Effect of systemically administered oxytocin on dose response for methylphenidate self-administration and mesolimbic dopamine levels

Abstract

The neuropeptide oxytocin (OT) alters behaviors related to the administration of drugs of abuse, including stimulants. OT also plays a key role in social bonding, which involves an interaction between OT and dopamine (DA) in the nucleus accumbens (NAc). The nature of the interaction between OT and DA in the striatum in the context of psychostimulants is unclear. We investigated the effect of OT, delivered intraperitoneally, on the methylphenidate (MP) dose–response function in rats. Food was used as a control condition. In a microdialysis study, we measured the effect of intraperitoneal OT on MP-stimulated striatal DA levels. Systemic OT pretreatment caused a downward shift in the MP dose–response function while having no effect on motor activity. OT also caused a reduction in food self-administration, although a significantly higher dose of OT was required for this effect compared to that required for a reduction of MP self-administration. Systemic OT pretreatment caused a potentiation of MP-stimulated DA levels in the NAc shell but not in the core. The significance of these findings is discussed, including the potential of OT as a therapeutic agent for addictive disorders.

Introduction

Oxytocin (OT) is a nine amino acid peptide, synthesized in the magnocellular neurons of the paraventricular (PVN), supraoptic (SON), and accessory magnocellular (AN) nuclei of the hypothalamus and released into the bloodstream from axon terminals of these neurons that are in the posterior pituitary. In this way OT acts as a hormone on peripheral targets to promote uterine contraction and lactation. OT also acts centrally via dendritic release from the PVN and SON ¹ and via fibers from the SON and PVN that project to mesocorticolimbic regions ^{2 3, 4}. Through these central pathways, OT promotes initiation of maternal behavior in virgin female rats ^{5, 6}, penile erection, ⁷ and grooming ⁸. Affiliative mammalian species such as the prairie vole (in contrast to non-affiliative species, e.g., the montane vole) were found to have OT receptors in brain areas responsible for reward processing, such as the nucleus accumbens (NAc), ⁹ and partner preference formation in this species was dependent on the interaction between OT and dopamine (DA) in the NAc shell ¹⁰. Moreover, in humans, exogenous administration of OT promotes various social approach, bonding, maternal, and stress-reducing behaviors [reviewed in Ref. 11].

The neurocircuitry underlying response to these natural rewards is thought to overlap with brain pathways that mediate the effects of drugs of abuse, including psychostimulants^{12, 13}, with response to both natural rewards and drugs of abuse involving mesocorticolimbic dopaminergic (DAergic) signaling^{14, 15}. In rodent studies, OT inhibited locomotor activity, self-administration, tolerance formation, conditioned place preference (CPP) acquisition, extinction, and reinstatement for several drugs of abuse, including psychostimulants, alcohol, and opiates [for reviews see Refs. 16 and 17]. In recent studies investigating the effect of drugs of abuse in animal models of social bonding such as the prairie vole, social bonding rendered amphetamine less rewarding¹⁸, while exposure to amphetamine decreased the capacity to form social bonds¹⁹ and exogenous OT reversed the amphetamine-induced deficits in social bonding²⁰. The mechanism for this effect involved increased DA concentration in the NAc shell²⁰.

The effect of OT on DA signaling in response to drugs of abuse in regions where OT and DA interact to promote motivated behaviors for natural reward is still unclear. OT reduced striatal DAergic signaling in response to cocaine; however, in that study ²¹, OT was administered centrally (i.c.v.) and only indirect measures of changes in extracellular DA concentrations were reported (dihydroxyphenylacetic and homovanillic acid to DA ratios). Intraperitoneal (i.p.) administration of OT in rodents resulted in reduced self-administration of methamphetamine ²² or cocaine ²³. However, only one psychostimulant dose was used in these studies.

