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Proc Natl Acad Sci U S A. Mar 20, 2007; 104(12): 5181–5186.
Published online Mar 9, 2007. doi:  10.1073/pnas.0610178104
PMCID: PMC1820518
Neuroscience

Prefrontal/accumbal catecholamine system determines motivational salience attribution to both reward- and aversion-related stimuli

Abstract

Recent evidence suggests that rewarding and aversive stimuli affect the same brain areas, including medial prefrontal cortex and nucleus accumbens. Although nucleus accumbens is known to respond to salient stimuli, regardless of their hedonic valence, with selective increased dopamine release, little is known about the role of prefrontal cortex in reward- and aversion-related motivation or about the neurotransmitters involved. Here we find that selective norepinephrine depletion in medial prefrontal cortex of mice abolished the increase in the release of norepinephrine by prefrontal cortex and of dopamine by nucleus accumbens that is induced by food, cocaine, or lithium chloride and impaired the place conditioning induced by both lithium chloride (aversion) and food or cocaine (preference). This is evidence that prefrontal cortical norepinephrine transmission is necessary for motivational salience attribution to both reward- and aversion-related stimuli through modulation of dopamine in nucleus accumbens, a brain area involved in all motivated behaviors.

Keywords: motivation, norepinephrine, prefrontal cortex, dopamine, place conditioning

Animals as well as humans have a propensity to seek out rewards and avoid punishments. This clearly adaptive behavior involves the ability to represent the value of rewarding and punishing stimuli, establish predictions about them, and use these predictions to guide behavior (1). Insofar as emotions can be defined as “states elicited by reinforcers (rewards and punishers)” (2), understanding the brain areas involved in processing motivationally salient rewarding or aversive stimuli may be relevant to understanding numerous emotional deficits in humans.

Recent data indicate that nucleus accumbens (NAc) and prefrontal cortex (pFC) constitute a common substrate for processing both rewarding and aversive stimuli (37). Ventral striatum (or NAc) is involved in processing the information underlying the motivational control of goal-directed behavior, and human and animal studies support a general role for this brain area in processing both rewarding and aversive stimuli, regardless of valence (38).

Moreover, a large body of evidence suggests that pFC is directly involved in goal-directed behavior as well as in affective processing (1, 9). However, although dopamine transmission in NAc has been proposed to mediate a shared “motivational salience” process in positive and negative valence (3, 6), the role of pFC in this process and the neurochemical substrate involved are still unknown.

Norepinephrine transmission in pFC is activated by aversive stimuli (10, 11) and by aversive and conditioned appetitive stimuli (12, 13). Moreover, norepinephrine in medial pFC (mpFC) was recently shown to be involved in the rewarding effects of some commonly abused drugs through its modulating action on dopamine release in NAc (14, 15). This suggests that prefrontal cortical norepinephrine transmission engages accumbal dopamine to process motivationally salient stimuli.

Here, using mice, we set out to assess whether the prefrontal cortical norepinephrine/mesoaccumbens dopamine system is a common neural substrate involved in processing affectively positive and negative stimuli. In particular, we investigated whether norepinephrine in mpFC, through its modulating action on the dopaminergic mesolimbic system, is necessary for attribution of motivational salience to reward- and aversion-related stimuli.

Because experience is a major determinant of the motivational impact of any given stimulus (7), we assessed the effects of first exposure to rewarding natural (palatable food, milk chocolate) and pharmacological (cocaine) stimuli and to an aversive pharmacological stimulus [lithium chloride (LiCl)] on prefrontal cortical norepinephrine and accumbal dopamine release by intracerebral microdialysis. In addition, to determine whether norepinephrine prefrontal transmission governs increased accumbal dopamine outflow induced by first exposure to these motivationally salient stimuli, we also evaluated the effects of selective norepinephrine depletion in mpFC on dopamine release in NAc and on norepinephrine release in mpFC induced by first exposure to these stimuli.

Finally, we investigated the effects of selective noradrenergic prefrontal depletion on conditioned place preference (CPP) induced by chocolate and cocaine and on conditioned place aversion (CPA) induced by LiCl. A place-conditioning procedure was chosen for this study because it permitted the assessment of the acquisition of conditioned appetitive and aversive properties to stimuli paired with primary rewards and aversive events and because a large body of evidence shows that it is a reliable measure of processes underlying motivational salience attribution to stimuli (3, 16).

Results

Experiment 1.

