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. Author manuscript; available in PMC: 2022 Dec 1.
Published in final edited form as: Exp Clin Psychopharmacol. 2020 Sep 17;29(6):567–572. doi: 10.1037/pha0000425

PKC inhibition decreases amphetamine-maintained responding under a progressive-ratio schedule of reinforcement

Rachel D Altshuler 1, Ryan C Mac 1, Margaret E Gnegy 1, Emily M Jutkiewicz 1
PMCID: PMC8611615  NIHMSID: NIHMS1731607  PMID: 32940488

Abstract

Protein kinase C (PKC) is important for the mechanism of action of amphetamine (AMPH). Inhibiting PKC blocks AMPH-stimulated increases in extracellular dopamine levels and AMPH-stimulated locomotor activity. This study examined the effects of PKC inhibition on the reinforcing properties of AMPH. Male Sprague-Dawley rats were trained to respond for infusions of 0.032 mg/kg/infusion AMPH or for sucrose pellets under a progressive-ratio (PR) schedule of reinforcement. Number of infusions earned, breakpoints, and session duration were recorded over consecutive sessions. Once AMPH-maintained responding stabilized, rats were treated with 0, 10, or 30 pmol of enzastaurin, a PKCβ-selective inhibitor, or 6 mg/kg 6c, a brain-permeable PKC inhibitor, 18 hr prior to a self-administration session. Pretreatment with 30 pmol enzastaurin or 6 mg/kg 6c decreased the number of AMPH infusions earned and breakpoints without altering sucrose-maintained behaviors. These data suggest that PKC inhibition decreases motivation for AMPH and, therefore, is worth pursuing as a potential treatment for AMPH-use disorder.

Keywords: Amphetamine, self-administration, progressive-ratio, protein kinase C

Introduction

AMPHs dysregulate dopamine levels in the brain. AMPHs enter dopaminergic neurons through the dopamine transporter (DAT) and reverse the function of DAT, resulting in neuronal dopamine release (Seiden, Sabol, &Ricaurte, 1993). Protein kinase C (PKC), a common signaling protein, has been shown to play a role in the mechanism of action of AMPH. Inhibition of PKC blunts the AMPH-stimulated increase in extracellular dopamine levels ex vivo and in vivo (Johnson, Guptaroy, Lund, Shamban, &Gnegy, 2005; Zestos, Mikelman, Kennedy, &Gnegy, 2016).Furthermore, pharmacological inhibition of PKC decreases AMPH-stimulated locomotor activity (Browman et al., 1998; Carpenter et al., 2017; Zestos et al., 2016). Additional studies indicate that the β isoform of PKC is particularly important for the action of AMPH (Chen et al., 2009; Johnson et al., 2005).

Our studies have demonstrated that selective pharmacological inhibition of the PKC isoform, PKCβ, reduces AMPH-stimulated locomotor activity as well as ongoing AMPH self-administration under a fixed-ratio (FR) schedule of reinforcement following an 18-hr pretreatment (Altshuler, Carpenter, Franke, Gnegy, & Jutkiewicz, 2019). The brain permeability of enzastaurin has not been published, thus all studies with enzastaurin utilized intracerebroventricular injections. We also found that a novel brain-permeable, non-selective PKC inhibitor, 6c, decreases responding for AMPH under an FR schedule of reinforcement after an 18-hr pretreatment (Carpenter et al., 2017). While an FR schedule of reinforcement is often a first step in screening the effectiveness of a novel therapeutic for substance use disorders, other schedules of reinforcement provide crucial additional information. Under a progressive-ratio (PR) schedule of reinforcement, the number of responses required for consecutive deliveries of a reinforcer increases and is considered to evaluate quantitatively the reinforcing strength or efficacy of drugs of abuse (Hodos, 1961; Richardson & Roberts, 1996). Furthermore, the PR schedule is often used to measure the motivation for drug-seeking (Markou et al., 1993).

