EJ has received financial support from the NovoNordisk Foundation. However, this does not alter the authors’ adherence to any of the journals policies on sharing data and materials. The remaining authors declare no conflict of interest.
Conceived and designed the experiments: EJ. Performed the experiments: JV DV. Analyzed the data: DV EJ. Contributed reagents/materials/analysis tools: EJ JAE. Wrote the paper: EJ DV JAE JV.
Amphetamine dependence, besides its substantial economical consequence, is a serious cause of mortality and morbidity. By investigations of the neurochemical correlates through which addictive drugs, such as amphetamine, activate the mesoaccumbal dopamine system unique targets for treatment of drug addiction can be identified. This reward link consists of a dopamine projection from the ventral tegmental area to the nucleus accumbens (NAc) suggesting that these brain areas are important for reward. The physiological function of gut-brain peptides has expanded beyond food intake modulation and involves regulation of drug reinforcement. A novel candidate for reward regulation is the anorexigenic peptide neuromedin U (NMU). We therefore investigated the effects of intracerebroventricular (icv) administration of NMU on amphetamine’s well-documented effects on the mesoaccumbal dopamine system,
Addiction to amphetamine, a common cause of mortality and morbidity, is a major cost for both the society and the individual [
The highly conserved neuropeptide NMU is detected along the gastrointestinal tract as well as in the brain. In the rat gut, NMU-like immunoreactivity has been demonstrated in nerve cell bodies as well as fibers in the submucosal and myenteric plexuses, as opposed to endocrine cells (for review see [
Adult post-pubertal age-matched male NMRI mice (8–12 weeks old and 25–40 g body weight; Charles River, Susfeldt, Germany) were used. The mice were allowed to acclimatize at least one week before the start of the experiment and were group housed and maintained at a 12/12 hour light/dark cycle, a temperature at 20°C and a 50% humidity. Tap water and food (normal chow; Harlan Teklad, Norfolk, England) were supplied
For studies investigating amphetamine-induced activation of the mesoaccumbal dopamine system in mice, dex-amphetamine sulphate (RBI, Natick, USA) was dissolved in vehicle (0.9% sodium chloride solution) and was administered intraperitoneal (ip) at a dose of 2 mg/kg 10 minutes prior to initiation of the experiments. NMU (Bionuclear, Bromma, Sweden) was diluted in Ringer solution (NaCl 140 mM, Ca Cl2 1.2 mM, KCl 3.0 mM and MgCl2 1.0 mM; Merck KGaA, Darmstadt, Germany). A dose of 1 μg in 1 μl for intracerebroventricular (icv) administration was selected since this dose previously was found to reduce food intake in mice [
In order to administer NMU or vehicle solution guide cannulas (stainless steel, length 10 mm, with an o.d./i.d. of 0.6/0.45 mm) were implanted four days prior to the experiments. The surgery was conducted as follows: The rodent was anesthetized with isofluran (Isofluran Baxter; Univentor 400 Anaesthesia Unit, Univentor Ldt., Zejtun, Malta), placed in a stereotaxic apparatus (David Kopf Instruments; Tujunga, CA, USA) and kept on a heating pad to prevent hypothermia. Two drops of Xylocain adrenalin (5 μg/ml; Pfizer Inic; New York, USA) were used for local anaesthesia. The skull bone was exposed after an incision and three holes were drilled, two for the guide cannula and one for the anchoring screw. The coordinates for the third ventricle (for icv administration) relative to bregma were AP -0.9 mm and ML ±0.0. The coordinates for NAc were AP +1.4 mm and ML ±0.6. The guide cannulas were placed 1 mm below the surface of the brain and they were subsequently anchored to the screw and the skull bone with dental cement (DENTALON® plus; Agntho’s AB, Lidingö, Sweden). After surgery the mice were injected with carprofen (Rimadyl
Locomotor activity was performed as previously described [
In the first experiment, the effects of NMU (1 μg, icv) on amphetamine-induced (2 mg/kg, ip) locomotor stimulation were investigated. Each mouse received one treatment combination (vehicle-vehicle, vehicle-amphetamine, NMU-vehicle or NMU-amphetamine) and was only subjected to one experimental trial.