Based on the typical inverted U-shaped dose–effect function of psychostimulant self-administration behavior, a reduction of self-administration behavior might result from both antagonism and potentiation of the psychostimulant reinforcing effects. Therefore, the effect of OT on the reinforcing efficacy of psychostimulants expressed by a dose–response curve is unknown. Thus, in order to understand the mechanisms of OT as a potential therapy for addictions, we investigated the effect of systemic OT (delivered i.p.) on rats trained to self-administer methylphenidate (MP) intravenously during single daily sessions. We chose MP as it is functionally similar to cocaine with respect to its effect on DAergic binding in the striatum ²⁴. We also examined, by brain microdialysis, the effect of OT (both systemically delivered and delivered to the NAc shell) on MP-stimulated extracellular levels of DA in the NAc, a brain area related to reward and reinforced behavior.

Materials and methods

Methylphenidate and food self-administration

Subjects.

Ten Sprague-Dawley male rats (weighing approximately 250 g upon arrival) were obtained from Envigo Inc. (Indianapolis, IN) and served as subjects after acclimation to the laboratory for at least one week. Animals were implanted with femoral vein catheters by Envigo before delivery and subjects were used as the patency allowed. Catheters were infused daily with 0.2 mL sterile saline solution containing heparin (30.0 IU/mL) and gentamicin (0.4 mg/mL) to minimize the likelihood of infection and the formation of clots and fibroids. A separate cohort of six male Sprague-Dawley rats without catheterization was also used. Food (Scored Bacon Lover Treats; Bio-Serv, Frenchtown, NJ) and tap water were available in their home cages. After acclimation, weights of rats were maintained at approximately 300 g by adjusting their daily food ration. The animal housing room was temperature and humidity controlled and maintained on a 12 h:12 h light/dark cycle, with lights on at 7:00 am. Care of the subjects was in accordance with the guidelines of the National Institutes of Health and the experiments were approved by the National Institute on Drug Abuse Intramural Research Program Animal Care and Use Program, which is fully accredited by AAALAC International.

Apparatus.

Experimental sessions were conducted with animals placed in operant-conditioning chambers (modified ENV-203; MED Associates, St. Albans, VT) that measured 25.5 × 32.05 × 25.5 cm and were enclosed within sound-attenuating cubicles equipped with a fan for ventilation and white noise to mask extraneous sounds. On the front wall of each chamber were two response levers, 5.0 cm from the midline and 4.0 cm above the floor. A downward displacement of the lever with a force equivalent to approximately 20 g defined a response, which always activated a relay mounted behind the front wall of the chamber, producing an audible feedback click. Three light-emitting diodes (LEDs) were located above each lever. A syringe infusion pump (model 22; Harvard Apparatus, Holliston, MA) placed above each chamber delivered injections of specified volumes and durations from a 10 mL syringe. The syringe was connected by Tygon tubing to a single-channel fluid swivel (375 Series Single Channel Swivels; Instech Laboratories, Inc. Plymouth Meeting, PA) that was mounted on a balance arm above the chamber. Tubing from the swivel was connected to the subject’s catheter using a tether provided by Envigo, equipped with a protective metal spring around the tubing.

Procedures.

Subjects were placed in chambers daily for experimental sessions, after which they were returned to their cages in the animal-housing room. The catheterized subjects were initially trained during 1-h sessions with a fixed-ratio (FR) 5-response schedule and a drug reinforcement of 1 mg/kg/infusion MP (each fifth response produced a drug infusion). Although subjects were provided with two levers, only the “active” lever (left lever for half of the subjects, right lever for the other half) produced reinforcements. During the sessions, the house light at the top of the chamber was illuminated to indicate that drug was available. Completion of five responses turned off the house light, illuminating the LEDs above the active lever and activated the infusion pump. Drug infusions were followed by a 20-s timeout period during which all lights were off and responses had no scheduled consequences. Following the timeout, the house light turned back on and responses again had scheduled consequences. Responses on both right and left levers were recorded throughout the entire session. After subjects responded at a sufficiently high rate with integrated responses (as determined by the research team) that was consistent across sessions, the dose of MP was progressively reduced by adjusting infusion volumes and duration to determine responses across a range of doses. The choice of doses was based on a log scale, and was sequentially reduced until a clear dose–effect curve was established. The selected MP doses for the curve were 0.01, 0.03, and 0.1 mg/kg/infusion delivered by infused volumes of 1.69, 5.06, and 16.9 μL and 0.0878, 0.263, and 0.878 s, respectively, based on a body weight of 0.3 kg. An infusion of 70 μL was given at the start of each session to fill the dead volume of the catheters, regardless of responses.

