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. 2019 Apr 17;10(4):1960-1969.
doi: 10.1021/acschemneuro.8b00259. Epub 2018 Nov 8.

Ruboxistaurin Reduces Cocaine-Stimulated Increases in Extracellular Dopamine by Modifying Dopamine-Autoreceptor Activity

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Ruboxistaurin Reduces Cocaine-Stimulated Increases in Extracellular Dopamine by Modifying Dopamine-Autoreceptor Activity

Alexander G Zestos et al. ACS Chem Neurosci. .
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Abstract

Cocaine is a highly abused drug, and cocaine addiction affects millions of individuals worldwide. Cocaine blocks normal uptake function at the dopamine transporter (DAT), thus increasing extracellular dopamine. Currently, no chemical therapies are available to treat cocaine abuse. Previous works showed that the selective inhibitors of protein kinase Cβ (PKCβ), enzastaurin and ruboxistaurin, attenuate dopamine overflow and locomotion stimulated by another psychostimulant drug, amphetamine. We now test if ruboxistaurin similarly affects cocaine action. Perfusion of 1 μM ruboxistaurin directly into the core of the nucleus accumbens via retrodialysis reduced cocaine-stimulated increases in dopamine overflow, measured using microdialysis sampling, with simultaneous reductions in locomotor behavior. Because cocaine activity is highly regulated by dopamine autoreceptors, we examined whether ruboxistaurin was acting at the level of the D2 autoreceptor. Perfusion of 5 μM raclopride, a selective D2-like receptor antagonist, before addition of ruboxistaurin, abrogated the effect of ruboxistaurin on cocaine-stimulated dopamine overflow and hyperlocomotion. Further, ruboxistaurin was inactive against cocaine-stimulated locomotor activity in mice with a genetic deletion in D2 receptors as compared to wild-type mice. In contrast, blockade or deletion of dopamine D2 receptors did not abolish the attenuating effect of ruboxistaurin on amphetamine-stimulated activities. Therefore, the inhibition of PKCβ reduces dopamine overflow and locomotor activity stimulated by both cocaine and amphetamine, but the mechanism of action differs for each stimulant. These data suggest that inhibition of PKCβ would serve as a target to reduce the abuse of either amphetamine or cocaine.

Keywords: D2 dopamine receptor knockouts; Protein kinase C beta; dopamine transporter; norepinephrine; protein kinase C beta inhibition.

