assumed modeling with Greenhouse–Geisser adjustment was applied. Simple effects were assessed by one-way ANOVA for each time point. Individual between group comparisons were carried out, where appropriate, by post-hoc test (Duncan multiple-range test).

Behavioral experiments

Conditioned Place Preference

For CPP experiments, statistical analyses were performed calculating the time (s) spent in center (Center), morphine- (Paired) and saline-paired (Unpaired) compartments on the test day. In the case of animals receiving saline pairing with both compartments, the Paired compartment was identified as the first they were exposed to.

Effects of Selective Prefrontal Cortical NE Depletion on CPP Induced by Morphine

Data from this CPP experiment were analyzed using repeated-measure ANOVA, with two between factors (pre-treatment, two levels: Sham, NE-depleted, and treatment, four levels: saline/saline (Sal); saline/morphine 5 mg/kg (Morph 5); saline/morphine 20 mg/kg (Morph 20); saline/morphine 30 mg/kg (Morph 30); and one within factor (pairing, three levels: Center, Paired, Unpaired). Because the important comparisons are those between Paired and Unpaired compartments (Mueller et al., 2002), mean comparisons of time spent in these chambers were made using repeated-measure ANOVA within each group.

Effects of Selective Prefrontal Cortical NE Depletion on Morphine-induced Reinstatement of Extinguished CPP

Data from CPP and extinction test, before surgery, were analyzed using repeated-measure ANOVA (pairing, three levels: Center, Paired, Unpaired), for each group. Data from the reinstatement test were analyzed using repeated-measure ANOVA, with one between factor (lesion, 2 levels: Sham, NE-depleted) and one within factor (pairing, three levels: Center, Paired, Unpaired). Mean comparisons of time spent in Paired and Unpaired compartments were made using repeated-measure ANOVA within each group.

One-way Passive Avoidance

Differences in the latencies (s) to enter the shock-paired chamber on the testing day between Sham and NE-depleted mice were analyzed by Student's t-test (two-tailed).

Results

NE Depletion in the mpFC

Statistical analyses were carried out on data from behavioral and microdialysis experiments. Two-way ANOVA for effects of prefrontal NE depletion on DA and NE tissue levels in the mpFC showed a significant lesion effect for NE only [F(1,66) = 6,79; P < 0.0005] but no effects of experiment.

Selective depletion of NE prefrontal cortical afferents produced an approximately 90% decrease in NE tissue levels (Sham = 702 ± 24; NE-depleted = 58 ± 16 ng/g wet tissue; P < 0.0005), whereas it spared DA tissue levels (Sham = 209 ± 12; NE-depleted = 193 ± 14 ng/g wet tissue).

Microdialysis

Intracerebral microdialysis was used to evaluate whether systemic morphine increased extracellular NE and DA in the mpFC. The average basal values of NE and DA for each group (saline, morphine 5, 10, 20 mg/kg) did not differ significantly. Therefore the values from all groups were combined (NE = 1.28 ± 0.14 ; DA = 0.52 ± 0.03 pg/20 μl). The effects of morphine on NE and DA release in the mpFC are shown in Figure 3A,B. Statistical analyses revealed a significant treatment × time interaction for both [NE = F(18,156) = 2.96; P < 0.005; DA = F(18,144) = 2.62; P < 0.05]. Simple effect analyses revealed a significant effect of time and a significant difference between morphine and saline (Sal). Morphine (10, 20 mg/kg, i.p.) increased extracellular NE (Fig. 3A) and DA (Fig. 3B) in the mpFC in a dose-dependent manner over the 120 min post-injection period. No significant effect was produced by the lowest dose (5 mg/kg) of morphine on NE release (Fig. 3A); only a small increase of DA outflow was evident at 40 min over that seen after saline (Fig. 3B).

Figure 3.

