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. Author manuscript; available in PMC: 2022 Sep 2.
Published in final edited form as: Pharmacology. 2021 Sep 2;106(11-12):597–605. doi: 10.1159/000518033

Neurokinin-1 antagonism distinguishes the role of norepinephrine transporter from dopamine transporter in mediating amphetamine behaviors

Padmanabhan Mannangatti a, Durairaj Ragu Varman a, Sammanda Ramamoorthy a, Lankupalle D Jayanthi a
PMCID: PMC8578286  NIHMSID: NIHMS1721788  PMID: 34515205

Abstract

Background:

Amphetamine and other psychostimulants act on norepinephrine (NE) transporter (NET) and dopamine (DA) transporter (DAT) and enhance NE and DA signaling. Both NET and DAT share anatomical and functional characteristics and are regulated similarly by psychostimulants and receptor-linked signaling pathways. We and others have demonstrated that NET and DAT are downregulated by amphetamine (AMPH) and substance P/neurokinin-1 receptor (NK1R)-mediated protein kinase C pathway.

Objectives:

Since both NET and DAT are downregulated by AMPH and NK1R activation and share high sequence homology, the objective of the study was to determine the catecholamine transporter specificity in NK1R modulation of AMPH-induced behaviors.

Methods:

The effect of NK1R antagonism on AMPH-induced conditioned place preference (CPP) as well as AMPH-induced NET and DAT downregulation were examined using NET and DAT knockout mice (NET-KO and DAT-KO) along with their wild-type littermates.

Results:

Aprepitant (5 mg/kg i.p.) significantly attenuated AMPH (2 mg/kg i.p.) induced CPP in the wild-type and DAT-KO, but not in the NET-KO. Locomotor activity measured during post-conditioning test (in the absence of AMPH) showed higher locomotor activity in DAT-KO compared to wild-type or NET-KO. However, the locomotor activity of all three genotypes remained unchanged following aprepitant. Additionally, in the ventral striatum (VST) of wild-type, the AMPH-induced downregulation of NET function and surface expression but not that of DAT was attenuated by aprepitant.

Conclusions:

The results from the current study demonstrates that aprepitant attenuates the expression of AMPH-induced CPP in DAT-KO mice, but not in NET-KO mice suggesting a role for NK1R-mediated NET regulation in AMPH-induced behaviors.

Keywords: NET-KO, DAT-KO, NK1 receptor, conditioned place preference, locomotor activity, aprepitant

Introduction

It is known that AMPH binds to both the norepinephrine transporter (NET) and the dopamine transporter (DAT) with nearly equal affinity and regulates their function and expression. These actions of AMPH are thought to maintain self-administration and discriminative stimulus effects (1). Noradrenergic and dopaminergic systems overlap anatomically and functionally and the rewarding properties of drugs of abuse are mediated by both DAT and NET (2). We and other investigators in the filed demonstrated that NET is regulated by signaling mechanisms downstream of receptor activation (3–5) and by drugs of abuse (6–10). While it is known that NET protein has several consensus phospho-sites, our laboratory has been in the forefront in demonstrating NET as a phosphoprotein and the regulation NET phosphorylation by PKC and p38 MAPK (5, 10, 11). Our laboratory has also discovered that psychostimulants also modulate NET regulation via phosphorylation of specific motifs in the NET protein (12, 13). For example, our studies identified NET-T258/S259 motif as a site involved in the regulation of NE transport by neurokinin 1 receptor (NK1R) and AMPH (5, 9). In addition, we showed that T258/S259-dependent physical interaction between NK1R and NET promotes subcellular distribution of NET following NK1R activation by its ligand substance P (SP) (14).

