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. Author manuscript; available in PMC: 2020 Nov 1.
Published in final edited form as: Psychopharmacology (Berl). 2019 May 27;236(11):3231–3242. doi: 10.1007/s00213-019-05278-0

The protein kinase Cβ-selective inhibitor, enzastaurin, attenuates amphetamine-stimulated locomotor activity and self-administration behaviors in rats

Rachel D Altshuler 1, Colleen A Carpenter 1, Timothy J Franke 1, Margaret E Gnegy 1, Emily M Jutkiewicz 1
PMCID: PMC6832797  NIHMSID: NIHMS1530281  PMID: 31134292

Abstract

Rationale

Pathological amphetamine (AMPH) use is a serious public health concern with no pharmacological treatment options. Protein kinase Cβ (PKCβ) has been implicated in the mechanism of action of AMPH, such that inhibition of PKCβ attenuates AMPH-stimulated dopamine efflux in vivo. With this in mind, inhibition of PKCβ may be a viable therapeutic target for AMPH use disorder.

Objective

The purpose of this study is to demonstrate that selective pharmacological inhibition of PKCβ alters AMPH-stimulated behaviors in rats.

Methods

Rats were administered intracerebroventricular (i.c.v.) injections of the PKCβ-selective inhibitor enzastaurin 0.5, 3, 6, or 18 hr before evaluating AMPH-stimulated locomotion (0.32-3.2 mg/kg). Rats were trained to make responses for different doses of AMPH infusions or sucrose under a fixed ratio 5 schedule of reinforcement, and the effects of enzastaurin pretreatment 3 or 18 hr prior to a self-administration session were determined. Also, the effect of enzastaurin on AMPH-stimulated PKC activity in the ventral striatum was evaluated.

Results

A large dose of enzastaurin (1 nmol) decreased AMPH-stimulated locomotor activity 0.5 hr following enzastaurin administration. Small doses of enzastaurin (10-30 pmol) attenuated AMPH-stimulated locomotor activity and shifted the AMPH dose-effect curve to the right following an 18-hr pretreatment. Rats pretreated with enzastaurin 18, but not 3, hr prior to a self-administration session showed a decrease in the number of responses for AMPH, shifted the ascending limb of the amphetamine dose effect curve, and produced no change in responses for sucrose. AMPH-stimulated PKC activity was decreased following a 0.5 or 18 hr pretreatment, but not a 3 hr pretreatment of enzastaurin.

Conclusions

These results demonstrate that inhibition of PKCβ will decrease AMPH-stimulated behaviors and neurobiological changes and suggest that PKCβ is potentially a viable target for AMPH use disorder.

Keywords: Amphetamine, protein kinas Cβ, behavior, self-administration, addiction

Introduction

Amphetamines (AMPHs) are a class of stimulants that are highly abused worldwide; this class includes amphetamine, methamphetamine, and 3,4-methylenedioxymethamphetamine (UNODC 2017). AMPHs are commonly prescribed to children in the form of Adderall for the treatment of attention deficit/hyperactivity disorders (Lakhan and Kirchgessner 2012) but are also commonly misused for non-medical purposes (Johnston et al. 2016; SAMHSA 2014). Long term use of AMPH in patients with attention deficit/hyperactivity disorder has been shown to be relatively safe when properly prescribed and used. However, long term misuse of AMPH may lead to cognitive deficits and psychosis (Janowsky and Risch 1979; Lakhan and Kirchgessner 2012; Ornstein et al. 2000). Acute effects of AMPH in humans include enhanced attention, alertness, and euphoria (Seiden et al. 1993). Similarly, in laboratory animals, small doses of AMPH produce increased locomotor activity and sustained attention (Grilly et al. 1989; Randrup et al. 1963; Wise and Bozarth 1987). Rodents and monkeys will self-administer AMPH, demonstrating its ability to act as a reinforcer in animal models (Balster and Schuster 1973; Pickens and Harris 1968). Despite the prevalence of AMPH abuse, current treatments are primarily psychological/behavioral in nature and no approved pharmacological interventions are available.

AMPHs elicit their rewarding effects in part by increasing extracellular dopamine levels in the brain via competition with dopamine for uptake into dopaminergic terminal (e.g., dopamine transporter (DAT) substrates). Once in neurons, AMPH reverses the function of DAT, evoking a release of dopamine into the synapse, instead of the removal of dopamine from the synapse (Seiden et al. 1993). AMPH administration also results in an increase in protein kinase C (PKC) activity (Giambalvo 1992). Activation of PKC can lead to increased extracellular dopamine levels through exocytosis and/or via DAT (Cowell et al. 2000; Giambalvo 1988). Inhibiting PKC in vitro, ex vivo, and in vivo attenuates AMPH-stimulated increases in extracellular dopamine levels (Johnson et al. 2005; Kantor and Gnegy 1998; Loweth et al. 2009; Zestos et al. 2016).