Figures

Figure 1:
Figure 1:
Microdialysis Probe placements targeting the core of the nucleus accumbens during microdialysis experiments for data in Figures 2–4.
Figure 2:
Figure 2:. Pretreatment with ruboxistaurin reduces effectiveness of cocaine in stimulating dopamine overflow and locomotor behavior.
At 30 min of baseline collection, vehicle (Veh) or 1 μM ruboxistaurin (Rubox) was perfused into the core of the nucleus accumbens for the remaining duration of the experiment. A 15 mg/kg i.p. injection of cocaine (Coc) was given 30 min later (at time zero) and fractions were collected for an additional h. Perfusion with ruboxistaurin attenuates cocaine-stimulated overflow of a. dopamine; b. 3-methoxytyramine (3MT); and d. locomotor activity. c. Neither cocaine nor ruboxistaurin affected dihydroxyphenylacetic acid (DOPAC) levels. Results are given as % Baseline or locomotor counts (counts) ± standard error of the mean (sem) (n=6). In post-hoc Bonferroni multiple comparison test, *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001 as compared to ruboxistaurin- treated samples.
Figure 3.
Figure 3.. Pretreatment with raclopride enhances cocaine-stimulated dopamine overflow (a) but inhibits cocaine-stimulated locomotor behavior (b).
In a,b, fifteen min of baseline were collected followed by perfusion of vehicle (Veh) or 5 μM raclopride (Rac) into the core of the nucleus accumbens for the remaining duration of the experiment (90 min). At 1 h (0 time), a 15 mg/kg injection of cocaine (Coc) was given i.p. and fractions were collected for an additional h. a. In post hoc Bonferroni multiple comparison test, * p < 0.05, ***, p < 0.001, n = 5. b. In post hoc Bonferroni multiple comparison test, *p < 0.02, n = 5. In c,d. pretreatment with raclopride blocks inhibition of cocaine-stimulated dopamine overflow (c.) and locomotor activity (d.) by 1 μM ruboxistaurin. Raclopride was perfused after 15 min (−45 min) and either vehicle (Rac + Veh, n=5) or 1 μM ruboxistaurin (Rac + Rubox, n=5) was perfused at −30 min). At 1 h (0 time), a 15 mg/kg i.p., injection of cocaine was given. Results are expressed as % baseline or locomotor counts in 1 h after addition of cocaine ± sem.
Figure 4:
Figure 4:. Pretreatment with raclopride did not change dopamine overflow (a) or locomotion (b) elicited by amphetamine and did not block inhibition by ruboxistaurin (c,d).
In a,b, 15 min of baseline were collected followed by perfusion of 5 μM raclopride (Rac) into the core of the nucleus accumbens for the rest of the experiment (105 min). At 1 h (time 0), a 2 mg/kg injection of amphetamine was given i.p. and fractions were collected for 1 additional h. a. vehicle n=6; raclopride n=6. b. vehicle n=6, raclopride, n=5. In c,d, pretreatment with raclopride did not block inhibition of amphetamine-stimulated dopamine overflow (c) and locomotor activity (d) by 1 μM ruboxistaurin. The experiment was conducted as above, but vehicle (Rac + Veh) or 1 μM ruboxistaurin (Rac + Rubox) was perfused at −30 min (n = 5). At 1 h (0 time), a 2 mg/kg i.p., injection of amphetamine was given. Results are expressed as % baseline or locomotor counts in 1 h ± sem. c. In post-hoc Bonferroni multiple comparison test, *p < 0.05, n = 5. d. In post-hoc Bonferroni multiple comparison test, *p < 0.05. n = 5.
Figure 5:
Figure 5:. The effects of amphetamine and cocaine administration on wildtype and D2- autoreceptor knockout mice.
Wildtype (WT) and D2-autoreceptor Knockout (KO) C57Bl mice were tested with cocaine (Coc), amphetamine (AMPH), ruboxistaurin (Rubox), and vehicle (Veh) and locomotor behavior was measured in a raturn. The animals were given an injection of 10 pmol ruboxistaurin or vehicle into the core of the nucleus accumbens and allowed to recover after 4 h. 30 min of basal locomotor activity was recorded followed by an injection of 2 mg/kg i.p. of amphetamine or 15 mg/kg i.p. of cocaine. The injection of ruboxistaurin attenuated cocaine-stimulated increases in locomotion in wildtype (a), but not in D2-autoreceptor knockout (b) mice. The injection of ruboxistaurin also attenuated amphetamine-stimulated locomotion in WT (c) mice and KO mice (d). Results are given as locomotor counts ± standard error of the mean (sem) (n=4). In post-hoc Bonferroni multiple comparison test, *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001 WT/D2R-KO mice, n=4.
Figure 6:
Figure 6:. Ruboxistaurin inhibits dopamine overflow elicited by amphetamine (AMPH) and cocaine by two different mechanisms. a. (Cocaine, left side).
Cocaine increases extracellular dopamine by blocking the dopamine transporter. Because cocaine blocks the reuptake of dopamine at the dopamine transporter that has been released by exocytosis, its effect will be regulated by D2-like autoreceptor activity. PKC activation normally inhibits dopamine autoreceptor activity by reducing surface expression of D2 receptors which will contribute to the concentration of extracellular dopamine–, . a. (Cocaine + rubox, right side) Ruboxistaurin inhibits PKCβ, increasing the surface expression of D2 autoreceptors by blocking their internalization thus enhancing their effectiveness in inhibiting exocytosis of dopamine. This leads to a reduction in extracellular dopamine (dopamine overflow). b. (AMPH, left side) The mechanism of amphetamine action is dependent on the dopamine transporter. Amphetamine is a substrate for the dopamine transporter and will elicit dopamine efflux through reversal of the transporter. Amphetamine activity does not depend on dopamine exocytosis and is not significantly regulated by dopamine autoreceptors. Amphetamine that is taken up through the transporter activates PKCβ which potentiates reversal of the transporter and dopamine efflux, . b. (AMPH + Rubox, right side) Blockade of PKCβ reduces the outward transport of dopamine but does not affect inward transport–, –, –. The end result of ruboxistaurin action on activation by either cocaine or amphetamine is a reduction in extracellular dopamine (or norepinephrine) and a reduction in stimulant-induced locomotor activity.

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