It is well known that both NET and DAT are downregulated by AMPH as well as following NK1R activation (3–5, 15–19). We demonstrated that aprepitant, an NK1R antagonist used in the treatment of nausea and vomiting associated with chemotherapy or surgery attenuates AMPH-induced reinforcing behaviors (20). Our earlier studies showing the requirement of NET-T258/S259 motif in NK1R and AMPH dependent NET downregulation (5, 9), and our recent study which demonstrated that intra-accumbal infusion of TAT-peptide containing the NET-T258/S259 motif attenuates AMPH-induced hyperactivity and conditioned place preference (CPP) (13) indicate the significance of NK1R modulated NET function in AMPH-induced behaviors. However, since both NET and DAT are downregulated by NK1R activation as well as AMPH and both transporters share homologous sequence surrounding T258/S259 motif, it is not clear whether AMPH-induced behaviors arise in part from NK1R regulation of NET or DAT or both. To address this, the effect of NK1R antagonism on AMPH-induced reinforcing behaviors was examined using the NET-KO and DAT-KO mice along with AMPH-induced downregulation of NET and DAT function and surface expression using ventral striatum of wild-type mice to identify the transporter specificity.

Materials and Methods

Animals

Wild-type, NET-knockout (NET-KO) and DAT-knockout (NET-KO) male mice (C57BL/6J background) of 8–9 weeks age and weighing around 25 g were used for the experiments. A total of 12 wild-type, 13 NET-KO and 7 DAT-KO mice were used for conditioning with AMPH (2 mg/kg i.p.) and for measuring CPP and locomotor activity on post-conditioning test day. In addition, a total of 16 wild-type mice were used for uptake and biotinylation (4 each for vehicle/saline, vehicle/AMPH, aprepitant/saline and aprepitant/AMPH). Mice were housed in groups of 4–5 in polypropylene cages with corn-cob bedding and had free access to food (Harlan Teklad) and tap water and maintained on a 12 h light/12 h dark cycle at an ambient temperature of 22°C and 42% humidity. Procedures involving the animal experiments were conducted according to National Institutes of Health guide for the Care and Use of Laboratory animals. Virginia Commonwealth University Institutional Animal Care and Use Committee has approved the protocols of this study.

Materials

D-Amphetamine hemisulfate (AMPH) was purchased from Sigma-Aldrich (St. Louis, MO). Aprepitant, (5-[[(2R,3S)-2-[(1R)-1-[3,5-bis(trifluoromethyl)phenyl]ethoxy]-3-(4-fluorophenyl)-4-morpholinyl]methyl]- 1,2-dihydro-3H-1,2,4-triazol-3-one) was from Merck & Co., (Kenilworth, NJ). All other chemicals used in this study were obtained from Sigma Chemical Co (St. Louis, MO) or Fisher Scientific (Pittsburgh, PA) unless otherwise indicated.

Drug administrations

Amphetamine was dissolved in injectable grade isotonic saline (0.9% NaCl) and injections of saline or AMPH (2 mg/kg, i.p.) were given in a volume of 10 μl/g body weight. Aprepitant was first dissolved in 100% dimethyl sulfoxide (DMSO) and then diluted with saline before injections so the final DMSO concentration is at 0.002%. The vehicle contained 0.002% DMSO. Vehicle or aprepitant (5 mg/kg, i.p.) was administered in a volume of 10 μl/g body weight. The doses of AMPH and aprepitant were selected as per previous publications (13, 20, 21).

Measurement of Behaviors

Schematic below shows our behavioral paradigm of simultaneous measurement of CPP and locomotor activity. Details of CPP and locomotor activity measurements are described in the schematic (Fig. 1).

Fig. 1. Schematic showing the drug administration and behavioral testing protocol:

Fig. 1.

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Following a period of acclimation with experimenter (2–3 days handling), mice were pretested for chamber preference on day 1 and then conditioned with saline or AMPH (2 mg/kg, i.p.) for 3 days (day 2–4) as described in the methods section. Mice were then tested for preference for drug-paired chamber on day 5 following vehicle injection in the morning and aprepitant (5 mg/kg, i.p.) injection in the afternoon. Locomotor activity of the mice was recorded simultaneously during postconditioning test.