Direct injection of PKC β-selective inhibitors into the nucleus accumbens or genetic deletion of PKCβ demonstrated the importance of the β-isomer of PKC in dopamine efflux in response to AMPH as well as its effect on AMPH-stimulated locomotor activity (Chen et al. 2009; Zestos et al. 2016). While there are data demonstrating that PKCβ modulates the neurochemical effects of AMPH, whether or not PKCβ inhibition alters the reinforcing effects of AMPH is unknown. In this study, we characterized the effects of a PKCβ inhibitor on AMPH-stimulated behaviors, namely locomotor activity and self-administration in rodents. We found that enzastaurin, a PKCβ-selective inhibitor (Faul et al. 2003), effectively decreased AMPH-stimulated locomotor activity and AMPH-maintained responding in a surmountable manner and attenuated PKC activity in the presence of AMPH in the striatum. We believe that these data provide proof-of-concept evidence demonstrating the feasibility of selectively targeting PKCβ for the treatment of AMPH abuse.

Methods

Subjects:

Male Sprague-Dawley rats (Harlan Laboratories, Indianapolis, IN) weighing approximately 300-350g at the start of the experiments were single-housed in a temperature and humidity controlled environment. Food was available ad libitum; however, rats used in AMPH and sucrose self-administration experiments were food restricted to approximately 80-90% of their free-feeding weight. All animals were on a 12-hr dark/light cycle with lights on at 0700 and all testing was done during the light phase. All 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’s Institutional Animal Care and Use Committee.

Drug Solutions and Delivery:

D-AMPH (National Institute on Drug Abuse, Bethesda, MD) was dissolved in saline and administered subcutaneously (s.c.) for locomotor studies or intravenously (i.v.) for self-administration studies. Ketamine (Hospira, Lake Forest, IL) and xylazine (Akorn, Lake Forest, IL) were administered intraperitoneally. Enzastaurin was obtained from Cayman Chemicals and dissolved in a vehicle solution containing 0.005% dimethyl sulfoxide (DMSO) in artificial cerebrospinal fluid (Durect, Cupertino, CA).

Enzastaurin or vehicle was administered intracerebroventricularly (i.c.v.) via a programmable pump that administered 10 μl over 1 hr at a rate of approximately 0.17 μl/min. An infusion cannula (2mm C313I, Plastics One, Roanoke, VA) was connected by Tygon tubing to a single channel plastic swivel (375/22PS, Instech Laboratories, Plymouth Meeting, PA) suspended in a counter-balanced arm (PHM-110-SAI, Med Associates, Fairfax, VT), which was attached to a 10 μl Hamilton syringe through Tygon tubing. The rats were awake and in their home cage throughout the course of the infusion.

Surgeries:

Cannula Implantation:

All rats were implanted with a guide cannula (28 gauge with 1.7 mm projection C313GRL/SPC, Plastics One, Roanoke, VA) to allow for i.c.v. injections. Rats were given 5 mg/kg carprofen (s.c.) and ketamine/xylazine (90:10 mg/kg i.p.) and placed in a stereotaxic instrument (Kopf Instruments, Tujunga, CA). The cannula was implanted relative to bregma (AP = −0.8mm, ML=+1.5mm, DV=−2.8mm (Paxinos and Watson 1998)). The guide cannula was held in place with dental cement (OrthoJet-BCA, Lang Dental Manufacturing Co., Wheeling, IL) and anchored with two steel screws (19010-10, Fine Science Tools, Foster City,CA). The guide cannulae were fitted with dummy cannulae with no extension. Following all experiments, cannulae were infused with methylene blue i.c.v. while the rats were under heavy anesthesia for 5 min. The rats were decapitated, and brain tissue was collected and examined for methylene blue distribution throughout the ventricles.

Catheter Implantation:

For AMPH self-administration studies, rats were also implanted with catheters into their left or right femoral vein during a separate surgery. Rats were anesthetized with ketamine/xylazine (90:10 mg/kg i.p.) and given 5 mg/kg carprofen (s.c.). Catheters made of Micro-Renathane tubing (MRE-040, Braintree Scientific, Braintree, MA) were attached to a backmount cannula guide (313-000BM-15-5UP/1/SPC, Plastics One, Roanoke, VA) that exited between the scapulae. Rats were allowed to recover for a minimum of 7 days. The catheters were flushed daily with 0.3-0.5 ml of heparinized-saline (50 U/ml), once daily during recovery and twice daily before and after each self-administration session. Intracranial cannulae were implanted approximately four weeks after catheter implantation.

Locomotor Activity:

Locomotor activity was measured in an acrylic cage (14” × 14” × 8”) containing infrared beams spaced 2.54 cm apart (Opto-M3 Activity Monitor). Experimental data were collected and analyzed using Multi Device Interface Software (Columbus Instruments, Columbus, OH). Rats received an injection of saline (s.c.) and were habituated to the locomotor boxes for 60 min. Following habituation, the rats were given a second injection of saline (s.c.) and placed back in the box for 30 min. AMPH (0.32, 1, or 3.2 mg/kg) was then administered (s.c.) and activity was recorded for an additional 2.5 hr. The rats were pretreated with 0 (vehicle), 1, 10, or 30 pmol or 1 nmol enzastaurin i.c.v. in their home cage either 0.5, 3, 6, or 18 hr before AMPH administration. The number of beam breaks were recorded every min and summed into 10 min bins.