Extracellular norepinephrine (A) and dopamine (B) in the medial prefrontal cortex of animals injected with saline (Sal; NE: n = 7; DA: n = 6) or morphine [Mor: 5 mg/kg (NE: n = 5; DA: n = 7), 10 mg/kg (NE: n = 9; DA: n = 8), 20 mk/kg (NE: n = 9; DA: n = 7)]. Results are expressed as percent changes from basal values of each experimental group. Statistical analyses were carried out on raw data. Drug was injected at time 0. All data are expressed as mean ± SE. *P < 0.01 in comparison with the corresponding time point of saline.

Figure 3.

Extracellular norepinephrine (A) and dopamine (B) in the medial prefrontal cortex of animals injected with saline (Sal; NE: n = 7; DA: n = 6) or morphine [Mor: 5 mg/kg (NE: n = 5; DA: n = 7), 10 mg/kg (NE: n = 9; DA: n = 8), 20 mk/kg (NE: n = 9; DA: n = 7)]. Results are expressed as percent changes from basal values of each experimental group. Statistical analyses were carried out on raw data. Drug was injected at time 0. All data are expressed as mean ± SE. *P < 0.01 in comparison with the corresponding time point of saline.

To determine whether cortical noradrenergic afferents control accumbal DA outflow induced by morphine challenge, we assessed the effects of selective noradrenergic depletion in the mpFC on the DA response in the NAc of animals challenged with morphine. The effects of selective prefrontal NE depletion on DA release induced by morphine in the NAc are shown in Figure 4. The average basal values of dopamine for each group did not differ significantly. Therefore the values from all groups were combined (1.36 ± 0.14 pg/20 μl).

Figure 4.

Effects of prefrontal cortical norepinephrine depletion on extracellular dopamine in the nucleus accumbens of animals [Sham (A), NE-depleted (B)] injected with saline (Sal) or morphine (Mor 10, 20 mg/kg, i.p.). Results are expressed as percent changes from basal levels of each experimental group (n = 6 for each group). Statistical analyses were carried out on raw data. Drug was injected at time 0. All data are expressed as mean ± SE. *P < 0.01 in comparison with the corresponding time point of saline.

Figure 4.

Effects of prefrontal cortical norepinephrine depletion on extracellular dopamine in the nucleus accumbens of animals [Sham (A), NE-depleted (B)] injected with saline (Sal) or morphine (Mor 10, 20 mg/kg, i.p.). Results are expressed as percent changes from basal levels of each experimental group (n = 6 for each group). Statistical analyses were carried out on raw data. Drug was injected at time 0. All data are expressed as mean ± SE. *P < 0.01 in comparison with the corresponding time point of saline.

Statistical analyses revealed a significant lesion × treatment × time interaction [F(12,180) = 4.99; P < 0.0005]. Simple effect analyses revealed a significant effect of time only for morphine, and a significant difference between morphine and saline. Morphine increased DA outflow in the NAc of control (Sham) animals in a dose related fashion. In animals with NE depletion of the mpFC, however, there was no morphine-induced increase in extracellular DA in the NAc at any of the doses tested (Fig. 4).

To allow assessment of whether NE depletion impaired morphine-induced NE and DA release, the effects of prefrontal NE depletion on NE and DA release induced by systemic administration of morphine in the mpFC are set out in Figure 5 (A,B). For NE, statistical analyses revealed a significant lesion × time interaction (F (6,84) = 3.9; P < 0.05). Simple effect analyses revealed a significant effect of time only for the Sham group and a significant difference between Sham and NE-depleted groups at all time points after morphine injection. Selective NE depletion in the mpFC impairs the increase in prefrontal cortical NE release induced by morphine, while it does not significantly affect basal extracellular NE (Sham: 1.32 ± 0.15; NE-depleted: 1.27 ± 0.17 pg/20 μl) (Fig. 5A). For DA, only a main effect of between factor [time: F(6,78) = 18.6; P < 0.0005] was evident. Selective NE depletion in the mpFC does not significantly affect basal and morphine-induced extracellular DA outflow (Sham: 0.57 ± 0.06; NE-depleted: 0.53 ± 0.07 pg/20 μl) (Fig. 5B).

Figure 5.

