Activation of Gβγ subunits in the NAc potentiates and inhibition of Gβγ subunits in the NAc or systemically blunts amphetamine-induced hyperlocomotion

First, we investigated the effect of mSIRK, a cell-permeable myristoylated peptide that specifically activates Gβγ subunits24,33,34, delivered bilaterally into the NAc on basal and psychostimulant-induced locomotor activity. A scrambled sequence of mSIRK, scr-mSIRK, was used as control. As depicted in Fig. 1a, b, locomotor activity did not differ significantly between groups after bilateral infusion of mSIRK (1 mM) or scr-mSIRK (1 mM) into the NAc. After intraperitoneal psychostimulant injection, distance traveled was significantly higher for both groups injected with amphetamine (3 mg/kg) compared to the two groups injected with saline (Fig. 1a, b; p < 0.0001). Importantly, the amphetamine-induced hyperlocomotion in rats infused with mSIRK was extended markedly beyond the transient hyperlocomotion in rats infused with scr-mSIRK (1 mM), resulting in a significant potentiation of the psychostimulant effect (Fig. 1a, b; p < 0.001).

Fig. 1: Activation of Gβγ subunits in the nucleus accumbens prolongs and inhibition of Gβγ subunits in the nucleus accumbens blunts amphetamine-induced hyperlocomotion. a Means ± s.e.m. of distance traveled across the 2 h testing period, depicted per 10 min block, for the scr-mSIRK-saline (n = 6), mSIRK-saline (n = 6), scr-mSIRK-amphetamine (n = 6) and mSIRK-amphetamine (n = 6) groups. Scr-mSIRK or mSIRK was infused into the nucleus accumbens (NAc) 30 min before saline or amphetamine injection. b Means ± s.e.m. of distance traveled by experimental period for the same groups. NAc infusion is the period immediately after intra-NAc infusion of scr-mSIRK, and intraperitoneal (i.p.) drug is the period immediately after saline or amphetamine injection. Habituation was performed in drug-free conditions. c Means ± s.e.m. of distance traveled across the 2 h testing period, depicted per 10 min block, for the vehicle-saline (n = 6), gallein-saline (n = 6), vehicle-amphetamine (n = 6) and gallein-amphetamine (n = 6) groups. Vehicle or gallein was infused into the NAc 30 min before saline or amphetamine injection. d Means ± s.e.m. of distance traveled by experimental period for the same groups. NAc infusion is the period immediately after intra-NAc infusion of vehicle of gallein, and i.p. drug is the period immediately after saline or amphetamine injection. Habituation was performed in drug-free conditions. *P < 0.05 between saline-treated and amphetamine-treated groups; #p < 0.05 between scr-mSIRK-amphetamine and mSIRK-amphetamine groups b or between vehicle-amphetamine and gallein-amphetamine groups d Full size image

Next, we determined whether intra-accumbal infusion of the Gβγ inhibitor gallein, a small molecule that binds to Gβγ and disrupts Gβγ signaling to effectors34,35,36, affects basal and psychostimulant-induced locomotor activity. Similar to our observations above, locomotor activity did not differ significantly between groups during baseline or after bilateral infusion of gallein (2 mM) or vehicle into the NAc (Fig. 1c, d). After drug injection, distance traveled once again was significantly higher in amphetamine-injected compared to saline-injected rats (p < 0.001), but the magnitude of the hyperlocomotion was blunted markedly for rats that had received infusion of gallein compared to rats infused with vehicle (p < 0.02; Fig. 1c, d).

To investigate the translational possibilities of targeting Gβγ to modulate the effect of amphetamine, we repeated the previous experiment, but administered gallein intraperitoneally (4 mg/kg). Similar to our findings when the administration of the Gβγ inhibitor was restricted to the NAc, systemic gallein treatment had no effect on basal locomotor activity, but it significantly attenuated the elevation in locomotor activity after amphetamine injection (p < 0.0001; Figure S1). Taken together, our findings show that manipulation of Gβγ activity in the NAc modulates amphetamine’s locomotor effects bi-directionally, and that systemic administration of the Gβγ inhibitor gallein is as effective in blunting amphetamine-induced hyperlocomotion as is intra-NAc administration.