In this study, we investigated the effects of the PKCβ inhibitor, enzastaurin, or the general PKC inhibitor, 6c, on responding for AMPH under a PR schedule of reinforcement. Our goal was to determine if PKC inhibition alters the reinforcing effects of AMPH under multiple schedules of reinforcement and motivation for AMPH, with the long-term objective of developing this potential target for treating AMPH-use disorder. As these two PKC inhibitors behaved similarly in our previous studies examining fixed-ratio self-administration, we predicted that both PKC inhibitors would similarly affect responding for AMPH under a progressive-ratio schedule of reinforcement. We found that enzastaurin and 6c decrease the breakpoint for AMPH self-administration compared with vehicle, without affecting the breakpoint for sucrose self-administration. These data indicate that PKC inhibition decreases the reinforcing effects and motivation for AMPH.

Methods

Subjects and surgeries: Male Sprague-Dawley rats (Envigo Laboratories, Indianapolis, IN) were singled-housed in a temperature- and humidity-controlled environment. The rats were food-restricted to 80–90% of their body weight. The rats were on a 12-hr light/dark cycle with lights on at 0700 and all testing was done during the light phase. Rats were implanted with intracranial cannulas and catheters as previously described in (Altshuler et al., 2019). The animal procedures were designed within the rules and regulations of the National Research Council Guide for the Care and Use of Laboratory Animals and approved by the University of Michigan Institutional Animal Care and Use Committee.

Drugs and solutions: d-AMPH was dissolved in saline and administered intravenously. Artificial cerebral spinal fluid (aCSF) was prepared according to a recipe produced by ALZET Osmotic Pumps (Durect Corporation, Cupertino, CA). Enzastaurin (Cayman Chemical, Ann Arbor, MI) was dissolved in a vehicle solution containing 0.005% dimethyl sulfoxide in aCSF and administered by intracerebroventricular (i.c.v.) injection. A total of 5 μl were administered through an infusion cannula (2 mm C313I, Plastics One, Roanoke, VA) at a rate of 1 μl/min, followed by a 2 min with the infusion cannula held in place. 6c (6c·2.5 HCl) was synthesized by the Vahlteich Medicinal Chemistry Core at the University of Michigan (Carpenter et al., 2016) and dissolved in a saline solution containing 5% Tween-80.

AMPH Self-Administration: The rats were trained to respond in nose poke devices for intravenous infusions of AMPH (0.1 mg/kg/infusion) on an FR 1 schedule of reinforcement during daily 60 min sessions in a procedure described in Altshuler et al. (2019). Once the rats reliably responded for more than 20 infusions per session (mean = 10 ± 4.5 d, range 3–28 d), the AMPH dose was decreased to 0.032 mg/kg/infusion. At this point, groups of rats receiving i.c.v. injections were implanted with cannulas (AP = −0.8 mm, ML = +1.5 mm, DV = −2.8 mm). After recovery from surgery, the rats continued daily AMPH self-administration sessions (0.032 mg/kg/infusion) under an FR1 schedule until responding was stable (Mean = 16.7 ± 10 d, range 5–46 d). Stable responding under an FR1 schedule of reinforcement was determined as no increasing or decreasing trend in responding for 3 consecutive days and responding varying less than 20% between two consecutive sessions. Once this was achieved, both groups began training under a PR schedule, in which the response requirement for a single infusion increased in an exponential manner (response requirement = 5e(0.2 x infusion number) - 5) as described in Robert and Richardson (1992). Self-administration sessions under the PR schedule could last 180 min but were terminated if a rat did not complete a ratio within 30 min. The final ratio completed is referred to as the breakpoint (Roberts &Goeders, 1989). The number of responses, reinforcers, and the final ratio completed were recorded at the end of each session. Stable responding under the PR schedule of reinforcement was determined as three consecutive sessions with a breakpoint within a range of one ratio (± 1) and no increasing or decreasing trends between sessions. Once stable responding under the PR schedule was achieved, cannulated rats were randomly divided into 4 groups and were given aCSF (Group 2), 10 (Group 3), or 30 pmol of enzastaurin (Group 4) i.c.v. 18 hr prior to the test session (session 4). The non-cannulated rats were divided into two groups and given vehicle (5% Tween-80 in saline) (Group 1) or 6 mg/kg 6c (Group 2) s.c. 18 hr prior to a test session (session 4). This time course was based on previous studies examining enzastaurin and 6c (Altshuler et al., 2019; Carpenter et al., 2017). During the test sessions, rats responded for AMPH infusions under a PR schedule and all cues remained present. In a separate group of rats (Group 1, Fig. 1a), vehicle pretreatments (i.c.v.) were administered and saline was substituted for AMPH for a single session to determine breakpoints for AMPH-paired cues in the absence of AMPH. After a single test session, AMPH-maintained responding was evaluated for an additional three days.