Self-Administration:

Apparatus:

For self-administration studies, rats were placed in operant chambers (ENV-008CT, Med Associates, St. Albans, VT) inside sound-attenuating chambers (ENV-018CT, Med Associates). The operant boxes were outfitted with two nose poke devices each containing a yellow light (ENV-114BM), which were located on either side of a pellet receptacle (ENV-200R7M. Med Associates) attached to a dispenser (ENV-203-45, Med Associates) filled with 45 mg sucrose pellets. A white house light was on the wall opposite the nose poke devices. Drug solutions were delivered via a variable infusion rate syringe pump (PHM-107, Med Associates) connected by Tygon tubing to a single channel plastic swivel (375/22PS, Instech Laboratories, Plymouth Meeting, PA) on a counter-balanced arm (PHM-110-SAI, Med Associates). The Tygon tubing inside the operant chamber was protected with a stainless-steel spring. Data were collected using MED-PC Software (SOF-735, Med Associates).

AMPH Self-Administration:

Rats with i.v. catheters were trained to respond in the nose poke device for infusions of AMPH (0.1 mg/kg/infusion) on a fixed-ratio 1 (FR1) schedule of reinforcement during 60 min daily sessions. Each session began with an infusion of 0.05 ml of drug solution to fill the catheters. The “active” nose poke was illuminated by a yellow light and responses into the active nose poke were recorded. The light in the “inactive” nose poke was not illuminated and responses in the inactive nose poke were recorded but had no scheduled consequence. Completion of a FR in the active nose poke resulted in an infusion (100 μl/kg over approximately 1 sec) with illumination of the house light. This was followed by a 10 sec blackout period during which all stimuli were turned off and responses during the blackout period were recorded but had no consequence. Once the animals responded in a consistent manner for AMPH infusions, the response requirement was gradually increased to an FR5 and the dose of AMPH was decreased to 0.032 mg/kg/infusion. Following stable AMPH self-administration (less than 20% variation in the number of responses and no increasing or decreasing trend in responding over 3 consecutive sessions), saline was repeatedly substituted for AMPH for 1-3 consecutive sessions until responding dropped to less than 30% of stable AMPH responding levels within a single session. All cues were present during the extinction tests. Once responding extinguished in the absence of AMPH, the rats were implanted with cannulae (as described above), then responding maintained by AMPH and extinction in the absence of AMPH were re-confirmed. The rats were pretreated with 10 pmol enzastaurin or vehicle (i.c.v.) 3 or 18 hr prior to an AMPH self-administration session.

A different group of rats were trained to self-administer 0.032 mg/kg/inf AMPH under an FR5 schedule of reinforcement as described above. Following stable AMPH self-administration, the rats were implanted with intracranial cannulae. Upon re-confirming stable responding for AMPH, the rats were switched to a multiple-dose self-administration session. Each daily session was comprised of five 25-min components with a 2-min blackout between each component. Responding during each component resulted in the delivery of different doses of amphetamine delivered in ascending order by altering the infusion duration: 0 (responding recorded with no consequence), 0.0032, 0.01, 0.032, and 0.1 mg/kg/inf AMPH. Following 2-3 consecutive sessions of stable responding, saline was substituted for AMPH for all 5 components in one day to extinguish responding. The AMPH dose effect curve was re-established and then the rats were pretreated with vehicle or 10 pmol enzastaurin (i.c.v.) 18-hr prior to the self-administration session.

Food Self-Administration:

Food self-administration studies were carried out with the same design as the AMPH self-administration sessions with a few exceptions. These rats had cannulae implanted but no catheters. The sessions lasted for 20 min and completion of a FR resulted in the delivery of a single 45 mg sucrose pellet. Instead of saline substitution, responding was extinguished by no delivery of the sucrose pellets. All other cues were present during the extinction test.

Protein Kinase C Activity:

Rats were administered 10 pmol or 1 nmol enzastaurin, or vehicle 0.5, 3, or 18 hr prior to an injection of 3 mg/kg AMPH (s.c.). The rats were euthanized 10-30 min following AMPH and the ventral striatum was dissected. The tissue was immediately frozen in liquid nitrogen, then 250 μl boiling 1% SDS was added to each sample. The samples were sonicated for 5 pulses at frequency of 20 kHz, amplitude 50% (sonic dismembrator, Fisher Scientific, Pittsburgh, PA) and then spun at 14,000 rpm at 4°C, saving the supernatant. The samples (75 μg) were separated by SDS-PAGE on a 12% polyacrylamide gel. The proteins were transferred to a nitrocellulose membrane at 100 mA for 16 hr. The membranes were blocked in a buffer (5% w/v milk, 150 mM NaCl, 10 mM Tris, 0.05% Tween20) before probing for rabbit anti-phosphoser41-growth associated protein 43 (pGAP43) (Santa Cruz Biotechnology Inc., Santa Cruza, CA) and goat anti-GAP43 (Santa Cruz Biotechnology) antibodies for 24 hr at 4°C on two separate membranes. Primary antibody binding was detected with secondary antibodies for 1 hr at room temperature: antibodies for goat anti-rabbit for pGAP43 and donkey anti-goat for total GAP43 (Santa Cruz Biotechnology). The antibodies were imaged with Chemiluminescent Western Substrate (EMD Millipore, Darmstadt, Germany) and band densities were quantified using Image J software.