A separate cohort of male rats without catheters (n = 6) was trained in a parallel manner with food reinforcement (20-mg food pellets; Bio-Serv) instead of MP. During daily 1-h sessions, subjects were trained under a FR 5-response schedule under which every fifth response produced a food pellet. Light and timeout conditions were the same as those in the MP self-administration (SA) sessions. As responses for food under these conditions were significantly higher than those for MP, subjects were given unrestricted access to chocolate-flavored pellets (1 g, supreme mini-treats, Bio-Serv) for 24 h prior to sessions in order to produce responses similar to those in the MP SA sessions.

Once response rates were stable across successive sessions, the effects of OT were tested. Drug doses and timing were based on previous experiments^{22, 25}. All OT (oxytocin acetate salt hydrate, Sigma-Aldrich, St. Louis, MO) solutions were prepared fresh in 0.9% NaCl and administered via i.p. injection 20 min before the start of SA sessions at doses of 0.1, 0.25, 0.5, 1, and 2 mg/kg OT. OT testing sessions were conducted at least 48 h apart and responses were monitored in the following days after treatment to ensure an approximate recovery of baseline levels before testing again. The effect of these doses of OT was tested for both food SA and the doses of MP that comprised the dose–effect curve (0.01–0.1 mg/kg/infusion MP).

Statistical analysis.

Response rates were analyzed in a two-way ANOVA with MP (0.01, 0.03, and 0.1 mg/kg/infusion)/food and OT (0, 0.1, 0.25, 0.5, 1, and 2 mg/kg) as within-subject factors. Where there were significant MP/food × OT interactions, Dunnett’s multiple comparisons test was used for post hoc pairwise comparisons for each MP/food condition comparing baseline versus OT condition.

In vivo brain microdialysis

Animals.

Male Sprague Dawley rats (Charles River, Wilmington, MA), experimentally naive at the start of the study and weighing 280 to 325 g, were doubly housed and had free access to food and water, except during testing sessions. Rats were housed in a temperature- and humidity-controlled room and were maintained on a 12 h:12 h light/dark cycle, with lights on from 7:00 am. All experiments were conducted during the light phase.

Probe preparation and surgery.

As extensively described previously ^26–31, concentric dialysis probes with an active dialysing surface of 1.8–2.0 mm were implanted under anesthesia with a mixture of ketamine and xylazine (60.0 and 12.0 mg/kg i.p., respectively) into the NAc shell or core (uncorrected coordinates, from the rat brain atlas ³²: shell, A = +2.0, L = ±1.0, V = 7.9; core, A = +1.6, L = ±1.9, V = 7.7; Anterior, A, and Lateral, L, mm from bregma; Vertical (V) mm from dura; see Fig. S1, online only). To allow for intravenous (i.v.) administration of drugs, a femoral vein catheter was implanted during the same microdialysis surgery session^{28, 31, 33, 34}. After the surgery, rats were placed in hemispherical CMA-120 cages (CMA/Microdialysis AB, Solna, Sweden) where they recovered overnight and then were tested during microdialysis sessions.

Sample collection and analytical procedure.

As detailed in several previous publications^{26–31, 35}, experiments on freely-moving rats started about 22 h after probe implantation. Ringer’s solution (147.0 mM NaCl, 2.2 mM CaCl₂, and 4.0 mM KCl) at a flow rate of 1 μL/min delivered by a BAS Bee Syringe Pump Controller (BAS West Lafayette, IN, USA) was infused through the microdialysis probes connected to fluid swivels (375/D/22QM, Instech, Plymouth Meeting, PA, USA). Dialysate samples of 10 μL were collected every 10 min and immediately analyzed in a high-performance liquid chromatography system equipped with a chromatographic column (MD-150X3.2, ESA, Chelmsford, MA) and a coulometric detector (Coulochem II, or Coulochem III, ESA, Chelmsford, MA) with analytical cell (5014B; ESA, Chelmsford, MA) electrodes set at +125 mV and −125 mV to quantify DA. The mobile phase^{28, 29, 31} (100 mM NaH₂PO₄, 0.1 mM Na₂EDTA, 0.5 mM n-octyl sulfate, 18% methanol; pH adjusted to 5.5 with Na₂HPO₄) was delivered at 0.50 mL/min by an isocratic pump (ESA 582, ESA, Chelmsford, MA).