Fig. 1. Enzastaurin decreases responding for AMPH under a PR schedule of reinforcement without altering responding for sucrose.

Fig. 1

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(a) Schematic showing the different test conditions and data reported for sessions 1–4 in this figure. (b) Responding for four different groups of rats for 0.032 mg/kg AMPH (PR schedule of reinforcement) across three consecutive sessions. The number of infusions earned (left y-axis) and the corresponding ratio completed (right y-axis) are shown. (c) 18 hr prior to the fourth session, enzastaurin or vehicle (aCSF) was administered to the rats (i.c.v.). Group 1 rats (open circles) received vehicle prior to saline self-administration, group 2 rats (open squares) received vehicle prior to AMPH self-administration, group 3 rats (closed circles) received 10 pmol enzastaurin prior to AMPH self-administration, and group 4 rats (closed triangles) received 30 pmol enzastaurin prior to AMPH self-administration. (d) Responding for three different groups of rats for sucrose (PR schedule of reinforcement) across three consecutive sessions. * p < 0.05 vs. vehicle + AMPH

The number of sucrose pellets earned (left y-axis) and the corresponding ratio completed (right y-axis) are shown. (e) 18 hr prior to a fourth session, enzastaurin or vehicle (aCSF) was administered to the rats (i.c.v.). Group 1 rats (open circles) received vehicle prior to session four, however no sucrose was delivered, group 2 rats (open squares) received vehicle prior to sucrose self-administration, and group 3 rats (closed triangles) received 30 pmol enzastaurin prior to sucrose self-administration. ** p < 0.01 vs. vehicle + sucrose (e), **** p < 0.0001 vs. vehicle + sucrose. n=6.

Sucrose Self-Administration: The sucrose self-administration studies were carried out under the same design as the AMPH self-administration, with a few differences. Completion of a response requirement resulted in the delivery of a 45 mg sucrose pellet. The training sessions under an FR schedule of reinforcement lasted for 20 min, but the sessions under a PR schedule could last for 180 min and were terminated if a ratio was not completed in 30 min. On the testing days, rats were pretreated with vehicle (Groups 5, 6) or 30 pmol enzastaurin (Group 7) (i.c.v.) 18 hr prior to a test session (session 4) where responding resulted in the delivery of a sucrose pellet (Groups 6, 7) or the conditioned stimuli alone (Group 5).

Statistics: Data were analyzed, and statistical analyses were performed using GraphPad Prism 8.0 (GraphPad Software Inc., San Diego, CA). Comparisons were made with two-way ANOVAs and post hoc analyses were completed with Tukey’s multiple comparisons tests. For all experiments, we have reported all measures, and we did not exclude any data points. Based on our typical experimental variance and expected effect sizes (Cohen’s d) of 1.5 or larger, we determined that 6–8 rats per experimental condition would be required.

Results

Enzastaurin decreases responding for AMPH but not sucrose under a PR schedule of reinforcement