Effects of selective prefrontal cortical norepinephrine depletion on extracellular norepinephrine (A) and dopamine (B) in the medial prefrontal cortex of animals receiving morphine (20 mg/kg, i.p.). Statistical analyses were carried out on raw data (NE, Sham: n = 9; NE-depleted: n = 7; DA, Sham: n = 7; NE-depleted: n = 8). Drug was injected at time 0. All data are expressed as mean (pg/20 μl) ± SE. *P < 0.01 in comparison with the corresponding time point of NE-depleted group.

Figure 5.

Effects of selective prefrontal cortical norepinephrine depletion on extracellular norepinephrine (A) and dopamine (B) in the medial prefrontal cortex of animals receiving morphine (20 mg/kg, i.p.). Statistical analyses were carried out on raw data (NE, Sham: n = 9; NE-depleted: n = 7; DA, Sham: n = 7; NE-depleted: n = 8). Drug was injected at time 0. All data are expressed as mean (pg/20 μl) ± SE. *P < 0.01 in comparison with the corresponding time point of NE-depleted group.

Behavioral Experiments

Conditioned Place Preference

Effects of Selective Prefrontal Cortical NE Depletion on CPP Induced by Morphine

The effects of increasing doses of morphine on CPP in Sham and NE-depleted mice are shown in Figure 6A,B. Repeated-measure ANOVA revealed only a main effect of the within factor [pairing: F(2,104) = 62.7; P < 0.0005]. Repeated-measure ANOVA within each group revealed a significant effect of pairing factor only for Sham animals injected with 20 mg/kg morphine [F(1,6) = 16.8; P < 0.05] (Fig. 6A). This group showed a significant preference for the morphine paired compartment (Paired), while 5 and 30 mg/kg morphine doses failed to induce any preference for the morphine-paired compartment. Animals that had experienced saline-pairing with both compartments showed no preference for either compartment, regardless of the lesion condition (Sham or NE-depleted). Moreover, the behavior of NE-depleted animals was similar to that of animals that experienced only the vehicle solution during training, namely, they did not show any preference for either compartment, regardless of the morphine dose (Fig. 6B).

Figure 6.

Effects of different doses of morphine (Sal, Mor 5, 20, 30 mg/kg, i.p.) in the conditioned place preference shown by Sham (A) and NE-depleted (B) mice (for details see Materials and Methods). All data are expressed as mean (s ± SE) time spent in paired, unpaired and center chamber. P < 0.05 in comparison with the unpaired chamber.

Figure 6.

Effects of different doses of morphine (Sal, Mor 5, 20, 30 mg/kg, i.p.) in the conditioned place preference shown by Sham (A) and NE-depleted (B) mice (for details see Materials and Methods). All data are expressed as mean (s ± SE) time spent in paired, unpaired and center chamber. P < 0.05 in comparison with the unpaired chamber.

Effects of Selective Prefrontal Cortical NE Depletion on Morphine-induced Reinstatement of Extinguished CPP

The effects of prefrontal NE depletion on morphine priming (5 mg/kg)-induced reinstatement of extinguished CPP are shown in Figure 7.

Figure 7.

Conditioned place preference (CPP) (20 mg/kg, i.p.), extinction (EXT) by saline pairing and reinstatement (RE) induced by priming injection of morphine (5 mg/kg, i.p.) shown by Sham (A) and NE-depleted (B) groups (n = 7 for each group) (for details, see Materials and Methods). All data are expressed as mean (s ± SE) time spent in paired, unpaired and center chamber. P < 0.005 in comparison with the unpaired chamber.

Figure 7.

Conditioned place preference (CPP) (20 mg/kg, i.p.), extinction (EXT) by saline pairing and reinstatement (RE) induced by priming injection of morphine (5 mg/kg, i.p.) shown by Sham (A) and NE-depleted (B) groups (n = 7 for each group) (for details, see Materials and Methods). All data are expressed as mean (s ± SE) time spent in paired, unpaired and center chamber. P < 0.005 in comparison with the unpaired chamber.