Experimental procedures.

All drugs or their vehicles were tested after stable DA values (less than 15% variability) were obtained for 2–4 consecutive samples. Pretreatments were administered i.p. with either OT (OT acetate salt hydrate, Sigma-Aldrich, St. Louis, MO) (1 and 2 mg/kg) or saline 10 minutes before the start (time = 0 in the figures) of the first intravenous (i.v.) administration of MP (Mallinckrodt, Saint Louis, MO). MP was administered at the time points of 0 (0.1 mg/kg), 30 (0.32 mg/kg) and 60 minutes (1.0 mg/kg). The effect of OT alone (2 mg/kg) was measured in an OT-only condition, in which saline infusions were given instead of MP. Using the same experimental conditions and the same schedule of MP injections, the experiment above was repeated with OT pretreatments in rats with microdialysis probes implanted in the NAc core. In a different set of experiments, OT dissolved in the Ringer’s solution (0, 10, and 50 μM) was administered through the probe by reverse dialysis into the NAc shell 30 minutes before the start of the first MP infusion.

Histology.

As described previously^{27–29, 31, 33, 35}, to identify the location of the probes, brains were collected, left to fix in 4% formaldehyde in saline solution for at least 5 days, and then sliced (Vibratome 1000 Plus, The Vibratome Company, St. Louis, MO, USA) in serial coronal slices (orientation as per Paxinos and Watson ³²). Probe track locations were verified using atlas brain sections³² as templates. Only the data obtained in animals showing a correct probe track within the boundaries of the targeted brain areas were included in the statistical analysis (see Fig. S1).

Behavioral activity.

Motor activity was assessed with a TSE InfraMot system (TSE Systems, Homburg, Germany), which uses infrared sensors to register activity of the subject by sensing infrared radiation from its body and its spatial displacement over time. The sensor assembly was mounted on the top of the microdialysis cage. The collected data provides a relative measure of the duration and intensity of the activity. The scanning interval was set to 5 min. We analyzed the first 30 min of counts after each MP dose treatment, including experiments where saline was administered instead of MP.

Statistical Analysis.

In the microdialysis study, results are presented as group means (± SEM), normalized and expressed as a percentage of basal DA values, which were calculated as means of 2–4 consecutive samples immediately preceding the OT or saline injection. Statistical analysis (Statistica software, Stat Soft, Tulsa, OK) was carried out using one-, two-, or three-way ANOVA for repeated measures over time applied to the data obtained from serial assays of dialysate DA normalized as percentage of basal values of each group. Significant results were subjected to post hoc Tukey`s tests. Statistical analysis of differences in basal DA values (fmol/10 μL sample ± SEM) between different experimental groups and brain areas was carried out with one-way ANOVA. Changes were considered to be significant when P < 0.05.

Results

Methylphenidate and food SA

There was a main effect of OT to reduce response rates for MP/Food (F_6,126 = 20.8, P < 0.0001) (Fig. 1). There was a main effect of MP/Food on response rates (F_3,126 = 25.9, P < 0.0001). There was a significant OT × MP/Food interaction on response rates (F_18,126 = 2.9, P = 0.0002). The percentage of total variation accounted for by OT was 32.9%, by MP/Food was 20.5%, and by their interaction was 13.9%. After post hoc analysis, there was a significant reduction in response rate from baseline at 0.03 mg/kg/infusion MP with pretreatments of 0.25, 0.5, 1, and 2 mg/kg OT (P = 0.0001), and a significant reduction from baseline was also observed at 0.1 mg/kg/infusion MP at 1 mg/kg OT (P = 0.043); however, there were no significant reduction after post hoc analyses for the effect of OT on SA of the 0.01mg/kg/infusion dose. Pretreatment with OT also resulted in a reduction in food SA (Fig. 2), and after post-hoc analysis there was a significant reduction only at the 2 mg/kg dose of OT (P = 0.033). Nonlinear regression analyses revealed that a significantly higher dose of OT was required for a 50% reduction in food SA (IC₅₀) as compared to MP (Fig. 3) under the same experimental conditions (P = 0.004).