Cocaine-induced hyperlocomotion is not affected by activation or inhibition of Gβγ subunits in the NAc

As a first step toward deciphering the mechanism through which manipulation of Gβγ subunits in the NAc modulates amphetamine-induced hyperlocomotion, we determined whether cocaine-induced hyperlocomotion is also sensitive to pharmacological manipulation of intra-NAc Gβγ with mSIRK or gallein. If the effects of manipulating Gβγ on amphetamine-induced hyperlocomotion are related to amphetamine’s ability to induce DA efflux through DAT, then these same Gβγ manipulations should not affect cocaine-induced locomotor activity. Figure 2a depicts that, as observed previously, infusion of mSIRK (1 mM) or scr-mSIRK (1 mM) had no effect on locomotor activity before psychostimulant injection. After drug injection, locomotor activity was significantly higher in both groups that received cocaine (20 mg/kg, intraperitoneally (i.p.)) than in their saline-injected counterparts (p < 0.0001). However, unlike our observations with amphetamine, the magnitude as well as the time course of the psychostimulant-induced hyperlocomotion was unaffected by mSIRK (Fig. 2a). A similar experiment with intra-NAc infusion of gallein (2 mM) replicated the lack of an effect of the Gβγ inhibitor on basal activity, and, importantly, on cocaine-induced hyperlocomotion (Fig. 2b). The differential effect of Gβγ manipulation on hyperlocomotion induced by amphetamine vs. cocaine is consistent with the hypothesis that the underlying mechanism of the modulatory effect involves amphetamine’s ability to cause DA efflux via DAT.

Fig. 2: Activation or inhibition of Gβγ subunits in the nucleus accumbens has no effect on cocaine-induced hyperlocomotion, whereas interference of Gβγ subunit binding with dopamine transporter (DAT) in the nucleus accumbens attenuates amphetamine-induced locomotor activity. a Means ± s.e.m. of distance traveled by experimental period for the scr-mSIRK-saline (n = 6), mSIRK-saline (n = 6), scr-mSIRK-cocaine (n = 6), and mSIRK-cocaine (n = 6) groups. Nucleus accumbens (NAc) infusion is the period immediately after intra-NAc infusion of scr-mSIRK, and intraperitoneal (i.p.) drug is the period immediately after saline or amphetamine injection. Habituation was performed in drug-free conditions. b Means ± s.e.m. of distance traveled by experimental period for the vehicle-saline (n = 6), gallein-saline (n = 6), vehicle-cocaine (n = 6), and gallein-cocaine (n = 6) groups. NAc infusion is the period immediately after intra-NAc infusion of vehicle of gallein, and i.p. drug is the period immediately after saline or cocaine injection. Habituation was performed in drug-free conditions. c Means ± s.e.m. of distance traveled by experimental period for the TAT-scr-DATct1-saline (n = 5), TAT-DATct1-saline (n = 5), TAT-scr-DATct1-amphetamine (n = 6), and TAT-DATct1-amphetamine (n = 5) groups. NAc infusion is the period immediately after intra-NAc infusion of TAT-scr-DATct1 or TAT-DATct1, and i.p. drug is the period immediately after saline or amphetamine injection. Habituation was performed in drug-free conditions. d Means ± s.e.m. of distance traveled by experimental period for the TAT-scr-DATct1-saline (n = 5), TAT-DATct1-saline (n = 5), TAT-scr-DATct1-cocaine (n = 5), and TAT-DATct1-cocaine (n = 5) groups. NAc infusion is the period immediately after intra-NAc infusion of TAT-scr-DATct1 or TAT-DATct1, and i.p. drug is the period immediately after saline or cocaine injection. Habituation was performed in drug-free conditions. *P < 0.05 between saline-treated and cocaine- or amphetamine-treated groups; #p < 0.05 between TAT-scr-DATct1-amphetamine and TAT-DATct1-amphetamine groups Full size image

Blocking Gβγ interaction with DAT in the NAc blunts amphetamine-induced but not cocaine-induced hyperlocomotion

To elucidate further the mechanism through which Gβγ inhibition modulates amphetamine’s locomotor effects, we examined whether amphetamine-induced hyperlocomotion involves a direct interaction between Gβγ and the carboxy terminus of DAT. We previously showed that residues 582 to 596 in the carboxy terminus of DAT bind directly to Gβγ, and that a TAT-fused peptide containing these residues in DAT, TAT-DATct1, prevented Gβγ–DAT interaction24. Therefore, we examined the effect of bilateral intra-NAc infusion of TAT-DATct1 on amphetamine-induced hyperlocomotion. A scrambled sequence of DATct1 fused to TAT, TAT-scr-DATct1, served as control. Similar to the observations with gallein, intra-NAc infusion of TAT-DATct1 (1 mM) had no effect on basal locomotor activity but markedly attenuated the magnitude of the increase in locomotor activity induced by amphetamine (Fig. 2c). Although higher than in saline-injected rats, distance traveled was significantly lower for amphetamine-injected rats that had received intra-NAC infusion of TAT-DATct1 compared to rats that had received intra-NAc infusion of TAT-scr-DATct1 (Fig. 2c; p < 0.0001). In sharp contrast, intra-NAc infusion of TAT-DATct1 vs. TAT-scr-DATct1 did not result in differential effects on elevated locomotor activity induced by cocaine (Fig. 2d). Immunoprecipitation of DAT did not result in the co-precipitation of Gβγ in tissue from animals receiving TAT-DATCt1 compared to control conditions (data not shown). Taken together with our findings above, these results support a role for Gβγ interaction with DAT in amphetamine-induced hyperlocomotion.