(a). Conditioned place preference (CPP)

Mouse CPP paradigm was followed as described in our previous studies (12, 20) using biased protocol. In brief, mice were placed in enriched environment and handled for three days prior to initiation of CPP testing. The CPP apparatus (Med-Associates, St. Albans, VT, ENV3013) consisted of white and black chambers (20 × 20 × 20 cm each), which differed in floor texture (white mesh and black rod: Med-Associates, ENV-3013WM and ENV-3013BR) to help the mice further differentiate between the two environments. Place conditioning chambers were separated by a smaller intermediate grey compartment with a smooth PVC floor and partitions that allowed access to the black and white chambers. On day 1, following 5 min acclimation time, mice were introduced into the chamber and their baseline preference for each chamber was recorded for 15 minutes. After testing for initial chamber preference on day 1 (preconditioning), mice were conditioned with 2.0 mg/kg AMPH. For conditioning, mice were given i.p. saline in the chamber where they showed higher initial preference and AMPH in the chamber where they showed higher initial preference on days 2, 3 and 4 (saline in the AM and AMPH in the PM once per day). After the conditioning period, CPP test (post-conditioning test) was conducted on day 5 by testing the mice for chamber preference by allowing the animal to freely explore both chambers. Postconditioning test was carried out in the AM following an injection of vehicle (saline containing 0.002% DMSO) given 15 min prior to CPP testing and also in the PM following an injection of aprepitant given 15 min prior to CPP testing. Preference scores that were measured in seconds represent the time the mice spent in the drug paired side during post conditioning test minus the time the mice spent in the drug paired side during preconditioning test (baseline preference).

(b). Locomotor activity

The locomotor activity of the mice was recorded as movement counts during CPP testing on postconditioning test day (day 5) following vehicle injection in the AM as well as following aprepitant injection in the PM as described by us previously (20). The movements of the mice were tracked using 16 evenly spaced infra-red (I/R) sources and sensors juxtaposed around the periphery of the four sides of the chamber.

Measurement of NET and DAT function and surface expression

Wild-type mice were subjected to conditioning with saline or AMPH as described above and in the schematic (Fig. 1). One half of saline or AMPH conditioned mice received vehicle (vehicle group) and the other half received aprepitant (aprepitant group) prior to post-conditioning test. Immediately following the post-conditioning test, animals were rapidly decapitated and brains were collected. Ventral striatum (VST) was dissected and synaptosomes were prepared as described previously (22). The synaptosomes were used immediately for measuring AMPH-mediated changes in NET and DAT activities and surface expression levels.

(a). DA uptake

NET and DAT -mediated DA uptake was measured as described previously (23). Briefly, synaptosomes (20 μg) were incubated in a total volume of 0.3 ml of Krebs-Ringer-HEPES (KRH) buffer (120 mM NaCl, 4.7 mM KCl, 2.2 mM CaCl2 10 mM HEPES, 1.2 mM MgSO4, 1.2 mM KH2PO4, 5 mM Tris, 10 mM D- glucose, ph 7.4) containing 0.1 Mm ascorbic acid, and 0.1 Mm pargyline at 37°C. Uptake was initiated by the addition of 40 nM [3H]DA and terminated after 5 min incubation at 37°C with the addition of 3 ml ice-cold PBS followed by rapid filtration over 0.3% polyethylenimine coated GF-B filters. Filters were washed rapidly with 5 ml cold PBS and radioactivity bound to filter was counted by liquid scintillation counter. Nonspecific uptake, defined as the uptake in the presence of 100 μM cocaine, was subtracted from total accumulation of [3H]DA to yield total specific DA uptake. The specific DAT blocker GBR 12909 (50 nM) and the norepinephrine transporter (NET) blocker nisoxetine (50 nM) were used to isolate NET and DAT -mediated [3H]DA uptake, respectively, from total specific [3H]DA uptake. [3H]DA uptake in the presence of GBR 12909 was subtracted from total specific DA uptake to define NET activity and [3H]DA uptake in the presence of nisoxetine was subtracted from total specific DA uptake to define DAT activity. All uptake assays were performed in triplicates and expressed as mean values of specific uptake ± S.D.