Figure 1.

Downward shift of methylphenidate (MP) dose–response curve. Oxytocin (OT) doses of 0.25, 0.5, 1.0, and 2.0 mg/kg reduce response rate for MP compared to baseline. ****P < 0.0001; *P < 0.05

Figure 2.

(A–C) Effect of oxytocin (OT) (vehicle, 0.1, 0.25, 0.5, 1.0, and 2.0 mg/kg) on response rate for food self-administration compared with methylphenidate (MP) self-administration at MP doses of (A) 0.01 mg/kg/infusion, (B) 0.03 mg/kg/infusion, and (C) 0.1mg/kg/infusion. (D-F) Effect of oxytocin (OT) (vehicle, 0.1, 0.25, 0.5, 1.0, and 2.0 mg/kg) on percentage of baseline response rate for food self-administration compared with methylphenidate (MP) self-administration at MP doses of (D) 0.01 mg/kg/infusion, (E) 0.03 mg/kg/infusion, and (F) 0.1mg/kg/infusion. For food, there was a significant reduction from baseline only at the 2 mg/kg dose of OT (*P < 0.05 ); ****P < 0.0001: significant reduction from baseline for MP, see Figure 1. B = baseline.

Figure 3.

A significantly greater dose of oxytocin (OT) was required for a 50% reduction in response rate for food compared to methylphenidate (MP) (P = 0.004).

In vivo microdialysis

Systemic OT (2 mg/kg, i.p.) alone had no significant effects on extracellular levels of DA in the NAc shell (Fig. 4). As expected, a sequence of injections of increasing doses of MP (0.1, 0.32, and 1.0 mg/kg i.v. spaced 30 min apart) resulted in a dose-dependent increase in NAc shell DA levels compared to baseline, with a maximum increase of 346% of baseline 10 minutes after administration of the highest dose. A two-way ANOVA for repeated measures over time revealed significant main effects of MP DOSE (F_3,20 = 15.34, P < 0.001), and TIME (F_2,40 = 40.21, P < 0.001) and a significant DOSE × TIME interaction (F_6,40 = 10.24, P < 0.001). When the injections of MP were preceded by pretreatment with OT (1 or 2 mg/kg i.p.) 10 minutes before the first MP injection, there was a significant dose-dependent enhancement of MP-stimulated DA levels in the NAc shell as compared to saline pretreatments (Fig. 4). Under these conditions, stimulation of DA levels reached a maxima of 583% and 758% after pretreatments with OT doses of 1.0 or 2.0 mg/kg, respectively. A two-way ANOVA for repeated measures over time showed a significant main effect of OT DOSE (F_2,14 = 6.32, P < 0.05) and TIME (F_12,168 = 72.55, P < 0.001), and a significant DOSE × TIME interaction (F_24,168 = 5.54, P < 0.01). When the same pretreatment/treatment was applied to animals with probes in the NAc core, there was no significant OT enhancement of the MP-induced stimulation of DA levels as compared with saline pretreatments (Fig. 5). Maximum MP-induced stimulation of NAc core DA levels obtained with saline or OT pretreatments were 339% and 381%, respectively, 10 minutes after injection of the 0.32 mg/kg dose of MP. A two-way ANOVA for repeated measures over time showed a main effect of TIME (F_12,108 = 31.17, P < 0.001) but no main effect of OT DOSE or TIME × OT DOSE interaction on MP-stimulated NAc core DA levels. To test for potential local effects of OT, the peptide was applied directly through the Ringer’s solution via reverse microdialysis to the NAc shell (Fig. 6). This local administration of OT followed by a sequence of i.v. saline injections (3 × 1ml/kg, spaced 30 min apart) did not significantly affect basal levels of NAc shell DA. A two-way ANOVA for repeated measures over time resulted in a nonsignificant main effect of TREATMENT (F_3,16 = 2.30, P > 0.05), and TIME (F_2,32 = 0.94, P > 0.05), and a nonsignificant TREATMENT × TIME interaction (F_6,32 = 1.40, P > 0.05). However, when saline injections were replaced with MP injections (0.1, 0.32, and 1.0 mg/kg i.v. every 30 min), there was an enhancement (564%) of MP-induced stimulation of DA levels in animals infused with the 50 μM concentration of OT. A two-way ANOVA for repeated measures over time showed a significant OT DOSE × TIME interaction such that OT affected the time course of MP-stimulated basal DA levels differently (F_24,144 = 1.62, P < 0.05). There was also a main effect of TIME (F_12,144 = 44.45, P < 0.001) and no main effect of OT DOSE (F_2,12 = 2.30, P > 0.05).