Inhibition of Gβγ activation or interaction with DAT decreases amphetamine-stimulated DA efflux in dopaminergic neurons in culture

The behavioral findings with the TAT-DATct1 peptide prompted us to investigate the role of Gβγ–DAT interaction in amphetamine-stimulated DA efflux in more depth. First, we examined the effects of the Gβγ inhibitor gallein and the TAT-DATct1 peptide on amphetamine-induced DA efflux in midbrain DA neurons in culture. Neurons were preloaded with [3H]-DA (20 nM) prior to treatment, and [3H]-DA release was assessed before and after amphetamine exposure. Amphetamine (10 μM) significantly increased DA efflux over control conditions (p < 0.0001; Fig. 3a). The effect of amphetamine on DA efflux was blunted in neurons pretreated with gallein (20 μM) (p < 0.01) or the TAT-DATct1 peptide (20 μM) (p < 0.001). The combined treatment of gallein and TAT-DATct1 peptide did not decrease further the amphetamine-induced efflux when compared to either gallein or TAT-DATct1 alone. Basal DA efflux was not affected by gallein, TAT-scr-DATct1, or TAT-DATct1 prior to amphetamine treatment, and the TAT-scr-DATct1 peptide had no effect on amphetamine-induced DA efflux (Fig. 3a). Thus, blocking Gβγ activation with gallein or blocking the Gβγ–DAT interaction with the TAT-DATct1 peptide reduces amphetamine-induced DA efflux.

Fig. 3: Activation or inhibition of Gβγ subunits or Gβγ–DAT interaction alters amphetamine-induced dopamine (DA) efflux in DA neurons in culture and nucleus accumbens tissue. a Inhibition of Gβγ subunits or Gβγ–DAT interaction blunts amphetamine-induced DA efflux in DA neurons preloaded with 20 nM3[H]-DA. Data represented as percent of control conditions for neurons treated with gallein (20 μM), TAT-scr-DATct1 (20 μM), TAT-DATct1 (20 μM), or TAT-DATct1 (20 μM) plus gallein (20 μM), ±amphetamine (10 μM) (n = 5/group). b Activation of Gβγ subunits increases amphetamine-induced DA efflux in nucleus accumbens (NAc) tissue. Mean ± s.e.m. of total DA efflux for tissue treated with vehicle (n = 8), scr-mSIRK (100 μM) (n = 10), or mSIRK (100 μM) (n = 10) before and after amphetamine (10 μM). c Inhibition of Gβγ subunits reduces amphetamine-induced DA efflux. Fractional release of the total DA of tissue perfused with amphetamine alone (10 μM) compared to gallein (20 μM)+amphetamine (10 μM) (n = 5/group). d Area under the curve (AUC) data for total DA efflux of tissue perfused with amphetamine alone compared to gallein+amphetamine.*P < 0.05 between amphetamine and gallein-amphetamine in NAc tissue. **p < 0.01 between mSIRK-amphetamine and vehicle-amphetamine in NAc tissue; ##p < 0.01 between amphetamine and gallein-amphetamine in DA neurons, ###p < 0.001 between amphetamine and TAT-DATct1-amphetamine in DA neurons, ****p < 0.0001 between control and amphetamine or scr-TAT-DATct1-amphetamine in DA neurons Full size image

Activation of Gβγ with mSIRK potentiates and inhibition of Gβγ with gallein reduces amphetamine-induced DA efflux in dorsal striatum and NAc ex vivo

To assess further the role of Gβγ–DAT interaction in the actions of amphetamine, we tested the effect of Gβγ activation on amphetamine-induced DA efflux using an ex vivo DA efflux assay. DA efflux did not differ in the dorsal striatal punches treated with vehicle (1% dimethyl sulfoxide (DMSO)), scr-mSIRK (100 μM), or mSIRK (100 μM) during the 20-min pretreatment, prior to amphetamine treatment (10 μM) (Figure S2). However, amphetamine (10 μM) administration significantly increased DA efflux in samples pretreated with mSIRK compared to samples pretreated with vehicle or scr-mSIRK (p < 0.0001). As was observed with the dorsal striatum, there was no difference in NAc tissue treated with vehicle, scr-mSIRK, or mSIRK prior to amphetamine treatment (Fig. 3b). After amphetamine administration, there was a significant increase in DA efflux in mSIRK-treated tissue compared to vehicle-treated tissue (p < 0.001). In summary, activation of Gβγ with mSIRK increased DA efflux after amphetamine exposure in the dorsal striatum and the NAc ex vivo.