We evaluated whether or not the PKCβ-selective inhibitor, enzastaurin, alters AMPH-maintained responding under a PR schedule of reinforcement. Fig. 1 shows data collected from the last 3 sessions prior to a test session (session 4) during which responding under the PR schedule was evaluated 18 hr following apretreatment with vehicle (Group 1, 2), 10 (Group 3), or 30 (Group 4) pmol enzastaurin (see schematic in Fig 1a). A repeated measures (across sessions) two-way ANOVA comparing pretreatments across the self-administration sessions 1–4 (Fig. 1b, c) demonstrates a significant interaction between pretreatment and session [F (9, 60) = 3.9, p = 0.0008] and a significant main effect of session [F (3, 60) = 21.5, p < 0.0001]. Post hoc analyses comparing across the different groups demonstrates no significant difference in the number of infusions during sessions 1–3, but a significant decrease in the number of infusions earned during session 4 following pretreatment with 30 pmol enzastaurin (Group 4) as compared with vehicle (Group 2) and 10 (Group 3) pmol enzastaurin pretreatments (Tukey’s multiple comparisons test p = 0.004, p = 0.03 respectively). In one group of rats pretreated with vehicle (Group 1), saline was substituted for AMPH to evaluate responding for AMPH-paired cues in the absence of AMPH. Under these conditions, the number of infusions earned decreased by approximately 50% as compared with infusions earned when AMPH was available [session 4 was statistically different from sessions 1–3 (Tukey’s multiple comparisons test p < 0.0001)]. Similarly, responding for AMPH following pretreatment with 30 pmol enzastaurin (Group 4) in session 4 was significantly decreased as compared with responding in sessions 1–3 (p<0.0001) Responding for saline infusions was not statistically different from responding for AMPH following pretreatment with 30 pmol enzastaurin (Group 1 vs Group 4) in session 4. A pretreatment with 10 pmol enzastaurin resulted in little change to the number of infusions of AMPH earned.

To determine if this dose of enzastaurin would decrease operant behavior in general, the effects of 30 pmol enzastaurin or vehicle on sucrose pellet-maintained behavior under a PR schedule of reinforcement were determined (see schematic in Fig 1a). A repeated-measures, two-way ANOVA shows a significant interaction ofpretreatment and session [F (6, 45) = 14, p <0.0001] with a significant main effect of session [F (3, 45) = 19, p < 0.0001] (Fig. 1d, e). Responding to sucrose-paired cues alone (Group 5) decreased as compared with responding for sucrose regardless of the pretreatment condition (Tukey’s multiple comparisons test p < 0.0001). A pretreatment of 30 pmol enzastaurin did not significantly alter the number of sucrose pellets earned as compared to vehicle pretreatment condition (Group 6 vs. 7).

6c decreases responding for AMPH under a PR schedule of reinforcement

Previous data demonstrated that the brain penetrant PKC inhibitor 6c decreases AMPH self-administration under an FR schedule of reinforcement (Carpenter et al., 2017); thus, we sought to determine the effects of 6c on AMPH-maintained responding under a PR schedule of reinforcement. These experiments followed the same timeline as shown in Fig 1a. Rats trained to self-administer AMPH under a PR schedule of reinforcement were given vehicle (Group 1) or 6 mg/kg 6c (Group 2) (s.c.) 18 h prior to the test session (session 4); data from the three sessions prior to the test are shown in Fig. 2a. An injection of 6c significantly decreased responding for AMPH under a PR schedule of reinforcement (repeated measures, two-way ANOVA [F (3, 30) = 21.4, p < 0.0001] with a significant main effect of session [F (3, 30) = 11.3, p < 0.0001]). Sidak’spost hoc analysis shows 6c significantly decreased the number of responses on the test day (session 4) as compared with the vehicle pretreatment on session 4 (p < 0.0001) and as compared to responding during sessions 1–3 (p < 0.0001).

Fig. 2. The brain permeable PKC inhibitor 6c decreases responding for AMPH under a PR schedule of reinforcement.

Fig. 2

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(a) Responding for two different groups of rats for 0.032 mg/kg AMPH (PR schedule of reinforcement) across three consecutive sessions. The number of infusions earned (left y-axis) and the corresponding ratio completed (right y-axis) are shown. (b) 18 hr prior to a fourth session, 6c or vehicle (saline) was administered to the rats (i.c.v.). Group 1 rats (open diamonds) received vehicle prior AMPH self-administration and group 2 rats (closed diamonds) received 6 mg/kg 6c prior to AMPH self-administration. *** p < 0.001 vehicle vs 6c. n=6.