To evaluate whether first exposure to both rewarding and aversive salient stimuli affects prefrontal norepinephrine and accumbal dopamine outflow, we evaluated, by intracerebral microdialysis, the effects of systemic cocaine or LiCl and chocolate consumption on norepinephrine release in mpFC and dopamine outflow in NAc. Moreover, to ascertain whether cortical noradrenergic transmission is necessary for accumbal dopamine outflow induced by first exposure to these motivationally salient stimuli, we assessed the effects of selective noradrenergic depletion on the accumbal dopamine response induced by cocaine, chocolate, and LiCl. Cocaine, chocolate, and LiCl produced a time-dependent increase in norepinephrine outflow in mpFC of sham-treated groups, reaching a maximal increase of ≈200% at 40 min, ≈70% at 120 min, and ≈100% at 60 min, respectively (Fig. 1a). Although increased norepinephrine release in the pFC in response to cocaine has been widely reported, to our knowledge this is the first report of increased norepinephrine outflow induced by first exposure to chocolate or systemic LiCl within mpFC. These stimuli also produced a parallel time-dependent increase in dopamine outflow in NAc of sham-treated animals (Fig. 1b), in agreement with the view that this area plays a major role in the processing of salient stimuli regardless of their valence (3, 6). The effects of this depletion on norepinephrine release in mpFC were also assessed. Prefrontal norepinephrine depletion was obtained by selective neurotoxic depletion of prefrontal cortical norepinephrine afferents (norepinephrine-depleted groups) in mpFC after protection of dopamine by a selective uptake inhibitor. This method produced a profound depletion of tissue levels of norepinephrine (≈90%), leaving tissue levels of dopamine virtually unaffected. Control animals (sham-treated groups) were subjected to the same treatment as the norepinephrine-depleted mice but received intracerebral vehicle. (Norepinephrine tissue levels were as follows: sham-treated group, 698 ± 26 ng/g wet tissue; norepinephrine-depleted group, 63 ± 17 ng/g wet tissue. Dopamine tissue levels were as follows: sham-treated group, 203 ± 18 ng/g wet tissue; norepinephrine-depleted group, 189 ± 16 ng/g wet tissue.)

Fig. 1.
Prefrontal cortical norepinephrine depletions on extracellular norepinephrine in mpFC and dopamine in NAc. Extracellular norepinephrine (NE) in mpFC (a) and dopamine (DA) in NAc (b) of sham-treated or norepinephrine-depleted animals injected with saline, ...

Selective norepinephrine depletion in mpFC impaired the increase in accumbal dopamine and prefrontal cortical norepinephrine release induced by both drugs and chocolate (Fig. 1), although it did not significantly affect basal extracellular dopamine in NAc (sham-treated group, 1.35 ± 0.15 pg per 20 μl; norepinephrine-depleted group, 1.29 ± 0.18 pg per 20 μl) or basal extracellular norepinephrine in mpFC (sham-treated group, 1.31 ± 0.18 pg per 20 μl; norepinephrine-depleted group, 1.26 ± 0.17 pg per 20 μl). The average basal values of dopamine in NAc and of norepinephrine in mpFC for each group [saline, cocaine (20 mg/kg), LiCl (3 meq/kg), and chocolate] did not differ significantly.

Our results indicate that intact noradrenergic transmission within mpFC is a necessary condition for the stimulation of dopamine release induced by both rewarding and aversive stimuli within NAc, thus strongly suggesting its major role in motivational salience.

Experiment 2.

To investigate whether norepinephrine prefrontal transmission is necessary for acquiring the conditioned appetitive and aversive properties to stimuli paired with primary rewards and aversive events, we assessed the effects of selective prefrontal norepinephrine depletion on place conditioning.

Prefrontal noradrenergic depletion abolished both cocaine- and chocolate-induced CPP as well as CPA induced by LiCl. Thus, although sham-treated animals showed a significant preference for the cocaine- or chocolate-paired compartment and a significant aversion to the LiCl-paired compartment (Fig. 2a), norepinephrine-depleted animals showed no preference for either compartment (Fig. 2b).

Fig. 2.
Prefrontal cortical norepinephrine depletion on place conditioning. Effects of food consumption (1 g of milk chocolate; sham-treated group, n = 8; norepinephrine-depleted group, n = 8) and systemic injection (i.p.) of saline (Sal) (sham-treated group, ...

Note that in preliminary experiments we observed that both CPP and CPA of sham-treated animals were indistinguishable from those of naïve animals. Animals that had experienced saline pairing in both compartments showed no preference for either compartment regardless of the lesion condition (sham treated or norepinephrine depleted). The behavior of norepinephrine-depleted animals treated with cocaine, chocolate, or LiCl was similar to that of animals that experienced only the vehicle solution during training; i.e., they showed no preference for either compartment.