In the second experiment, the effects of NMU (0.3 μg, icv) on amphetamine-induced (2 mg/kg, ip) locomotor stimulation were explored. Each mouse received one treatment combination (vehicle-vehicle, vehicle-amphetamine, NMU-vehicle or NMU-amphetamine) and was only subjected to one experimental trial.
The third experiment was conducted to establish a dose for intra-NAc administration without no effect per se on locomotor activity. The effects of local administration of vehicle or NMU (250, 125 or 62.5 ng in 0.5 μl per side) bilaterally into the NAc were studied in mice.
In the fourth experiment in separate mice the role of accumbal NMUR2 for amphetamine-induced reward was investigated. Therefore, the effects of intra-NAc NMU (62.5 ng in 0.5 μl) administration on amphetamine-induced (2 mg/kg, ip) locomotor stimulation were evaluated. Each mouse received one treatment combination (vehicle-vehicle, vehicle-amphetamine, NMU-vehicle or NMU-amphetamine) and was only subjected to one experimental trial.
The present experiment investigates the role of central NMU for amphetamine-induced activation of the mesoaccumbal dopamine system. For measurements of extracellular dopamine levels, mice were implanted with a microdialysis probe positioned in the shell of NAc. Surgeries were performed as previously described [
The effect of central administration of NMU (1 μg, icv) on amphetamine-induced (2 mg/kg, ip) accumbal dopamine release was investigated using microdialysis in freely moving mice. On the day of the experiment the probe was connected to a microperfusion pump (U-864 Syringe Pump; AgnThós AB) and perfused with Ringer solution at a rate of 1.5 μl/minute. After one hour of habituation to the microdialysis set-up, perfusion samples were collected every 20 minutes. The baseline dopamine level was defined as the average of first three consecutive samples (-40 min until 0 min). After baseline samples, NMU or vehicle was administered (0 minutes). Amphetamine was administered at 20 minutes later (20 minutes), creating the following treatment groups: vehicle-amphetamine and NMU-amphetamine. Eight consecutive 20-minute samples were thereafter collected. The challenge-induced increase in accumbal dopamine was calculated as the percentage of increase from the baseline samples.
Dopamine was separated and quantified using two different high-performance liquid chromatography apparatuses with electro chemical detection as described previously [
The CPP experiments were designed to evaluate the effects of NMU, icv or accumbal, on the expression of amphetamine-induced CPP. The CPP test was performed in mice as previously described [
Following each experiment the location of the probe (located in the NAc shell) and/or guide cannulas (located in the third ventricle (icv,
(A) A coronal mouse brain section showing five representative guide cannula placements (illustrated by vertical lines) aiming at the third ventricle (icv) [
The locomotor activity experiments as well as microdialysis experiments were evaluated by a two-way ANOVA followed by Bonferroni post-hoc test for comparisons between different treatments and specifically at given time points. The CPP data were evaluated by an unpaired t-test. In addition, the effect of treatment on time spent in drug-paired compartment was analysed with a one-way ANOVA followed by a Bonferroni post-hoc test for comparisons between different treatment. Data are presented as mean ± SEM. A probability value of P<0.05 was considered as statistically significant.