In the case of CPP testing carried out before surgery, statistical analyses revealed a significant pairing effect for both groups [F(2,12) = 19.24; P < 0.005; F(2,12) = 12.55; P < 0.005]. Both groups showed a significant preference for the morphine-paired compartment [F(1,6) = 12.7; P < 0.05; F(1,6) = 7.13; P < 0.05]. Extinction test results revealed no preference for either previously morphine- and saline-paired compartments for each group (Fig. 7). Finally, as far as the reinstatement test is concerned, statistical analyses revealed a significant pairing × lesion interaction [F(2,24) = 8.03; P < 0.005]. After the extinction procedure, morphine priming reactivated the place preference in Sham mice [F(1,6) = 20.84; P < 0.005], while morphine failed to induce any preference for the morphine-paired compartment in the NE-depleted group.

One-way passive avoidance

No significant differences were found in the passive avoidance test between Sham and NE-depleted mice in the latency to enter the shock-paired chamber on the test day (Sham = 137 ± 14 s; NE-depleted = 146 ± 16 s; mean ± SE), a result that rules out any associative or memory impairment in NE-depleted animals.

Discussion

The first major finding in the present study is that morphine enhances NE outflow in the mpFC in a dose-dependent fashion. These are the first results to show the ability of opiates to increase NE release in the mpFC. Conflicting results have been reported concerning the effects of acute morphine administration on NE release and metabolism in cortical areas. Thus, a low dose of morphine was found to produce a very mild decrease in NE release in the prefrontal cortex of rats implanted with horizontal microdialysis probes (Devoto et al., 2002), which cross prefrontal areas different from those crossed by the vertical probes used in our study. However, it has been reported that morphine challenge increases prefrontal NE metabolism in mice as shown by levels of 3-methoxy-4-hydroxyphenylethyleneglycol (MOPEG), thus indicating an increase in NE release (Airio and Ahtee, 1999). Although morphine is known to reduce the electrical activity of noradrenergic neurons in the locus coeruleus which project into the mpFC, it has been reported that morphine increases periodic bursting of LC neurons in awake monkeys (Aston-Jones et al., 1992). Moreover, it must be taken into account a possible stimulation of LC by projections from the mpFC (Jodo et al., 1998) that could in turn produce enhanced cortical NE activity.

It is also possible that local controls on the NE terminals in the prefrontal cortex are exerted by other neurotransmitter and/or neurohormone systems (Berridge and Waterhouse, 2003). Thus, a number of studies showed a complex modulation of neuronal activity by opioid systems in the mpFC (see Steketee, 2003 for review), indicating that GABAergic and glutamatergic neurons in this brain area respond in heterogeneous way (either inhibitory or excitatory) to mu receptor stimulation, and their responses could affect through a pre-synaptic action NE release in the mpFC. It may be also that morphine-induced increased DA release in the mpFC leads to increased NE release, possibly involving D1 receptors, as recently demonstrated (Pan et al., 2004), thus producing a local DA-mediated action that would add to that postulated previously through VTA–LC interactions (Deutch et al., 1986). In addition, a role of glia may be envisaged, since glia has been recently shown to modulate LC neurons excitability (Alvarez-Maubecin et al., 2000). Finally, other brain stem areas may be involved in stimulatory effect of opioids on noradrenergic projections to the mpFC (Johnson et al., 2002).

The second important finding of this study is that enhanced NE release in the mpFC was critical for the effects of morphine on mesoaccumbens DA. Thus, in the Sham group systemic morphine produced a large and significant increase in DA outflow in the NAc. Conversely, no significant increase was observable in the NAc of NE-depleted animals, thus pointing to intact noradrenergic transmission within the mpFC being a necessary condition for stimulated DA release within the NAc by morphine, in agreement with previous reports showing reduced DA release potential in the NAc of rats pretreated with DSP-4 (Haidkind et al., 2002). It is worth noting that our method allows selective NE depletion in the mpFC while other methods, such as neurotoxic lesions of the dorsal noradrenergic bundle, are likely to affect other brain areas in addition to mpFC (Sadile et al., 1995; Valentini et al., 2004), as well as other neurotransmitter systems (Eller and Harro, 2002; Haidkind et al., 2002; Harro et al., 2003).