Discussion

Here we report evidence that prefrontal cortical norepinephrine transmission, through modulation of dopamine in NAc, is a necessary condition for motivational salience attribution to both reward- and aversion-related stimuli.

First, because prior experience is a major determinant of the motivational impact of any given stimulus (7), we evaluated, by intracerebral microdialysis, the effects of first exposure to systemic cocaine or LiCl as well as the effects of chocolate consumption on norepinephrine or dopamine release in mpFC and NAc, respectively. Cocaine, chocolate, and LiCl produced a time-dependent increase in the accumbal dopamine as well as in the prefrontal norepinephrine outflow of sham-treated groups. A significant increase of norepinephrine overflow was evident in the mpFC of sham-treated animals within 20 min of receiving chocolate; the overflow subsequently returned to baseline levels and was followed by a large sustained increase. Although this biphasic chocolate-induced increase of norepinephrine in mpFC did not parallel the increase of dopamine in NAc throughout, the initial increase was likely related to the impact of palatable food and to the increase of dopamine in NAc. On the other hand, the second large increase might represent a neurochemical correlate of the cortical arousal required for processing spatial information related to searching and locating the food reward (17). In fact, it has been suggested that increased norepinephrine outflow serves to signal the presence of stimuli with high motivational salience (17). Thus, this increased norepinephrine outflow might permit the selective attention required to search for additional palatable food and might help in the acquisition of conditioned appetitive properties to stimuli paired with food. However, postingestive effects of food intake on norepinephrine cannot be ruled out.

Although increased dopamine release in NAc induced by rewarding or aversive stimuli and increased norepinephrine release in the pFC in response to cocaine have been widely reported, to our knowledge this is the first report of increased norepinephrine outflow induced by chocolate exposure or LiCl within mpFC. Most importantly, we show here that prefrontal cortical noradrenergic transmission is necessary for accumbal dopamine outflow induced by the first exposure to these motivationally salient stimuli. In fact, no significant increase in both prefrontal norepinephrine and accumbal dopamine outflow, induced by these stimuli, was evident in norepinephrine-depleted mice. Norepinephrine in mpFC might activate mesoaccumbens dopamine release through excitatory prefrontal cortical projection to ventral tegmental area dopamine cells (18, 19) and/or through corticoaccumbal glutamatergic projections (20). Moreover, a role for pFC projections to the locus coeruleus in exerting an excitatory influence can be envisaged because this nucleus has been shown to activate ventral tegmental area dopamine neurons (2123), which could lead to increased dopamine release in NAc.

Thus, our results, in agreement with previous reports, show that both unconditioned rewarding and aversive stimuli increase norepinephrine outflow in mpFC (1015) as well as dopamine release in NAc (3, 24). Most importantly, however, they demonstrate that intact noradrenergic transmission within mpFC is a necessary condition for the stimulation of dopamine release induced by both rewarding and aversive pharmacological and natural stimuli within NAc. Therefore, they point to prefrontal norepinephrine and accumbal dopamine transmission as a neural system whose activation by unconditioned rewarding and aversive stimuli is likely a substrate for motivational salience. This view is supported by results from behavioral experiments on the effects of prefrontal norepinephrine depletion on cocaine-, chocolate-, or LiCl-induced place conditioning.

Thus, the second important finding of this study is that prefrontal cortical norepinephrine depletion impairs both CPP induced by cocaine or food and CPA induced by LiCl. Although sham-treated animals showed a significant preference for the cocaine- or chocolate-paired compartment and a significant aversion to the LiCl-paired compartment, norepinephrine-depleted animals showed no preference for either compartment, thus demonstrating that intact prefrontal cortical norepinephrine transmission is necessary for the acquisition of conditioned properties to stimuli paired with primary rewarding or aversive events in a place-conditioning procedure.

The present results indicate that, in prefrontal cortical norepinephrine-depleted mice, the lack of norepinephrine release induced by exposure to rewarding and aversive stimuli (cocaine, food, or LiCl, unconditioned stimulus) prevented motivational salience attribution to conditioned stimulus (spatial pattern) during the pairing sessions. Also, note that prefrontal norepinephrine depletion did not interfere with either associative or mnemonic processes because, as previously shown, norepinephrine-depleted animals proved capable of learning a passive avoidance task (15) and of associating the context with the drug effects (14). However, further investigation is necessary to understand the precise nature of impairments of prefrontal cortical norepinephrine-depleted animals.