Statistical Analysis:

Statistical analyses were performed using GraphPad Prism 8.0 (GraphPad Software Inc, San Diego, CA). Locomotor activity data are presented as beam breaks over time and as the area under the curve (AUC) of beam breaks over time for the first 40 min following AMPH administration. Comparisons were made with t-tests, one-way ANOVAs or the Kruskal-Wallis test (for nonparametric tests with uneven group size), two-way ANOVAs, and three-way ANOVAs, as indicated in the results section and/or figure legends. Alpha level was set at 0.05. A Dunnett’s multiple comparison post hoc tests were performed for the dose-effect of enzastaurin studies and the time course of the different pretreatment times, Dunn’s multiple comparison post hoc tests for the different pretreatment time AUC graph, and Sidak’s multiple comparison post hoc tests were used for the locomotor activity time courses following different doses of AMPH, AMPH dose-effect curve for locomotor activity, and single-dose self-administration studies. A Tukeys multiple comparison post hoc test was used for the AMPH dose-effect curve with self-administration.

Results

An 18 hr pretreatment of enzastaurin decreased AMPH-stimulated locomotor activity

A previous study demonstrated that the PKCβ inhibitor enzastaurin administered directly into the nucleus accumbens rapidly reduced AMPH-stimulated locomotor activity (Zestos et al. 2016). To further characterize the effect of PKCβ inhibition on AMPH-stimulated behaviors, we examined whether enzastaurin given i.c.v. altered AMPH-mediated locomotor activity. In the current study, the data are expressed as the number of beam breaks over time (Fig. 1a,c,e) and also summarized as the AUC of the beam breaks over time (Fig. 1b,d,f). A small dose of enzastaurin (10 pmol) administered i.c.v. as a 30 min pretreatment did not alter AMPH-stimulated locomotor activity; however, 1 nmol enzastaurin given i.c.v. as a 30 min pretreatment significantly decreased AMPH-stimulated locomotor activity as compared with a vehicle pretreatment (Fig. 1a,b). A two-way ANOVA demonstrated a significant interaction [F (24,168) = 2.19, p = 0.002] and significant main effects of time [F (12, 168) = 61.86, p < 0.0001] and enzastaurin dose [F (2,14) = 8.36, p = 0.004] (Fig. 1a). In data converted to AUC, a one-way ANOVA revealed rats treated with 1 nmol enzastaurin had significantly lower levels of locomotor activity compared with vehicle treated rats [F (2,14) = 6.02, p = 0.01] (Fig. 1b).

Fig. 1.

Fig. 1

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Time- and dose-dependent reduction of AMPH-stimulated locomotion by enzastaurin. Baseline activities for time course graphs (a, c, e) were calculated from the average number of beam breaks/10 min following an injection of saline prior to receiving AMPH. Data are presented as the number of beam breaks in 10 min bins over time. (a) Rats were injected i.c.v. with vehicle (open circles), 10 pmol enzastaurin (closed circles), or 1 nmol enzastaurin (closed triangles) 0.5 hr prior to 1 mg/kg AMPH s.c. (n = 5-6). * p < 0.05 1 nmol vs. vehicle. (b) Data are summarized as the AUC of beam breaks over 40 min following AMPH administration. (c) Rats were injected with vehicle (open circles) or 10 pmol enzastaurin (i.c.v.) 0.5 (closed triangles), 3 (gray diamonds), 6 (gray triangles), and 18 (closed circles) hr prior to 1 mg/kg AMPH s.c. (n = 6-7 for all groups except vehicle n = 20). * p < 0.05, **** p < 0.0001 18 hr vs. vehicle pretreatment. Data with 10 pmol enzastaurin pretreatment at 0.5 h are the same data shown in Figure 1a. (d) Data are further summarized as AUC of beam breaks over time. * p < 0.05 vs vehicle. (e) Rats were injected with 0 (vehicle-open circles), 1 (closed squares), 10 (closed circles), or 30 (closed diamonds) pmol of enzastaurin i.c.v. 18 hr prior to 1 mg/kg AMPH s.c. (n = 6-7). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 10 pmol vs. vehicle pretreatment, $ p < 0.05, $$ p < 0.01, $$$ p < 0.001 30 pmol vs. vehicle pretreatment. Data with 10 pmol enzastaurin pretreatment are the same 18 hr pretreatment data shown in Fig 1c. (f) Enzastaurin dose-effect summarized as AUC. * p < 0.05, ** p < 0.01 vs. vehicle.

To assess any slow onset or delayed effects of PKCβ inhibition, vehicle or 10 pmol enzastaurin was administered i.c.v. to rats 0.5, 3, 6, and 18 hr prior to injection of 1 mg/kg AMPH i.p. The levels of AMPH-stimulated locomotor activity in vehicle-pretreated rats did not significantly change with the pretreatment time and were compiled into the group labeled vehicle. Interestingly, an 18 hr pretreatment, but not 0.5, 3, or 6 hr pretreatment), with 10 pmol enzastaurin significantly decreased AMPH-stimulated locomotor activity. A two-way ANOVA indicated a significant interaction between the enzastaurin pretreatment time and time post-AMPH [F (48,492) = 1.79, p = 0.001] and significant main effects of time post-AMPH [F (12,492) = 112.4, p < 0.0001] and enzastaurin pretreatment time [F (4,41) = 2.63, p = 0.05] (Fig. 1c). In data converted to AUC, locomotor activity in rats administered enzastaurin 18 hr (p=0.03) but not 0.5, 3, or 6 hr, prior to AMPH was significantly different from rats pretreated with the vehicle [Kruskal-Wallis test (p = 0.05)] (Fig. 1d).