Discussion

We previously demonstrated that PKC inhibitors decrease ongoing AMPH self-administration under an FR schedule of reinforcement (Altshuler et al., 2019; Carpenter et al., 2017). In this study, we sought to evaluate further the effects of PKC inhibitors on AMPH-maintained behaviors under a complex schedule of reinforcement. Our findings show for the first time that the PKCβ-selective inhibitor enzastaurin and the structurally-distinct, brain-permeable PKC inhibitor 6c decrease responding for AMPH under a PR schedule of reinforcement without altering responding for the non-drug reinforcer, sucrose. Rats pretreated with enzastaurin or 6c earned fewer infusions and had a lower breakpoint for AMPH compared with their previous levels of AMPH-maintained responding and compared with rats that received vehicle pretreatments. In addition, enzastaurin and 6c pretreatments decreased AMPH-maintained responding to levels observed in the absence of AMPH. This suggests that PKC inhibition may change the reinforcing strength of AMPH and/or the motivation to work for AMPH infusions (Hodos, 1961; Richardson & Roberts, 1996; Roberts & Richardson, 1992).

Interestingly, both enzastaurin and 6c attenuated AMPH-maintained responding following a long pretreatment time, regardless of their route of administration. This is consistent with our previous studies demonstrating that an 18 hr pretreatment of enzastaurin or 6c is required to decrease AMPH self-administration (Altshuler et al., 2019; Carpenter et al., 2017). This could indicate that a second mechanism indirectly due to PKC inhibition may be responsible for the long-lasting effects of the drugs. A possible explanation is for this delayed mechanism is a change in protein level and/or function downstream of PKC signaling. Alternatively, a metabolite may be an additional explanation for the time course of the PKC inhibitors; however, this is unlikely due to the structurally different PKC inhibitors used in the present study and the pharmacokinetics of enzastaurin. The half-life of enzastaurin following oral administration is 2 hr (personal communications with Denovo Biopharma and Eli Lilly), indicating that the concentration of enzastaurin will be minimal at an 18 hr time point. Indeed, microdialysis studies have demonstrated that the concentration of 6c in the nucleus accumbens is minimal an hour following administration of the drug (Carpenter et al., 2017).

Altogether, these studies demonstrate that two structurally different PKC inhibitors previously shown to decrease AMPH self-administration under an FR schedule of reinforcement also decrease AMPH self-administration under a PR schedule of reinforcement. These findings suggest that PKC inhibition may decrease the reinforcing strength of AMPH and/or the motivation to work for AMPH infusions. These drugs do not decrease responding for sucrose, suggesting that the doses tested are not inhibiting general behavior or inducing locomotor deficits or adverse effects. One limitation to the study is the inclusion of a single dose of 6c. Multiple doses are necessary to determine in what direction 6c shifts the dose-effect curve for self-administration. We believe that 6c would act in a similar manner to enzastaurin, due to the shared mechanism of PKC inhibition, to shift the dose-effect curve to the right. This will be evaluated in future studies. Further work should also evaluate the effect of PKC inhibition on additional doses of AMPH, other drugs of abuse, and other models of drug seeking behaviors such as the extinction/reinstatement paradigm. These findings are a promising step towards the development of PKC inhibitors as therapeutic targets for AMPH-use disorder.

Public Health Significance:

This work demonstrates that protein kinase C inhibitors decrease AMPH self-administration under a progressive-ratio schedule of reinforcement. These findings have implications on the role of protein kinase C on AMPH-mediated behaviors and motivation. Furthermore, this study demonstrates the feasibility of targeting protein kinase C as a therapeutic intervention for AMPH-use disorders.

Acknowledgements:

Rachel Altshuler, Margaret Gnegy, and Emily Jutkiewicz designed all experiments and contributed to the writing and editing of the manuscript. Rachel Altshuler and Ryan Mac conducted all experiments. All authors have read and approved the final manuscript.

Disclosures:

This research was supported by National Institutes of Health grant R01 DA11697, National Institutes of Health Training Grant T32-GM007767, Benedict and Diana Lucchesi Graduate Education Fellowship.

Footnotes

The authors have no conflicts of interest to disclose.

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