Dopaminergic transmission within NAc is considered to mediate the hedonic impact of reward or some aspects of reward learning (see ref. 25 for review). Our results, in agreement with a different view (3), show that dopamine transmission in NAc plays a role in both positively and aversively motivated behavior; most importantly, however, they demonstrate that this motivational process is governed by prefrontal cortical norepinephrine. In fact, selective prefrontal norepinephrine depletion produces the block of both cocaine- or chocolate-induced CPP and LiCl-induced CPA and the impairment of dopamine release in NAc induced by these salient stimuli in control mice, thus demonstrating that noradrenergic prefrontal transmission, through modulation of dopamine release within NAc, is a necessary condition for the motivational processing of both reward- and aversion-related stimuli.

Taken together, the present results from behavioral and microdialysis experiments demonstrate that prefrontal norepinephrine transmission not only mediates the rewarding properties of commonly abused drugs, as suggested by previous studies (14, 15), but is necessary for motivational salience attribution to both reward- and aversion-related stimuli, further showing that addictive drugs, as well as aversive pharmacological stimuli, exploit the same neurobiological mechanism as natural stimuli.

In conclusion, our data extend previous findings that point to the mesolimbic dopaminergic system as a “salience system” involved in all motivated behaviors (3, 6, 26). They also demonstrate that this system is under norepinephrine prefrontal cortical control, thus supporting the view that rewarding and aversive stimuli affect similar pathways in the CNS (7).

Our results provide insights on the neurobiology of reward and aversion because they show that processing both rewarding and aversive salient stimuli involve the same brain areas; i.e., they point to prefrontal noradrenergic and accumbal dopaminergic transmission as a common neural system. Understanding the neurotransmitter systems activated by affectively rewarding or aversive stimuli and their molecular mechanisms will help to provide a basis for elucidating the functioning of neural systems involved in positive as well as negative emotions.

Materials and Methods

Animals.

Male mice of the inbred C57BL/6JIco strain (Charles River Laboratories, Wilmington, MA), which are commonly used in neurobehavioral phenotyping, 8–9 weeks old at the time of the experiments, were housed as previously described (14, 15). Each experimental group consisted of six to eight animals. All experiments were conducted in accordance with Italian national law (Decreto Legislative no. 116, 1992) governing the use of animals for research.

Drugs.

Chloral hydrate, 6-hydroxydopamine (6-OHDA), GBR 12909, cocaine hydrochloride, and LiCl were purchased from Sigma–Aldrich (St. Louis, MO). Cocaine (20 mg/kg), LiCl (3.0 meq/kg), chloral hydrate (350–450 mg/kg), and GBR 12909 (15 mg/kg) were dissolved in saline (0.9% NaCl) and injected i.p. in a volume of 10 ml/kg. 6-OHDA was dissolved in saline containing sodium metabisulfite (0.1 M). For food experiments, the reward was milk chocolate (1 g; Nestlé, Vevey, Switzerland).

Microdialysis.

Animals were anesthetized with chloral hydrate (450 mg/kg), 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 outer diameter of 0.38 mm; Metalant AB, Stockholm, Sweden) in mpFC or in NAc (14, 15). 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 methods of Franklin and Paxinos (27)] were as follows: +2.52 anteroposterior and 0.6 lateral for mpFC and +1.60 anteroposterior and 0.6 lateral for NAc [mostly including the shell subdivision (27)]. The probe (dialysis membrane length of 2 mm for mpFC and 1 mm for NAc and outer diameter of 0.24 mm, MAB 4 cuprophane microdialysis probe; Metalant AB) was introduced 24 h before microdialysis experiments. The animals were lightly anesthetized with chloral hydrate (350 mg/kg) to facilitate manual insertion of the microdialysis probe into the guide cannula. Animals were returned to their home cages. The outlet and inlet probe tubing was protected by locally applied Parafilm. The membranes were tested for in vitro recovery of dopamine and norepinephrine (relative recovery was as follows: dopamine, 10.7 ± 0.82%; norepinephrine, 12.2 ± 0.75%; n = 20) on the day before use to verify recovery.