Different doses of enzastaurin (0-30 pmol) were given to rats i.c.v. 18 hr before 1 mg/kg AMPH s.c. The larger doses of enzastaurin (10 and 30 pmol) significantly decreased AMPH-stimulated locomotor activity. There was a significant interaction between pretreatment dose and time [two-way ANOVA: F (36,252) = 1.82, p=0.005] (Fig. 1e). As compared with vehicle-treated rats (Fig. 1f), enzastaurin given 18 hr prior to AMPH decreased AMPH-stimulated locomotor activity [one-way ANOVA: F (3,21) = 6.02, p = 0.004] at 10 pmol (p=0.003) and 30 pmol (p=0.04).

Large doses of AMPH surmounted inhibition induced by enzastaurin

AMPH-stimulated locomotor activity displays an inverted U-shaped dose-effect curve (Rosenzweig-Lipson et al. 1997). The effect of enzastaurin on locomotor stimulation produced by different doses of AMPH was investigated in order to determine potential shifts in the AMPH dose-effect curve (Fig. 2a, b, c, d). These data were further summarized as the AUC of the locomotor activity data over time to generate AMPH dose-effect curves (Fig. 2e). Enzastaurin significantly altered AMPH-stimulated locomotor activity at 1.0 mg/kg AMPH [F(15,180) = 2.321, p = 0.0048] (Fig. 2c) and 3.2 mg/kg AMPH (F(15,150) = 2.341, p = 0.0049] (Fig. 2d). A two-way ANOVA of the dose-effect curve (Fig. 2e) determined that there was a significant interaction between dose of AMPH and enzastaurin pretreatment [F(3,42) = 12.05, p < 0.0001]. Enzastaurin did not alter locomotor activity in the absence of AMPH. Locomotor activity was significantly decreased at 1 mg/kg AMPH in rats pretreated with enzastaurin (p < 0.0001) and significantly increased at 3.2 mg/kg AMPH in rats pretreated with enzastaurin (p = 0.006) as compared with vehicle. Overall, enzastaurin appeared to produce a rightward shift in the AMPH dose response curve.

Fig. 2.

Fig. 2

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Enzastaurin pretreatment shifts the dose effect curve of AMPH. Rats were pretreated with vehicle (open circles) or 10 pmol enzastaurin (closed circles) i.c.v. 18 hr prior to AMPH. Locomotor activity was measured for 2 hr following administration of (a) saline, (b) 0.32, (c) 1, or (d) 3.2 mg/kg AMPH (s.c.) (n = 6-7 per treatment). Data are presented in number of beam breaks in 10 min bins over time. (e) Beam breaks over time were converted to AUC following AMPH or saline administration. ** p < 0.01, *** p < 0.001, **** p < 0.0001 vehicle vs. enzastaurin.

Enzastaurin reduced AMPH-maintained responding without altering responding for sucrose

To determine if a PKCβ inhibitor altered the reinforcing effects of AMPH, we examined the effect of enzastaurin on AMPH-maintained responding. Rats were trained to respond for a 0.032 mg/kg/infusion AMPH on an FR5 schedule of reinforcement and to extinguish responding in the absence of AMPH. Following acquisition of responding criteria, pretreatments of vehicle or 10 pmol enzastaurin (i.c.v.) were administered 3 and 18 hr prior to a self-administration session. Following an 18-hr pretreatment of enzastaurin (Fig. 3a), the number of responses in the active nose poke were decreased by 80% when compared with vehicle pretreatment and with stable AMPH-maintained responding in the absence of a pretreatment. A two-way ANOVA demonstrated a significant interaction between enzastaurin pretreatment and self-administration condition/stage [F(2,18)=8.4, p= 0.003]. In the absence of AMPH, responding significantly decreased (p<0.0001). An 18 hr pretreatment of enzastaurin significantly decreased AMPH-maintained responding as compared with vehicle pretreatment (p<0.001) to levels similar to that observed in the absence of AMPH. There were no significant differences in the number of active responses following a 3-hr pretreatment of enzastaurin or vehicle as compared with stable AMPH-maintained responding (Fig. 3b). There were no significant differences in inactive responses between rats pretreated with enzastaurin or vehicle at 3 or 18 hr prior to the session (Fig. 3a, b).

Fig. 3.

Fig. 3

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Enzastaurin decreased AMPH-maintained responding. The figures demonstrate responding maintained by AMPH (0.03 mg/kg/injection) under an FR5 schedule of reinforcement and during saline substitution (no AMPH). After achieving stable responding for AMPH and rapid extinction of responding in the absence of AMPH, separate groups of rats were pretreated with vehicle (Veh) or 10 pmol enzastaurin (Enza) either 18 (a) or 3 (b) hr prior to an AMPH self-administration session. Active responses are shown in the top graphs and inactive responses are shown in the bottom graphs. ### p < 0.001 vs. vehicle *** p < 0.001 and **** p < 0.0001 vs. AMPH alone, $$$$ p< 0.0001 vs. No AMPH (n = 5-6).

In control experiments, rats were trained to respond for sucrose pellets under similar conditions to the AMPH self-administration experiments, and we evaluated the effects of enzastaurin administered 18 hr prior to a sucrose self-administration session. A two-way ANOVA revealed a significant main effect of self-administration condition/stage [F(2,20)=125.8, p<0.0001) indicating that responding significantly decreased in the absence of sucrose (Fig. 4). However, there was no significant interaction, demonstrating that enzastaurin pretreatment did not significantly alter sucrose-maintained responding as compared with vehicle pretreatment. There were also no significant differences in the number of inactive responses in rats pretreated with enzastaurin or vehicle.