Figure 4.

Effects of oxytocin (OT) pretreatments (1.0 and 2.0 mg/kg i.p.) on methylphenidate (MP; 0.1, 0.3, and 1 mg/kg IV, spaced 30 min apart)-induced stimulation of DA levels from NAc shell dialysates in rats. Oxytocin dose-dependently significantly enhanced methylphenidate (MP)-stimulated DA levels in the NAc shell as compared with saline-pretreated rats. * = significant increase compared with baseline, that is, prior to OT or saline (SAL) injection. Post-hoc analysis: the 70 minute time point of the OT 2 mg/kg condition was significantly greater than that in the OT 1mg/kg and saline condition, which were equivalent. Results are means, with vertical bars representing SEM, of the amount of DA in 10-min dialysate samples, expressed as percentage of basal values.

Figure 5.

Oxytocin (OT) pretreatments had no significant effect on the time course of methylphenidate (MP)-stimulated basal DA levels in the NAc core, in contrast with its effects on the NAc shell (see Fig. 4) under the same experimental conditions.

Figure 6.

Local administration of oxytocin (OT; 10 and 50 μM in Ringer) delivered through reverse microdialysis into the NAc shell 30 min before the first injection of MP yielded, at the highest dose tested, a significant difference in the time course of methylphenidate (MP)-stimulated basal DA levels.

The sequence of injections of increasing doses of MP (0.1, 0.32, and 1.0 mg/kg i.v. every 30 min) during the microdialysis procedure that resulted in a dose-dependent increase in DA levels also significantly increased the behavioral activity counts in rats, about 24,500 in 30 min after the highest MP dose, as compared with saline injections, about 5,100 in 30 min (one-way ANOVA for repeated measures, F_3,27 = 31.09, P < 0.001) (Fig. 7). Behavioral activity counts obtained after MP or saline injections during the microdialysis procedures were unaffected by OT pretreatments, about 21,000 in 30 min for the highest dose of MP after OT (1.0 or 2.0 mg/kg), and about 5,000 for saline after OT (2.0 mg/kg) (Fig. 7). A two-way ANOVA for repeated measures showed a significant main effect of MP DOSE (F_2,34 = 47.81, P < 0.001), no significant effect of OT pretreatment (F_2,17 = 0.62, P > 0.05), and no significant MP DOSE × OT pretreatment interaction (F_4,34 = 0.66, P > 0.05).

Figure 7.

Activity counts of the rats during the microdialysis experiments. There was no significant effect of oxytocin (OT) on the time course of increasing behavioral activity over the course of three methylphenidate (MP; 0.1, 0.3, and 1 mg/kg i.v., spaced 30 min apart) injections.