Our results cannot be explained by reduced basal DA levels in the NAc. In fact, basal extracellular DA levels in the NAc of NE-depleted mice were no different from those of Sham animals. Moreover, the present results cannot be ascribed to increased or reduced levels of extracellular DA in the mpFC induced by prefrontal NE depletion, since basal or morphine-induced extracellular DA values shown by NE-depleted mice in dialysate were not different from those of Sham animals. A body of evidence suggests that DA in the prefrontal cortex is co-released with NE from noradrenergic terminals (Devoto et al., 2001, 2002). Moreover, it has been reported that DA in this brain area is normally cleared by NE transporter (Tanda et al., 1997; Moròn et al., 2002). Our results, showing a lack of effects of the lesion on basal extracellular DA, suggest that the likely reduction of DA released from destructed noradrenergic terminals is compensated by augmented availability of DA due to its reduced uptake from these terminals. However, NE-depleted animals showed an increase of morphine-induced DA release similar to that exhibited by Sham animals, thus suggesting that prefrontal noradrenergic and dopaminergic projections are functionally uncoupled.

It is worth noting that although the experimental procedure used to induce neurotoxic lesion of medial prefrontal cortex (Ventura et al., 2003a, 2004a,b) produced a dramatic decrease in NE tissue levels in the mpFC, basal extracellular NE values shown by NE-depleted mice in dialysate did not differ from those of Sham animals. These results suggest a compensatory response by spared cortical afferents in the NE-depleted mice 7 days following surgery that, however, fails to enable the response to pharmacological challenge. Moreover, our experimental procedure did not produce trauma or rough damage that could interfere with microdialysis experiments, as supported by histological analysis. In fact, both DA and NE basal levels in the mpFC of Sham animals were non different from those of naïve (not submitted to lesion procedure) animals (DA: Naïve = 0.52 ± 0.03; Sham = 0.57 ± 0.06; NE: Naïve: 1.28 ± 0.14; Sham: 1.32 ± 0.15 pg/20 μl; NS, Student's t-test), thus indicating clearly that neurotoxin microinjection procedure did not damage significantly brain tissue to bias microdialysis experiments.

Morphine-induced increase in NE outflow may participate in morphine-promoted DA release within the NAc through a number of possible mechanisms. First, it might activate the excitatory prefrontal cortical projection to the ventral tegmental area (Shi et al., 2000). Second, it might activate cortico-accumbal glutamatergic projections stimulating AMPA/kainate and NMDA presynaptic receptors located on DA nerve terminals, thus facilitating release (Darracq et al., 2001). Third, it might facilitate the ionotropic receptor-mediated activation of efferent inhibitory GABAergic neurons, which participate in a double inhibition loop involved in the control of DA cells activity by local GABAergic neurons (Darracq et al., 2001). The marked effect of cortical NE depletion on accumbal DA release by morphine in our experiments points to the involvement of all three mechanisms. Taken together, the disruption of these various mechanisms by mpFC NE depletion may converge to inhibit the activity of DA neurons.

The third finding of this study is that prefrontal cortical NE depletion impairs conditioned preference for morphine-paired contexts. Morphine induces a conditioned place preference only at 20 mg/kg dose in Sham animals, while both the lowest (5 mg/kg) and the highest (30 mg/kg) doses of morphine are not able to induce a significant preference for morphine-paired compartment. While Sham animals trained with 20 mg/kg during conditioning phase exhibited a significant preference for the morphine-paired compartment, no preference was evident in NE-depleted mice, regardless of the morphine dose. The results obtained with the dose of 20 mg/Kg are consistent with recent evidence that α1-subtype adrenergic receptor stimulation is necessary for morphine-induced CPP (Drouin et al., 2002).