Fig. 4.

Fig. 4

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Enzastaurin did not decrease sucrose-maintained responding. Figures show responding maintained by sucrose pellets (Food) under an FR5 schedule of reinforcement and during the absence of sucrose (No Food). After achieving stable responding for sucrose and rapid extinction of responding in the absence of sucrose, separate groups of rats were pretreated with vehicle (Veh) or 10 pmol enzastaurin (Enza) 18 hr prior to a sucrose self-administration session. Active responses are shown in the top graph and inactive responses are shown in the bottom graph. **** p < 0.0001 vs. Food alone, $$$$ p< 0.0001 vs. No Food (n = 5-6).

As with locomotor activity, the dose-effect curve for AMPH-maintained responding also displays an inverted-U shape, making it necessary to assess how enzastaurin alters the AMPH dose-effect curve. Two groups of rats were trained to self-administer 0-0.1 mg/kg/inf AMPH across five within-session, sequential components (Fig. 5a, b). The rats were pretreated with vehicle or 10 pmol enzastaurin i.c.v. 18 hr prior to the subsequent self-administration session (Fig. 5c). A three-way ANOVA demonstrated a significant interaction between dose, time (responding before and after the pretreatment within subject), and enzastaurin/vehicle pretreatment [F(4,4) = 7.2, p < 0.0001], and significant interactions between time and pretreatment [F(1,4) = 10.6, p = 0.002] and dose and pretreatment [F(4,4) = 4.2, p = 0.004]. Responding for 0.01 mg/kg/inf AMPH was significantly decreased in enzastaurin-treated rats compared to vehicle (p < 0.0001) and compared to levels of responding prior to the pretreatment (p < 0.0001). The peak level of responding in rats pretreated with enzastaurin was significantly different before and after the enzastaurin pretreatment (p = 0.002). These results indicate that enzastaurin pretreatment results in a rightward and downward shift the dose-effect curve for AMPH self-administration.

Fig. 5.

Fig. 5

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Enzastaurin altered the dose-effect curve for AMPH self-administration. (a) Figure shows the number of responses maintained by AMPH under an FR5 schedule of reinforcement for three consecutive sessions prior to an enzastaurin (Group1-open squares) or vehicle (Group2-open circles) pretreatment. The data show responding over five 25 min components, separated by two min between each component. Responding during each component results in the delivery of 0, 0.0032, 0.01, 0.032, 0.1 mg/kg/inf AMPH, sequentially. (b) Saline was substituted in for AMPH for Group1 (half-filled squares) and Group2 (half-filled circles). (c) Group1 was pretreated with 10 pmol enzastaurin (closed squares) and Group2 was pretreated with vehicle (closed circles) 18 hr prior to responding for AMPH. **** p < 0.0001 enzastaurin vs. vehicle (n = 6).

Enzastaurin decreased PKC activity in AMPH-treated rats

We evaluated whether or not PKC activity was altered at time points corresponding to the enzastaurin-induced changes in AMPH-stimulated locomotor activity and responding for AMPH. Rats were pretreated with vehicle, 10 pmol, or 1 nmol enzastaurin (i.c.v.) at 0.5, 3 or 18 hr prior to receiving AMPH s.c. (Fig. 6). PKC activity was determined by phosphorylation of GAP43 at serine41 (pGAP43), a substrate site selective for PKC (Oehrlein et al., 1996). pGAP43 levels were significantly decreased following 10 pmol by more than 40% and 1 nmol enzastaurin by more than 50% compared with vehicle [one-way ANOVA: F(2,17) = 7.44, p = 0.005] (Fig. 6a). pGAP43 was decreased by over 50% in AMPH-treated rats following an 18-hr pretreatment of 10 pmol enzastaurin as compared with vehicle pretreatment (Fig. 6c) [t-test: p = 0.02]. We did not observe a significant change in GAP43 phosphorylation following a 3-hr pretreatment of 10 pmol enzastaurin as compared with vehicle (Fig. 6b). There was no significant difference in total levels of GAP43 in the striatum after each pretreatment.

Fig. 6.

Fig. 6

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A 30 min and 18-hr, but not a 3-hr, pretreatment with enzastaurin decreased PKC activity in the ventral striatum of AMPH-treated rats. Levels of phosphoser41GAP43 (pGAP43) over total GAP43 (tGAP43) are shown in the ventral striatal tissue of AMPH-treated rats given vehicle/enzastaurin (10 pmol or 1 nmol) 30 min (a), 3 hr (b), or 18 hr (c) prior to collecting the tissue. Data are presented as O.D. pGAP43 /tGAP 43 (% vehicle). * p < 0.05, ** p < 0.01 vehicle vs. enzastaurin (n = 6-7).