Discussion

We report that OT, delivered systemically, resulted in a downward shift in the dose response of MP-maintained self-administration behavior in rats. OT also reduced the response for food. The OT dose required for the latter effect was significantly higher as compared with that required for a reduction in MP SA. This was confirmed by significant statistical differences in the shift in IC₅₀ for MP- versus food-maintained behavior. It is unlikely that the reduction in MP and food SA was due to nonspecific behavioral effects, such as reduction in mobility, as the dose-dependent increase in activity counts after MP injections in the microdialysis experiments was unaffected by OT administration. To our knowledge, this is the first study on the effect of OT on the dose–response function for a psychostimulant.

These results have clinical relevance with respect to OT as a putative therapeutic agent for stimulant use disorders. That is, the downward shift in the dose–response function in the self-administration study underscores the potential value of OT as a therapeutic agent for psychostimulant addiction. Vertical (upward) shifts in the dose–response curve predict a vulnerable phenotype for drug use disorder as these individuals would consume the highest quantity of drug and would be at risk to develop high levels of use when both low and high doses are available ³⁶. In addition, compulsive use, characterized by allostatic shifts in reward function, are accompanied similarly by an upward shift in the dose–response curve ³⁷. Therefore, this preclinical finding, if it translates to human behavior, indicates that OT may be especially effective both in those vulnerable to developing addiction and in those already exhibiting compulsive use.

In an additional experiment, the same dose of OT that reduced all doses of MP self-administration also potentiated MP-stimulated DA levels in the NAc shell, without affecting basal DA levels, when given in the absence of MP (Fig. 4). These effects were replicated when OT was delivered into the NAc shell by reverse microdialysis (Fig. 6). In contrast, systemic OT pretreatments did not potentiate MP stimulation of NAc core DA levels.

The results of the microdialysis study highlight the context-dependent effect of OT on DA signaling selectively in the NAc shell, a region that is involved in appetitive learning ³⁸. First, OT alone had no effect on DA levels in the NAc shell, which is consistent with preclinical studies indicating that OT in the doses used in this study are not reinforcing³⁹. However, in the context of reinforcing doses of MP, OT both potentiated MP-stimulated DA levels in the NAc shell and decreased MP SA. If these effects translate to humans, this suggests that in the context of stimuli that prime striatal DAergic signaling such as psychostimulant use or the exposure to drug-related cues ⁴⁰, OT may reduce psychostimulant use. In addition, to the extent that the effect of MP on attention deficit hyperactivity disorder symptoms is dependent on increasing dopamine levels in the NAc, the combination of MP and OT may have some therapeutic role, perhaps to augment the effect of MP, obviating the need for MP dose increases and attendant side effects.

The mechanism by which OT regulates motivated behaviors via DAergic signaling in the NAc is not well understood. Activation of OT receptors in the ventral tegmental area (VTA) by OT is required for social reward in hamsters ⁴¹. OT injected into the VTA causes penile erection and increases extracellular DA levels in the NAc and PVN in male rats ⁷. Recent tracing studies have reported that OT neurons in the PVN, but not in the SON, project to the VTA, where OT receptors are located on DAergic and GABAergic neurons ⁴. In the VTA, OT receptors predominate on DAergic neurons. Optogenetic stimulation of these OT fiber terminals increases DA neuron firing ⁴ in the VTA, where OT mediates social reward ⁴². In other regions, such as the substantia nigra, these projections modulate DAergic neurons via stimulation of GABAergic interneurons, thus reducing DAergic neuron firing. OT receptors are also present on neurons projecting to the NAc from diverse regions, such as anterior olfactory nucleus, paraventricular thalamus, basolateral and central amygdala, hippocampus, dorsal raphe, and VTA ³. Therefore, OT acts presynaptically in the NAc via its receptor modulating input from other brain regions. Lastly, OT receptor D2 receptor heterodimers facilitate OT-DA interactions in the ventral striatum that underlie pair bonding⁴³ and in the amygdala to mediate anxiolytic effects⁴⁴. These studies have begun to elucidate the mechanism underlying how hypothalamic OT regulates DA neurons to promote motivation for natural reward.