The microdialysis probe was connected to a CMA/100 pump (Carnegie Medicine, Stockholm, Sweden) through PE 20 tubing (Metalant AB) and an ultralow torque dual-channel liquid swivel (model 375/D/22QM; Instech Laboratories, Plymouth Meeting, PA) to allow free movement. Artificial cerebrospinal fluid (147 mM NaCl/1 mM MgCL/1.2 mM CaCl2/4 mM KCl) 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) and with home cage bedding on the floor. Dialysis perfusion was started 1 h later. After the start of dialysis perfusion, mice were left undisturbed for ≈2 h before the collection of baseline samples. The mean concentration of the three samples collected immediately before treatment (<10% variation) was taken as basal concentration. Before microdialysis experiments started, the mice were assigned to one of the different treatments (saline, cocaine, chocolate, or LiCl) within each group (sham treated or norepinephrine depleted). For experiments with food, the animals were placed on a food-deprivation schedule (28) 4 days before experiments started.

Dialysate was collected every 20 min for 120 (for cocaine and LiCl groups) or 160 (for food groups) min. Only data from mice with a correctly placed cannula are reported. Placements were judged by methylene blue staining. Twenty microliters of the dialysate samples were analyzed by HPLC. The remaining 20 μl was kept for possible subsequent analysis. Concentrations (pg per 20 μl) were not corrected for probe recovery. The HPLC system consisted of an Alliance HPLC system (Waters, Milford, MA) and a coulometric detector (model 5200A; Coulochem II, ESA, Chelmsford, MA) provided with a conditioning cell (M 5021) and an analytical cell (M 5011). The conditioning cell was set at 400 mV, electrode 1 was set at 200 mV, and electrode 2 was set 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 (14, 15). The assay detection limit was 0.1 pg.

Norepinephrine Depletion in mpFC.

Anesthesia and surgical set are described above. Animals were injected with GBR 12909 (15 mg/kg) 30 min before 6-OHDA microinjection to protect dopaminergic neurons. Bilateral injection of 6-OHDA (1.5 μg per 0.1 μl for 2 min for each side) was made into mpFC [coordinates were +2.52 anteroposterior, ±0.6 lateral, and −2.0 ventral with respect to bregma (27)] through a stainless steel cannula (outer diameter of 0.15 mm; Unimed, Lausanne, 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-treated animals were subjected to the same treatment but received intracerebral vehicle. Animals were used for microdialysis or behavioral experiments 7 days after surgery.

Norepinephrine and dopamine tissue levels in mpFC were assessed as previously described (14, 15) to evaluate the extent of depletion.

Place Conditioning.

Behavioral experiments were performed by using a place-conditioning apparatus (14, 15, 29). 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. Animals were used for behavioral experiments 7 days after surgery. Before conditioning, the mice were assigned to one of the different treatments (saline, cocaine, chocolate, or LiCl) within each group (sham treated or norepinephrine depleted).

The training procedure for place conditioning was described previously (14, 15). 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 place conditioning with pharmacological stimuli, one of the patterns was consistently paired with saline and the other with cocaine (20 mg/kg i.p., CPP) or LiCl (3.0 meq/kg i.p., CPA) during the conditioning phase. These doses were chosen on the basis of previous studies showing that C57BL/6JIco mice display a stronger CPP at a cocaine dose of 20 mg/kg (30, 31) and a trend toward aversion in CPA test at a LiCl dose of 3.0 meq/kg (32). For animals in the control group, both chambers were paired with saline. For CPP with food, one of the patterns was consistently paired with standard food (1 g of mouse standard diet) and the other with palatable food (1 g of milk chocolate). The animals were placed on a food-restriction schedule (28) 4 days before conditioning started. This schedule lasted throughout conditioning.

For all place-conditioning experiments, pairings were balanced so that for half of each experimental group the unconditioned stimulus (cocaine, chocolate, or LiCl) was paired with one of the two patterns; for the other half of each group, the unconditioned stimulus was paired with the other pattern. Testing for the expression of CPP or CPA was conducted on day 10 by using the pretest procedure. Behavioral data were collected and analyzed by the EthoVision fully automated video tracking system (Noldus, Wageningen, The Netherlands). 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 to the computer's memory. Later, the digital data are analyzed by means of the EthoVision software to obtain “time spent” (in seconds), which is used as raw data for preference scores in each sector of the apparatus by each subject.

Statistics.

Place conditioning.

For place-conditioning experiments, statistical analyses were performed by calculating the time (in seconds) spent in center (Center), drug/chocolate-paired (Paired), and saline/standard food-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 one to which they were exposed.

Effects of selective prefrontal cortical norepinephrine depletion on place conditioning.