Discussion

A role for PKCβ in the behavioral effects of AMPH has been demonstrated using PKC inhibitors in rats (Browman et al. 1998; Carpenter et al. 2017; Zestos et al. 2016) and genetic deletion of PKCβ in mice (Chen et al. 2009). In this study, we sought to establish the consequences of selective inhibition of the PKCβ isoform on the reinforcing effects of AMPH. A highly soluble, brain permeable PKCβ inhibitor would be an ideal tool; however, due to a dearth of such drugs, we chose to administer the PKCβ-selective inhibitor enzastaurin centrally (i.c.v.) to evaluate its effects on amphetamine-induced locomotor activity, amphetamine-maintained behavior, and to take note of any adverse events. We performed an investigation of dose and time dependence of PKCβ inhibition on AMPH-stimulated locomotor activity. Acute enzastaurin treatment attenuated AMPH-stimulated locomotor activity and GAP43 phosphorylation. Interesting, we also discovered that small doses of enzastaurin, which were ineffective at altering acute AMPH-stimulated behaviors, demonstrated delayed effects on AMPH-induced locomotion. An 18-hr pretreatment of 10 pmol enzastaurin i.c.v. decreased AMPH-stimulated locomotor activity. Importantly, we then demonstrated that this same dose of enzastaurin (10 pmol) with an 18-hr pretreatment decreased AMPH-maintained responding to levels observed with saline substitution; however, an 18-hr pretreatment of enzastaurin did not alter sucrose-maintained responding in a separate group of rats. We previously showed that a novel non-selective PKC inhibitor decreased AMPH-maintained responding in rats (Carpenter et al. 2017). All together, we have shown that pharmacological inhibition of PKCβ is effective at decreasing AMPH-stimulated behaviors.

These findings suggest that enzastaurin decreased the reinforcing effects of AMPH. To further probe this interpretation, we evaluated the effects of enzastaurin on an AMPH dose-effect curve determined within session. The ability of enzastaurin to decrease AMPH-maintained behavior was surmountable at high doses of AMPH, but this was not a parallel rightward shift in the amphetamine dose-effect curve. The shift in the AMPH dose-effect curve was either a downward shift or a shift in the ascending limb of the AMPH dose-effect function. This complex shift in the AMPH dose-effect curve may be an artifact of the within session design (and the extent of drug accumulation with multiple injections) and/or due to the complex interaction between the site of action of AMPH and PKCβ. Few studies have evaluated or characterized shifts in drug dose-effect curves following inhibition of one component of the intracellular signaling pathway. These interactions are likely to be multifaceted and potentially dose-dependent. For example, small doses of AMPH may be more dependent on PKCβ signaling mechanisms, whereas large AMPH doses may invoke multiple signaling molecules, cellular actions, and neurocircuits overwhelming the effects of PKCβ. However, future studies would need to explore these effects and shifts in the AMPH dose effect curve in more detail. Altogether, these findings strongly support the hypothesis that inhibition of PKCβ reduces AMPH-mediated behaviors and reinforcing effects without altering sucrose-maintained responding.

A possible alternative explanation for the effect of PKCβ inhibition on the reinforcing effects of AMPH is through the impairment of memory. PKCβ is important for memory through its role in promoting LTP (Colley and Routtenberg 1993; Lovinger et al. 1986; Nogues 1997; Routtenberg et al. 1986; Weeber et al. 2000), and pharmacological inhibition of PKC can cause memory impairment, especially with drug-associated memories (Aujla and Beninger 2003; Cervo et al. 1997; Takashima et al. 1991). This raises the possibility that decreased responding for AMPH in the presence of enzastaurin may be due to impairment of memories associated with self-administration training (e.g., operation of the nose poke device and/or reinforcer contingencies). However, we did not observe alterations in responding for sucrose under similar experimental conditions, suggesting that we are not impairing memory retrieval under these experimental conditions.

We also showed that pharmacological inhibition of PKCβ produced a rightward shift in the AMPH dose effect curve without altering baseline locomotor activity (Fig. 2). Vehicle-pretreated rats displayed the typical inverted-U shaped AMPH dose-effect curve typically seen in adult rats (Campbell et al. 1969). At the measured times, 10 pmol enzastaurin i.c.v. were only effective at decreasing AMPH-stimulated locomotor activity 18 hrs following administration. Notably, the normal inverted U-shaped dose effect curve was not evident in enzastaurin-treated rats, although larger AMPH doses would be needed to probe the shape of this dose effect curve. Increased locomotor activity with the highest dose of AMPH in these rats was likely due to decreased levels of stereotypy in the enzastaurin-treated rats (Del Rio and Fuentes 1969). Possibly, a higher dose of AMPH could reveal the inverted U-shaped curve. These results suggest that 10 pmol enzastaurin shifts the dose-effect curve of AMPH-stimulated locomotor activity to the right, consistent with data from PKCβ deletion or inhibition (Chen et al. 2009). The different shifts in the AMPH dose effect curves between the locomotor and self-administration studies may be due to the differences in AMPH and the experimental design (e.g., single dose bolus s.c. for locomotion vs multiple injections per dose of four doses i.v. for self-administration). However, in both experiments, the effects of enzastaurin were surmounted by larger AMPH doses.