The effect of Gβγ inhibition with gallein on amphetamine-induced DA efflux was also examined in dorsal striatal and NAc tissue (Fig. S2 and 3c, d). Amphetamine (10 μM) alone caused a significant increase in DA efflux in dorsal striatal tissue. However, there was a significant reduction in DA efflux in tissue pretreated with gallein (20 μM) compared to tissue treated with amphetamine only (p < 0.05) (Figure S2). Similarly, in the NAc, there was a significant reduction in amphetamine-induced DA efflux with tissue treated with gallein prior to amphetamine compared to tissue treated with amphetamine alone (p < 0.05) (Fig. 3c, d). These data show that Gβγ inhibition reduces amphetamine-induced DA efflux in ex vivo dorsal striatum and NAc tissue.

Activation of Gβγ with mSIRK potentiates and inhibition of Gβγ with gallein reduces amphetamine-induced increases in extracellular DA in the NAc of freely moving rats

Next, we determined the effects of Gβγ manipulation on amphetamine-induced increases in extracellular DA in the NAc of awake, freely moving rats using in vivo microdialysis. First, we assessed the effects of Gβγ activation with mSIRK on amphetamine-stimulated increase in extracellular DA. There was no difference in extracellular DA levels between groups during either the baseline period or the scr-mSIRK vs. mSIRK (1 mM) pretreatment period, i.e., prior to amphetamine (3 mg/kg) administration (Fig. 4a, b). However, amphetamine induced a significant increase in DA overflow in rats pretreated with mSIRK compared to rats pretreated with the control peptide scr-mSIRK (p < 0.001) (Fig. 4a, b). The present results show that Gβγ activation potentiates amphetamine’ s effects on DA overflow in the NAc of awake rats.

Fig. 4: Activation or inhibition of Gβγ subunits alters amphetamine-induced extracellular dopamine (DA) levels in the nucleus accumbens of freely moving rats. a Activation of Gβγ subunits increases amphetamine-induced extracellular DA levels in the nucleus accumbens (NAc). Data represented as percent increase of baseline extracellular DA levels per 10 min for rats treated with scr-mSIRK (1 mM)–amphetamine (3 mg/kg) (n = 4) or mSIRK (1 mM)–amphetamine (3 mg/kg) (n = 5) over 210 min of testing. scr-mSIRK or mSIRK was perfused through the microdialysis probe by reverse dialysis for 1 h (pretreatment period) before an intraperitoneal (i.p.) injection of amphetamine (3 mg/kg) (treatment period) and continually perfused through the 2 h treatment period. b Area under the curve (AUC) of total extracellular DA levels during the baseline, pretreatment, and treatment periods. c Inhibition of Gβγ subunits reduces amphetamine-induced extracellular dopamine levels in the NAc of freely moving rats. Data represented as percent increase of baseline extracellular DA levels per 10 min for rats treated with gallein (4 mg/kg)–saline (1 ml/kg) (n = 4), vehicle (25% dimethyl sulfoxide (DMSO) in saline)–amphetamine (3 mg/kg) (n = 7) or gallein–amphetamine (n = 7) over the 180 min testing period. The i.p. injections of vehicle or gallein occurred during the pretreatment period followed by an i.p. injection of amphetamine or saline during the 2 h treatment period. d AUC analysis of total extracellular DA levels during the baseline, pretreatment, and treatment periods. ***P < 0.001 denotes AUC between scr-mSIRK-amphetamine and mSIRK-amphetamine during the treatment period, ****p < 0.0001 denotes AUC between gallein-saline and vehicle-amphetamine during the treatment period, ####p < 0.0001 denotes AUC between vehicle-amphetamine and gallein-amphetamine during the treatment period Full size image

We also tested the effect of Gβγ inhibition on amphetamine-stimulated increases in extracellular DA. Again, there was no difference in extracellular DA levels during the baseline period, or the gallein (4 mg/kg) vs. vehicle pretreatment period (Fig. 4c, d). During the drug treatment period, amphetamine (3 mg/kg) significantly increased extracellular DA levels in rats that had received vehicle pretreatment, and the amphetamine-induced increase in extracellular DA was blunted in rats pretreated with gallein (p < 0.0001) (Fig. 4c, d). Thus, inhibition of Gβγ activation with gallein significantly reduced amphetamine-stimulated extracellular DA in awake rats. This outcome is consistent with the effect of gallein on DA efflux in ex vivo tissue samples and on amphetamine-induced locomotor activity.