It must be pointed out that prefrontal NE depletion did not interfere with either associative or mnemonic processes since NE-depleted animals proved capable of learning a passive avoidance task. Instead, the effects of NE depletion on the CPP test appear specifically to affect the rewarding effects of the addictive drug. In this regard, it is worth noting that the CPP has become a widely utilized measure of the rewarding effects of drugs (Tzschentke, 1998). Moreover, drug-induced CPP has been shown to depend on mesoaccumbens DA transmission, which is generally considered a main component of the brain reward system (McBride et al., 1999). This is strong evidence that the effects of prefrontal NE depletion on morphine-induced CPP are dependent on the blockade of morphine-induced mesoaccumbens DA release.

Finally, a major role of NE in the mpFC has been recently shown for other reinforces such amphetamine (Ventura et al., 2003a) and cocaine (Ventura et al., 2003b).

The conditioned place preference paradigm was used also to study relapse to drug dependence (Lu et al., 2002; Mueller et al., 2002; Shaham et al., 2003; Wang et al., 2002). Reinstatement induced by priming injection of drugs is widely accepted as a model for investigating the mechanisms involved in relapse (Stewart, 1984; De Vries et al., 1998).

Although knowledge of the neural mechanisms involved in opiate addiction is still incomplete, there is some evidence that the mesolimbic DA system is involved in reinstatement induced by priming injections of opiates (Di Chiara and North, 1992; Stewart, 1984; Wang et al., 2002). Moreover, increasing evidence points to a major involvement of the mpFC in drug relapse behavior (Neisewander et al., 2000; See 2002; Capriles et al., 2003). Our data suggest, for the first time, that prefrontal cortical NE transmission plays a critical role in the reinstatement to morphine-seeking behavior. Thus, the last set of evidence showed that morphine priming in a low dose, which is non-effective in inducing CPP in naive animals, reactivated place preference in Sham animals following the extinction procedure, in agreement with previous data (Lu et al., 2002; Mueller et al., 2002; Wang et al., 2000, 2002). This effect of the priming dose is very likely to depend on sensitization to the opiate (Lu et al., 2002; De Vries et al., 1998) produced by repeated drug administration. However, the effects of morphine priming on conditioned place preference reinstatement were completely abolished in NE-depleted mice. Thus, selective NE depletion in the mpFC carried out after extinction of CPP to morphine impaired the reinstatement of the extinguished CPP that was, by contrast, evident in Sham animals.

Although there is evidence that the neuronal events underlying drug-induced reinstatement are to some degree different from those involved in the acute rewarding effects (Shaham, 2003), as reported for cocaine (Shalev et al., 2002), it has been suggested that priming injections of drugs reinstate drug seeking after extinction because they activate brain systems involved in their rewarding effects (Stewart, 1984). The present results provide evidence to support the latter view. Thus, they indicate a common mechanism that mediates both the rewarding and priming effects of morphine.

In conclusion, our results suggest that intact prefrontal cortical NE transmission, by allowing increased DA release in the NAc induced by morphine, is a necessary condition for morphine-induced reward as well as for morphine primed reinstatement of an extinguished CPP. It is worth noting that imaging studies in humans suggest that the prefrontal cortex is activated during reinforced and conditioned response and during compulsive drug intake (Volkow and Fowler, 2000). Moreover, the present data suggest a major role of the mesocorticolimbic catecholaminergic circuit in which the prefrontal noradrenergic and accumbal dopaminergic transmission is synergistically coupled in order to control the addictive properties of morphine.

We thank Dr E. Catalfamo for his skilful assistance. This research has been supported by Ministero della Ricerca Scientifica e Tecnologica (COFIN 2003), Università ‘La Sapienza’ Ateneo (2002/2003), Ministero della Salute (Progetto Finalizzato RF00.96P and RF03.182P).

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Author notes

1Dipartimento di Psicologia, Università ‘La Sapienza’, via dei Marsi n. 78, 00185 Rome, Italy and 2IRCCS Fondazione Santa Lucia, Rome, Italy