In contrast, previous preclinical studies reported that OT reduced motivation for drugs of abuse and alcohol (reviewed in Ref. 17). Systemically administered OT exerts its effects centrally to mediate behaviors relevant to addiction. OT administered i.p., in similar doses used in this study (1–2 mg/kg), reduced self-administration and reinstatement of methamphetamine-seeking behavior ^{22, 25}. These behavioral changes were accompanied by central changes: a reduction in methamphetamine-induced c-Fos expression in subthalamic nucleus and NAc core ²⁵. Several studies have reported that administration of an OT receptor antagonist into the NAc reverses the effect of OT on CPP for methamphetamine ^45–47. We conducted a clinical study administering a single intranasal dose of OT to individuals in inpatient treatment for cocaine use disorder and in long-term abstinence ⁴⁸. As is common with intranasal OT clinical studies, there was a context-dependent effect, where OT removed the significant positive correlation between state anger and cue-induced craving. In the absence of drug cues, OT increased patients’ desire to use. Since we studied long-term abstinent inpatients who were not on MP and had no opportunity to use, the effects of this single-dose challenge study may have been due to an anxiolytic effect of OT that has been well described ⁴⁹. There are no clinical trials to date with intranasal OT as a treatment for psychostimulant use disorder. This may be due to the poor central nervous system penetrance of OT when administered by the intranasal route⁵⁰.

We report here that OT administered intraperitoneally reduced self-administration of MP and we demonstrated this effect at all doses comprising the dose–effect curve for MP self- administration. Similar doses of OT, administered intraperitoneally or by local infusion directly into the NAc shell, enhanced MP-stimulated DA levels in the NAc shell; this effect was absent in the NAc core after systemic OT administration. This suggests that OT receptors in the NAc shell are mediating this effect; however, local infusion of OT may elicit effects on OT receptors located in brain areas surrounding the NAc shell that may contribute to its effects on DA levels. Nevertheless, the neurocircuitry involving the OT receptors that mediates this effect is not known.

In the mouse VTA, OT receptors are expressed on DAergic and more prominently on glutamatergic neurons that project to the NAc and other mesolimbic targets ⁵¹. OT infused into the NAc increased extracellular glutamate there ⁵². Therefore, OT may act on OT receptors located in the NAc on glutamatergic terminals to increase glutamate, and may also increase DA in the NAc by this same mechanism as there are midbrain DA neurons that co-release DA and glutamate in the striatum ⁵³. OT-stimulated increases in glutamate may also activate glutamate receptors on midbrain DA neurons, enhancing DA release, particularly under conditions of DA transporter blockade, as with MP. Removal of presynaptic glutamatergic feedback through mGluR2 deletion had the opposite effect that OT had: mGluR2 deletion reduced both cocaine-stimulated DA and glutamate levels in the NAc and shifted the cocaine dose–response curve upward ⁵⁴. The effect of OT to reduce cue-induced reinstatement of cocaine seeking was dependent on mGluR2/3 signaling ⁵².

OT may also negatively modulate stimulated transmission at the postsynaptic DA receptor, resulting in reduction of DA-related self-administration behavior. This reduction of postsynaptic transmission removes negative feedback and enhances DA release. Importantly, these effects are analogous to the results reported here where enhanced DA release occurs when DA levels are already stimulated, but not under resting conditions, as shown by administration of OT alone which did not significantly modify basal DA levels. Also, OT did not modify MP-induced stimulation of DA levels in the core, an effect that could be related to the absence of potentiation of MP-stimulated behavioral activity counts ⁵⁵.

In conclusion, we have shown that systemic OT reduces the reinforcing effects of MP, a typical psychostimulant drug pharmacologically similar to cocaine. Taken together, our results suggest that OT efficacy as a potential medication for psychostimulant use disorder would address more the motivational/reinforcing (drug-taking) effects of this class of drugs than their stimulant (ambulatory activity) actions. More research is needed to understand the mechanism underlying the effect of OT on the neurocircuitry of psychostimulant response; however, these results suggest that OT may have promise therapeutically for psychostimulant use disorders.

Supplementary Material

Supp figS1

Figure S1. Histological analysis of brain slices from rats used in the experiments described in the main text.