Data from place-conditioning experiments were analyzed by using repeated-measures ANOVA with one between factor (pretreatment, two levels: sham treated and norepinephrinedepleted) and one within factor (pairing, three levels: Center, Paired, and Unpaired) for each treatment [saline/saline, saline/cocaine (20 mg/kg), saline/LiCl (3 meq/kg), and standard food/chocolate]. Because the important comparisons are those between Paired and Unpaired compartments, mean comparisons of time spent in these chambers were made by using repeated-measures ANOVA within each group.

Two-way ANOVA revealed significant pretreatment × pairing interaction for cocaine [F (2, 28) = 3.47; P < 0.05], LiCl [F (2, 28) = 4.55; P < 0.05], and chocolate [F (2, 28) = 3.5; P < 0.05].

Repeated-measures ANOVA within each group revealed a significant effect of the pairing factor only for sham-treated animals injected with cocaine [F (1, 14) = 24.3; P < 0.0005], LiCl [F (1, 14) = 10.3; P < 0.01], or chocolate [F (1, 14) = 7.31; P < 0.05].

Norepinephrine depletion in mpFC.

The effects of prefrontal norepinephrine depletion on tissue levels of dopamine and norepinephrine in mpFC were analyzed by two-way ANOVA. The factors were as follows: lesion (two levels: sham treated and norepinephrine depleted) and experiment (two levels: behavioral experiment and microdialysis experiments). Individual between-groups comparisons were carried out when appropriate by post hoc test, Duncan's multiple-range test. Statistical analyses were carried out on data from behavioral and microdialysis experiments. Two-way ANOVA for effects of prefrontal norepinephrine depletion on dopamine and norepinephrine tissue levels in mpFC showed a significant lesion effect for norepinephrine only [F (1, 188) = 2.02; P < 0.0005], but no experimental effects.

Microdialysis.

Statistical analyses were carried out on raw data (concentrations of pg per 20 μl). The effects of prefrontal norepinephrine depletion on norepinephrine release in mpFC or on dopamine outflow in NAc of animals challenged with cocaine (20 mg/kg) or LiCl (3 meq/kg) were analyzed by repeated-measures ANOVA with two between factors (pretreatment, two levels, sham treated and norepinephrine depleted; and treatment, three levels, saline, cocaine, and LiCl) and one within factor (time, seven levels, 0, 20, 40, 60, 80, 100, and 120). The effects of prefrontal norepinephrine depletion on norepinephrine release in mpFC or on dopamine outflow in NAc of animals exposed to chocolate were analyzed by repeated-measures ANOVA with one between factor (pretreatment, two levels, sham treated and norepinephrine depleted) and one within factor (time, nine levels, 0, 20, 40, 60, 80, 100, 120, 140, and 160). Simple effects were assessed by one-way ANOVA for each time point. Individual between-group comparisons were carried out when appropriate by post hoc test, Duncan's multiple-range test.

Statistical analyses for the effects of pharmacological stimuli on prefrontal norepinephrine outflow revealed a significant pretreatment × treatment × time interaction [F (12, 180) = 4.98; P < 0.005]. Statistical analyses for effects of chocolate consumption on norepinephrine release revealed a pretreatment × time interaction [F (8, 80) = 7.77; P < 0.005]. Simple effect analyses revealed a significant effect of time only for the sham-treated group and a significant difference between sham-treated and norepinephrine-depleted groups after cocaine or LiCl injection as well as after chocolate consumption.

Statistical analyses for effects of pharmacological stimuli on accumbal dopamine outflow revealed a significant pretreatment × treatment × time interaction [F (12, 180) = 10.02; P < 0.0005]. Statistical analyses of chocolate data revealed a significant pretreatment × time interaction [F (8, 80) = 2.12; P < 0.05]. Simple effect analyses revealed a significant effect of time only for the sham-treated groups and a significant difference between sham-treated and norepinephrine-depleted groups after drug (cocaine or LiCl) injection as well as after chocolate consumption.

Acknowledgments

We thank Dr. E. Catalfamo for skillful assistance. This research was supported by Ministero della Ricerca Scientifica e Tecnologica (PRIN 2005), Università “La Sapienza” Ateneo (2004/2005), and Ministero della Salute (Progetto Finalizzato RF03.182P).

Abbreviations

NAc
nucleus accumbens
pFC
prefrontal cortex
mpFC
medial pFC
CPP
conditioned place preference
CPA
conditioned place aversion
6-OHDA
6-hydroxydopamine.

Footnotes

The authors declare no conflict of interest.

This article is a PNAS direct submission.

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