(b). Surface biotinylation

Synaptosomes (300 μg) were subjected to surface biotinylation and isolation of avidin-bound and -unbound fractions as described by us (1, 24, 25) Aliquots from total extracts (50 μl) and entire eluted fractions were separated by SDS-PAGE (10%), transferred to membrane, and probed with mouse NET antibody or DAT antibody. Blots were stripped and reprobed with anti-calnexin antibody. NET or DAT proteins were visualized using ECL or ECL Plus reagent followed by exposure to Hyperfilm ECL. Multiple exposures of immunoblots were taken to ensure that the band development on the film was within the linear range. Band densities were quantified by scanning and analyzed using NIH ImageJ (version 1.48j) software. Anti-calnexin antibody was used to validate the surface biotinylation of plasma membrane proteins. NET or DAT band densities from total were normalized using total levels of respective calnexin and biotinylated (representing the surface pool) fractions were normalized using respective total levels of NET or DAT.

Statistical analysis

Prism software (GraphPad, San Diego, CA) was used for statistical analyses of the data and values are expressed as mean ± S.D. Two-way ANOVA with Tukey’s multiple comparisons test was used for examining the effect of conditioning drug (AMPH) as well as the effect of aprepitant administration on AMPH-induced CPP. Similar method was used in the analysis of locomotor activity, NET or DAT mediated DA uptake and NET or DAT surface expression levels. A value of p ≤ 0.05 was considered statistically significant.

Results

Aprepitant attenuates AMPH-induced CPP expression in the wild-type and DAT-KO mice but not in the NET-KO mice.

Conditioning with 2 mg/kg AMPH for 3 days induced the expression of CPP in wild-type, NET-KO and DAT-KO mice. The mean preference scores (2 mg/kg AMPH-induced CPP) were 160.77 (vehicle) and 54.77 (aprepitant) for WT; 183.73 (vehicle) and 176.01 (aprepitant) for NET-KO; 153.22 (vehicle) and 30.61 (aprepitant) for DAT-KO. In wild-type and DAT-KO mice, AMPH induced CPP was significantly lower following aprepitant administration compared to vehicle administration (p < 0.0001) (Fig. 2). However, in NET-KO mice, AMPH-induced CPP remained unchanged following aprepitant administration compared to vehicle administration (p = 0.872) (Fig. 2). Two-way ANOVA showed significant genotype effect (F2, 58 = 5.632; p = 0.0058) and treatment effect (F1, 58 = 14.82; p = 0.0003). There was no significant drug-drug interaction between AMPH conditioning and aprepitant treatment (F2, 58 = 2.25; p = 0.1143).

Fig. 2. Effect of aprepitant on AMPH-induced CPP in the WT, NET-KO and DAT-KO mice:

Fig. 2.

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CPP scores recorded during postconditioning testing, given as means ± SD, show significant genotype effect in mice (wild-type (n12), NET-KO (n13) and DAT-KO (n6)) conditioned with AMPH 2 mg/kg (# p < 0.05). Treatment with aprepitant (5 mg/kg) significantly reduced AMPH CPP in WT and DAT-KO (* p < 0.05), but not in NET-KO mice.

Aprepitant does not affect the locomotor activity of wild-type, DAT-KO or NET-KO mice following AMPH conditioning.

The locomotor activity measured during post-conditioning test (in the absence of AMPH) showed that DAT-KO mice exhibit higher locomotor activity of compared to wild-type or NET-KO mice. Although there was significant difference between the genotypes (F2, 58 = 20.18; p < 0.0001), the locomotor activity of wild-type, NET-KO or DAT-KO did not change significantly following aprepitant administration (F1, 58 = 1.433; p = 0.2362) (Fig. 3). There was no significant drug-drug interaction between AMPH conditioning and aprepitant treatment (F2, 58 = 0.4173; p = 0.6608) (Fig. 3).