Previous studies and our current results demonstrate that enzastaurin and other PKC inhibitors can have immediate effects on AMPH-induced behaviors when given in large enough doses or when delivered directly into a select brain region (Browman et al. 1998; Loweth et al. 2009; Zestos et al. 2016). Interestingly, our data repeatedly demonstrated that small doses of enzastaurin, given i.c.v. had delayed effects on AMPH-mediated behaviors. This raises the possibility that different mechanisms may mediate the decrease in AMPH-mediated behaviors observed at 18 hr and following acute administration. In order to take a closer look at drug action following the different pretreatment times, we evaluated PKC activity following enzastaurin administration. Previous studies demonstrated that AMPH increases phosphorylation of GAP43, a substrate of PKC, in rat striatal tissue and that PKC inhibitors block this effect (Iwata et al. 1997a, Iwata et al. 1997b). We used AMPH-stimulated GAP43 phosphorylation as a readout of PKC activity to determine how long and short pretreatments of enzastaurin effect PKC activity. Although most PKC isozymes will phosphorylate GAP43, phosphorylation of the protein is especially robust with PKCβ (Oehrlein et al. 1996, Sheu et al. 1990, Young et al. 2002). While it might be assumed that a PKC inhibitor would inhibit all substrates of PKC equally, that is not necessarily true (Carpenter et al. 2017). Because GAP-43 is readily phosphorylated in response to AMPH treatment both in vivo and in vitro (Iwata et al. 1997a, Iwata et al. 1997b), inhibition of AMPH-stimulated PKC activity may be more directed to GAP-43 than to other substrates, as demonstrated by Carpenter et al. (2017). We found that AMPH-stimulated PKC phosphorylation of GAP43 is decreased by i.c.v. enzastaurin administration following a 30 min pretreatment and an 18 hr pretreatment, but not following a 3 hr pretreatment - the same time points when enzastaurin decreases locomotor activity.

While 30 min pretreatment with 10 pmol enzastaurin decreased PKC activity in the ventral striatum, it had no effect on amphetamine-stimulated locomotor activity. There are a couple possible explanations for these unusual results. The assay may not be sensitive enough to differentiate fine changes in PKC activity, or PKCβ inhibition alone in the nucleus accumbens (as measured here) may not be sufficient to decrease amphetamine-stimulated locomotor activity. Locomotor activity is regulated by complex neurocircuitry, and the ventral striatum is only a small snapshot of the brain. Therefore, the dose-dependent actions of enzastaurin in the nucleus accumbens and/or other brain regions may be important for modulating locomotor activity.

It was surprising that enzastaurin decreased PKCβ activity 18 hr after administration. It is unlikely that these delayed effects were due to a long-lasting metabolite of enzastaurin because 1) there was no change in PKCβ activity at 3 hr post-administration, and 2) this is greater than 4-5 times the half-life of the metabolite in rats. While human studies indicated enzastaurin has long-lasting, active metabolites (Carducci et al. 2006), pharmacokinetic studies in Fischer 344 rats following oral administration found the half-life of enzastaurin to be around 2 hr and the half-life of its active metabolite to be 3.6 hr with the lowest dose tested (personal communications with Denovo Biopharma and Eli Lilly). However, metabolism cannot be entirely ruled out, due to differences in route of administration between the current study and the pharmacokinetic studies. Since another structurally-unrelated PKC inhibitor also displays a similar time course of action (Carpenter et al. 2017), we believe that the delayed effects is more likely related to some mechanism of action and not pharmacokinetics. It is possible that the mechanism underlying the long pretreatment effect is due to changes downstream of PKC signaling that ultimately affect PKC activity. Some groups have demonstrated that PKCβ inhibition with enzastaurin can decrease PKCβ promotor activity and PKCβ mRNA levels, which is one possible explanation for a PKC inhibitor’s secondary effect on AMPH-mediated behaviors (Liu et al. 2004). We will be exploring this possibility in future studies.

Taken together, our results show that PKCβ inhibition may be a viable therapeutic option to treat AMPH abuse. A major concern in developing a PKCβ inhibitor as a therapeutic is potential toxic effects due to the ubiquitous nature of PKC. However, clinical studies with enzastaurin and other PKCβ inhibitors demonstrated safe profiles (Vinik et al. 2005; Welch et al. 2007), and our own studies have shown that enzastaurin does not suppress baseline or conditioned behaviors. Other isozymes of PKC may be compensating for the reduction in PKCβ activity in ways that lessen the impact and toxicity.

While we have shown that enzastaurin decreased AMPH-maintained behaviors, we did not evaluate its effects on AMPH-seeking behaviors and motivation for AMPH. Future work will utilize reinstatement procedures and progressive ratio schedules of reinforcement to further evaluate the behavioral effects PKCβ inhibition. More work will also need to be done to further understand the time course and mechanism of PKCβ inhibitors. Additional studies to assess the effect of PKCβ inhibition on AMPH sensitization, additional schedules of reinforcement, and extended-access self-administration will also be useful in assessing PKCβ inhibitors as potential therapeutics. In conclusion, the present findings provide proof-of-concept selective PKCβ inhibitors may be useful for therapeutic interventions for AMPH abuse.

Acknowledgments

Funding: National Institutes of Health grant R01 DA11697, National Institutes of Health Training Grant T32-GM007767, Benedict and Diana Lucchesi Graduate Education Fellowship

Abbreviations:

AMPH

Amphetamine

AUC

Area under the curve

PKC

Protein kinase C

Footnotes

Publisher's Disclaimer: This Author Accepted Manuscript is a PDF file of a an unedited peer-reviewed manuscript that has been accepted for publication but has not been copyedited or corrected. The official version of record that is published in the journal is kept up to date and so may therefore differ from this version.

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