Inhibition of Gβγ subunits in the NAc blunts amphetamine-conditioned place preference but has no effect on cocaine-conditioned place preference

To determine whether the modulation of amphetamine’s neurochemical and locomotor effects by manipulation of Gβγ activity or its interaction with DAT in the NAc extends to the rewarding effects of amphetamine, we investigated the effect of bilateral intra-NAc infusion of gallein on amphetamine-mediated conditioned place preference. As shown in Fig. 5a, rats that received intra-NAc infusions of vehicle solution before amphetamine (1.5 mg/kg, i.p.) conditioning trials exhibited a significant, robust preference above pre-conditioning level for the amphetamine-paired compartment (p < 0.005). In contrast, rats that received intra-accumbal infusion of gallein (2 mM) before amphetamine conditioning trials displayed neither a preference for nor an aversion of the amphetamine-paired compartment. The lack of the amphetamine-conditioned place preference in the latter group did not appear to stem from aversive effects of gallein, because gallein alone had no effect on place conditioning, as indicated by the absence of conditioned place preference or aversion by rats that received intra-accumbal infusion of gallein in the absence of amphetamine conditioning trials (saline conditioning trials only, Fig. 5a).

Fig. 5: Inhibition of Gβγ subunits in the nucleus accumbens blunts amphetamine-induced place preference but has no effect on cocaine-induced place preference. a Means ± s.e.m. of conditioned place preference index for the gallein-saline (n = 9), vehicle-amphetamine (n = 10), and gallein-amphetamine (n = 10) groups. Vehicle or gallein was infused directly into the nucleus accumbens (NAc) 30 min before saline or amphetamine injection (intraperitoneally (i.p.)) on days 1, 3, and 5. b Similar data as shown in a, except that vehicle or gallein was administered i.p. 30 min before saline or amphetamine injection. The group sizes were identical to those in a. Animals that received gallein, whether by intra-NAc infusion (a) or i.p. injection (b), displayed neither a place preference nor an aversion, regardless of whether they underwent amphetamine conditioning or served as gallein-only controls. c Means ± s.e.m. of the conditioned preference index for cocaine-treated animals (N = 7 for both groups). Vehicle or gallein was administered i.p. 30 min before cocaine injection (i.p.) on days 1, 3, and 5. Animals displayed a place preference for the cocaine-paired compartment, regardless of whether they were pretreated with gallein or vehicle. *P < 0.05 between group average and 0.0 (no conditioning-induced shift in preference); #p < 0.05 between the vehicle-amphetamine-treated group and either of the two gallein-pretreated groups Full size image

To determine whether these observations extend to conditions with relatively greater translational relevance, we repeated the same experiment described above, but administered gallein systemically before drug conditioning trials. Once again, amphetamine-conditioned rats pretreated with vehicle solution developed a significantly positive preference index (Fig. 5b; p < 0.01), and, similar to our observations with gallein administration restricted to the NAc, rats that received systemic administration of gallein (4 mg/kg, i.p.) before amphetamine conditioning trials failed to develop an increase in preference for the amphetamine-paired compartment (Fig. 5b). Repeated exposure to systemic gallein alone did not produce an aversion, as indicated by a preference index near 0.0 by the gallein-treated group that received saline trials only.

We reported above that gallein or the TAT-DATct1 peptide had no effect on cocaine-induced hyperlocomotion, an outcome that suggested that Gβγ modulates amphetamine’s actions on DA efflux via DAT. To test whether the differential effect of Gβγ inhibition on amphetamine- vs. cocaine-induced locomotor behavior also extends to psychostimulant-induced reward seeking, we exposed rats to cocaine place preference conditioning using essentially the same conditioning paradigm, except that the experiment entailed only two groups, cocaine-conditioned rats pretreated with vehicle solution and cocaine-conditioned rats pretreated with gallein on psychostimulant conditioning days. Figure 5c shows that both vehicle- and gallein-pretreated rats developed a robust preference for the cocaine-paired compartment (both p < 0.05), and that the level of place preference did not differ between the two groups. Taken together, these results demonstrate that Gβγ inhibition, with gallein within the NAc or systemically, specifically modulates not only amphetamine-induced locomotor activity, but also amphetamine-conditioned place preference, an indicator of reward seeking.