Fig. 3. Effect of aprepitant on locomotor activity of the WT, NET-KO and DAT-KO mice following AMPH conditioning:

Fig. 3.

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Movement counts as an index of locomotor activity recorded simultaneously during postconditioning testing, given as means ± SD, show significant genotype effect in mice (wild-type (n12), NET-KO (n13) and DAT-KO (n6)) conditioned with AMPH (2 mg/kg) (# p < 0.05). Treatment with aprepitant (5 mg/kg) did not affect locomotor activity measured on the day of postconditioning testing following 3 day AMPH conditioning.

Aprepitant attenuates AMPH-induced NET down-regulation but not DAT down-regulation in the VST of wild-type mice.

In vehicle treated group, AMPH conditioned animals showed significantly reduced DAT-specific DA uptake as well as NET-specific DA uptake compared to saline conditioned animals (Fig. 4) (DAT: F1,38 = 111.6; p < 0.0001) (NET: F1,36 = 45.58; p < 0.0001). In aprepitant treated group, while DAT-specific DA uptake was still significantly reduced in AMPH conditioned animals compared to saline conditioned animals (Fig. 4A) (F1,38 = 6.12; p = 0.0179), there was no significant difference in NET-specific DA uptake between saline and AMPH conditioned animals (Fig. 4B) (F1,36 = 3.55; p = 0.0677) (Fig 4). While there was no significant drug-drug (AMPH-aprepitant) interaction in DAT-specific DA uptake (F1,38 = 0.116; p = 0.7357), there was significant drug-drug interaction in the analysis of NET-specific DA uptake (F1,36 = 8.87; p = 0.0052).

Fig. 4. Effect of aprepitant on NET- and DAT- mediated DA uptake in WT mice (n=4/group run in triplicates) following AMPH conditioning:

Fig. 4.

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DAT-mediated DA uptake data (A) given as % vehicle show significant effect of AMPH (2 mg/kg) in both vehicle and aprepitant treated animals (****p < 0.0001). Tukey’s multiple comparisons test showed significant differences between vehicle/saline and vehicle/AMPH, between vehicle/saline and aprepitant/AMPH, between vehicle/AMPH and aprepitant/saline, and between aprepitant/saline and aprepitant/AMPH (****p < 0.0001). NET-mediated DA uptake data (B) given as % vehicle show significant effect of AMPH (2 mg/kg) only in vehicle treated animals (****p < 0.0001). Tukey’s multiple comparisons test showed significant differences between vehicle/saline and vehicle/AMPH (****p < 0.0001), between vehicle/saline and aprepitant/AMPH (**p = 0.008), between vehicle/AMPH and aprepitant/saline (****p < 0.0001), and between vehicle/AMPH and aprepitant/AMPH (*p = 0.012).

Similar to transport functional regulation, in vehicle treated group, AMPH conditioning compared to saline conditioning resulted in significantly reduced surface expression of DAT (F1,12 = 38.52; p < 0.0001) and NET (F1,12 = 6.079; p = 0.0279) (Figs. 5A,B&C). Quantified band densities expressed as % vehicle showed that in aprepitant treated group while DAT surface expression was still significantly reduced in AMPH conditioned animals compared to saline conditioned animals (Fig. 5D) (F1,12 = 4.815; p = 0.0486), there was no significant difference in NET surface expression between saline and AMPH conditioned animals (F1,12 = 3.837; p = 0.0738) (Fig 5G). While there was no significant drug-drug (AMPH-aprepitant) interaction with respect to DAT surface expression (F1,12 = 1.522; p = 0.2409), there was significant drug-drug interaction with respect to NET surface expression (F1,36 = 8.87; p = 0.0052). Total DAT and NET protein expression (Fig. 5B) and quantified band densities expressed as % vehicle (Figs. 5E&H) showed no significant differences among all groups and no significant drug-drug interaction. Reflecting the changes in surface band densities, there were concomitant increases in intracellular DAT and NET following AMPH (compared to saline) in vehicle treated animals (Fig. 5C) (DAT: F1,12 = 110.4, p < 0.0001) (NET: F1,12 = 13.7, p = 0.0003). Quantified intracellular band densities expressed as % vehicle (Figs. 5F&I) showed that while the increase in intracellular DAT following AMPH was intact in aprepitant treated animals (F1,12 = 6.595, p = 0.0246), the increase in intracellular NET was abolished in aprepitant treated animals (F1,12 = 4.05, p = 0.067).