Analysis of baseline locomotor activity showed no overall main effect of treatment (F(3,41) = 1.18,
An overall main effect of treatment (F(3,252) = 64.97,
(A) Amphetamine-induced (2 mg/kg, ip) locomotor stimulation was blocked by a central injection of NMU (1 μg, icv) at time point 15–35 minutes. Central NMU administration had no effect
Accumbal microdialysis measurements of dopamine in mice revealed an overall main effect of treatment (F(1,117) = 23.47, P<0.0001), time (F(12,117) = 5.415, P<0.0001) and of treatment x time interaction (F(12,117) = 2.209, P<0.0154) (
The amphetamine-induced (2 mg/kg) (amphetamine-vehicle, n = 8) CPP was significantly attenuated by an acute icv administration of NMU (1 μg, icv) (amphetamine-NMU, n = 7) on the post-conditioning day (
Analysis of baseline locomotor activity showed no overall main effect of treatment (F(3,42) = 1.51,
An overall main effect of treatment (F(3,252) = 201.3,
Amphetamine-induced (2 mg/kg, ip) locomotor stimulation was reduced, but not blocked, by a central injection of a lower does of NMU (0.3 μg, icv). Central administration of this lower dose of NMU had no effect
The locomotor activity data in
Analysis of baseline locomotor activity showed no overall main effect of treatment (F(3,42) = 1.63,
There was an overall effect of treatment (F(3,252) = 15.18,
Analysis of baseline locomotor activity showed no overall main effect of treatment (F(3,66) = 0.55,
An overall main effect of treatment (F(3,396) = 36.70,
(A) Amphetamine-induced (2 mg/kg, ip) locomotor stimulation was attenuated by an accumbal injection of NMU (62.5 ng per side). Accumbal NMU administration had no effect
Mice receiving bilateral vehicle administration into NAc (
The amphetamine-induced (2 mg/kg) (amphetamine-vehicle, n = 11) expression of CPP was not affected by intra-NAc administration of NMU (62.5 ng/side) (amphetamine-NMU, n = 14) on the post-conditioning day (
The present study revealed three principal sets of findings. Firstly, we show for the first time that activation of the central NMU receptor signalling system inhibited the well-documented effects of amphetamine on the mesoaccumbal dopamine system [
Secondly, we demonstrated that NMU administration into the NAc blocked the ability of amphetamine to induce a locomotor stimulation, suggesting that accumbal NMUR2 modulate amphetamine-induced activation of the mesolimbic dopamine system. Supportively preclinical findings show: i) amphetamine-induced locomotor stimulation is mediated, at least in part, via its ability to increase accumbal dopamine releases [
Thirdly, we showed in mice that icv administration of NMU, but not directly into the NAc, attenuates the expression of CPP that may reflect a measure of reward-related contextual cues associated with a drug experience [
The downstream mechanisms through which central NMU signalling reduces amphetamine-induced reward remain unknown and need to be further investigated. The possibility that NMU attenuates amphetamine-induced reward is secondary to its effect on the hypothalamus-pituitary-adrenal (HPA) stress axis should be deliberated. Indeed, central administration of NMU, in higher doses than presently used, increases stress-like behaviours such as face washing, scratching and grooming [
During the last years the traditional role of gut-brain peptides as modulators of energy homeostasis has been extended and it has been shown that several of these peptides regulate reward (for review see [
Collectively the present series of experiments show that central NMU signalling regulates amphetamine-induced reward as well as reward-related contextual cues associated with drug experience in mice. Moreover, accumbal NMUR2 regulate amphetamine induced locomotor stimulation but not the expression of CPP in mice. We therefore argue that the role of central NMUR2 in drug dependence should be investigated further. Nevertheless, the multiple functions of NMU should be taken into account when considering targeting NMU signalling for treatment of drug addiction.
Britt-Mari Larsson and Kenn Johannessen are gratefully acknowledged for expert and valuable technical assistance. The study is supported by grants from the Swedish Research Council (2009–2782 and 2011–4646 and 2015–03219), Swedish Society for Medical Research, The Swedish brain foundation, LUA/ALF (grant no. 148251) from the Sahlgrenska University Hospital, Torsten Söderberg, Alcohol research council of the Swedish alcohol retailing monopoly and the foundations of Adlerbertska, Fredrik and Ingrid Thuring, Tore Nilsson, Längmanska, Wilhelm and Martina Lundgren, Knut and Alice Wallenberg, Magnus Bergvall, Anérs, Jeansons, Åke Wiberg, NovoNordisk, Gothenburg Psychiatry Research Foundation, the Swedish Society of Medicine. The funding sources had no role in the collection, analysis and interpretation of data, in the writing of the report or in the decision to submit the article for publication.