Fig. 5. Effect of aprepitant on NET and DAT surface expression in WT mice (n=4)following AMPH conditioning:

Fig. 5.

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Representative immunoblots (A, B, C) show total, biotinylated and non-biotinylated DAT (~68 kDa) and NET (~65 kDa). The intracellular calnexin bands (~90 kDa) were shown under respective blots. Quantified DAT and NET band intensities are as shown. Total DAT or NET normalized to total calnexin (D&G), biotinylated surface DAT or NET normalized to total DAT or NET (E&H), and non-biotinylated intracellular DAT or NET normalized to non-biotinylated calnexin (F&I). Quantified surface DAT band density given as % vehicle is shown in (D) show significant effect of AMPH (2 mg/kg) in vehicle and aprepitant treated animals. Tukey’s multiple comparisons test showed significant differences between vehicle/saline and vehicle/AMPH (**p = 0.0027), between vehicle/AMPH and aprepitant/saline (*p = 0.0123), vehicle/saline and aprepitant/AMPH (****p < 0.0001), and between aprepitant/saline and aprepitant/AMPH (**p = 0.0003). Quantified surface NET band density (G) given as % vehicle show significant effect of AMPH (2 mg/kg) only in vehicle treated animals. Tukey’s multiple comparisons test showed significant differences between vehicle/saline and vehicle/AMPH (*p = 0.018), between vehicle/AMPH and aprepitant/saline (*p = 0.038), and between vehicle/AMPH and aprepitant/AMPH (*p = 0.034). Quantified total DAT or NET normalized to total calnexin (E&H) showed no significant differences among all groups. Quantified intracellular DAT normalized to intracellular calnexin (F) given as % vehicle show significant effect of AMPH (2 mg/kg) in vehicle and aprepitant treated animals. Tukey’s multiple comparisons test showed significant differences between vehicle/saline and vehicle/AMPH (****p < 0.0001), between vehicle/AMPH and aprepitant/saline (****p < 0.0001), and between aprepitant/saline and aprepitant/AMPH (***p = 0.0002), Quantified intracellular NET normalized to intracellular calnexin (I) given as % vehicle show significant effect of AMPH (2 mg/kg) only in vehicle treated animals (**p = 0.003). Tukey’s multiple comparisons test showed significant differences between vehicle/saline and vehicle/AMPH (**p = 0.003), between vehicle/AMPH and aprepitant/saline (**p = 0.008), and between vehicle/AMPH and aprepitant/AMPH (*p = 0.023).

Discussion/Conclusion

Previously, we demonstrated that a single injection of aprepitant prior to post conditioning test attenuates AMPH-induced CPP in C57BL/6J wild-type mice and conditioning with aprepitant alone does not induce CPP (20). Both NET and DAT are regulated by AMPH treatment (8, 9, 24) and also following NK1R activation by SP (5, 25). Therefore, it is possible that NK1R mediated regulation of NET and DAT may play a role in AMPH-induced behaviors. NET-KO mice exhibit higher cocaine-induced CPP and super-sensitivity to psychostimulants (26). DAT-KO mice, similar to wild-type, exhibit intact AMPH-induced CPP at 2.5 mg/kg (27). We found that AMPH-induced CPP expression was similar in all three genotypes following vehicle administration and the preference scores observed in this study are similar to those observed in previously published studies by us and others (20, 27). However, this AMPH-induced CPP expression in the wild-type and DAT-KO was significantly attenuated to similar extent following aprepitant administration. The AMPH-induced CPP expression in the NET-KO was intact following aprepitant administration. It is possible that DAT (for example in NET-KO mice where NET is completely absent) might be solely responsible for AMPH-induced CPP and hence NET-KO mice show intact AMPH-induced CPP similar to wild-type. While DAT-KO mice lack functional DAT, they retain functional NET. On the other hand, NET-KO mice lack functional NET, but retain functional DAT. Therefore, the fact that aprepitant failed to block AMPH-induced CPP in NET-KO suggests that NK1R-mediated NET regulation plays a role in AMPH-induced behaviors.

It is known that DAT-KO mice are hyperactive even in the absence of psychostimulants (28). We found that DAT-KO mice exhibit higher locomotor activity on post-conditioning test day (in the absence of AMPH) compared to wild-type or NET-KO mice. Nonetheless, the locomotor activity of wild-type, NET-KO or DAT-KO remained unaffected following aprepitant administration. Thus, NK1 antagonism specifically attenuates AMPH-induced CPP (Fig. 2) and is not due to any non-specific effects on animal movement (Fig. 3).

In conclusion, current study demonstrates that aprepitant attenuates AMPH-induced CPP expression by the wild-type and DAT-KO mice, but not by the NET-KO mice. Additionally, aprepitant was able to attenuate AMPH-mediated NET downregulation (both function and surface expression) but not the DAT downregulation in the VST (Figs. 4 & 5) and also in the prefrontal cortex (data not shown) providing compelling evidence that NK1R-mediated NET downregulation and not DAT downregulation contributes to AMPH-evoked CPP modulation. It is known that DAT mediates DA uptake and AMPH facilitates DA efflux from DA neurons contributing to its behavioral effects of AMPH (29, 30). It is also known that NET plays a key role in regulating DA homeostasis in important brain areas where DAT is absent or deficient (31, 32). In DAT-KO mice, basal DA levels are high, and it is possible that AMPH enhances extraneuronal DA by interfering with NET-mediated DA uptake. It is also possible that AMPH-mediated NE release from NE terminals could modulate DA signaling contributing to AMPH-mediated behaviors because studies have shown that NE system contributes to mesolimbic DA signaling (33–36). Given that NET can clear DA from extraneuronal space, NK1R mediated NET regulation could modulate NET-mediated DA clearance and consequently alter AMPH triggered extraneuronal DA, DA neurotransmission, and CPP behavior. Collectively, the findings presented here along with our previous studies (20) unequivocally demonstrate a major role for NET in the NK1 receptor regulation of amphetamine actions.

NE and NET mediated behaviors are implicated in addiction and NE directed therapeutics are well-recognized in the therapy for addiction (37–39). Substance P is expressed in brainstem monoaminergic nuclei, and SP-NK1R system mediates behaviors linked to stress and addiction (40). Both NK1 and NE anatomically and physiologically exist closely in the brain (41, 42) and NK1 directed therapeutics are also of clinical interest (43, 44). Thus, the findings from current study not only underscore the importance of NK1R-mediated NET regulation in psychostimulant-mediated behaviors, but also highlight the significance of NE/NK1 directed therapies in addiction treatment strategies.

Acknowledgements

The authors thank Dr. Marc G Caron, Departments of Cell Biology, Neurobiology, and Medicine, Duke University Medical Center, Durham, NC for kindly providing the DAT-KO and NET-KO mice.

Funding Sources

This work was supported by the National Institutes of Health grants DA039451 and DA045888 (LDJ).

Footnotes

Statement of Ethics

All procedures involving the animal experiments were conducted according to National Institutes of Health guide for the Care and Use of Laboratory animals. Virginia Commonwealth University Institutional Animal Care and Use Committee has approved the protocols (AD10000476) of this study.

Conflicts of Interest

The authors have no conflicts of interest to declare.

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