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. Author manuscript; available in PMC: 2022 Dec 15.
Published in final edited form as: Neuropharmacology. 2021 Oct 7;201:108830. doi: 10.1016/j.neuropharm.2021.108830

Ibudilast attenuates cocaine self-administration and prime- and cue-induced reinstatement of cocaine seeking in rats

Lianwei Mu 1, Xiaojie Liu 1, Hao Yu 1,2, Mengming Hu 1, Vladislav Friedman 1, Thomas J Kelly 1, Li Zhao 1,2, Qing-song Liu 1
PMCID: PMC8656241  NIHMSID: NIHMS1749369  PMID: 34626665

Abstract

Ibudilast is a non-selective phosphodiesterase (PDE) inhibitor and glia cell modulator which has shown great promise for the treatment of drug and alcohol use disorders in recent clinical studies, but it is unknown whether and how ibudilast affects cocaine seeking behavior. Here we show that systemic administration of ibudilast dose-dependently reduced cocaine self-administration under fixed- and progressive-ratio reinforcement schedules in rats and shifted cocaine dose-response curves downward. In addition, ibudilast decreased cocaine prime- and cue-induced reinstatement of cocaine seeking. These results indicate that ibudilast was effective in reducing the reinforcing effects of cocaine and relapse to cocaine seeking. Chronic cocaine exposure induces cAMP-related neuroadaptations in the reward circuitry of the brain. To investigate potential mechanisms for ibudilast-induced attenuation of cocaine self-administration, we recorded from ventral tegmental area (VTA) dopamine neurons in ex vivo midbrain slices prepared from rats that had undergone saline and cocaine self-administration. We found cocaine self-administration led to a decrease in inhibitory postsynaptic currents (IPSCs), an increase in the AMPAR/NMDAR ratio, and an increase in the excitation to inhibition (E/I) ratio. Ibudilast pretreatments enhanced GABAergic inhibition and did not further change cocaine-induced potentiation of excitation, leading to normalization of the E/I ratio. Restoration of the balance between excitation and inhibition in VTA dopamine neurons may contribute to the attenuation of cocaine self-administration by ibudilast.

Keywords: ibudilast, cocaine, self-administration, reinstatement, ventral tegmental area, synaptic plasticity

1. Introduction

Ibudilast (3-isobutyryl-2-isopropylpyrazolo-[1,5-a] pyridine) is a non-selective phosphodiesterase (PDE) inhibitor and glia cell modulator with anti-inflammatory activity (Huang et al., 2006; Schwenkgrub et al., 2017). This small molecule drug is used clinically for treatment of asthma in Japan due to its efficacy as a bronchodilator, and it has received “Fast Track” designation by the FDA as a therapy for neuroinflammatory conditions (Dolgin, 2016). Chronic exposure to drugs of abuse such as psychostimulants and opioids causes neuroinflammation resulting from increased glial cell activation and proinflammatory cytokine production in the brain (Beardsley and Hauser, 2014; Hofford et al., 2019; Namba et al., 2021). Accumulating evidence suggests that neuroinflammation contributes to the development of addictive behavior, and drugs that dampen glial activity and neuroinflammation may produce anti-addiction effects (Beardsley and Hauser, 2014; Hofford et al., 2019; Namba et al., 2021). In support of this idea, recent small-scale clinical studies indicate that the glial activity modulator ibudilast reduced cravings for heroin, tobacco, and cocaine and withdrawal-related symptoms following opioid discontinuation (Cooper et al., 2016; Metz et al., 2017). Ibudilast is orally active and well-tolerated in this patient population (Cooper et al., 2016; Metz et al., 2017), and may be repurposed to effectively treat substance use disorders. In animal studies, ibudilast and its analog AV1013 reduced methamphetamine-induced increases in locomotor activity and sensitization (Snider et al., 2012), methamphetamine self-administration (Snider et al., 2013), and prime- and stress-induced methamphetamine relapse in rats (Beardsley et al., 2010). Additionally, ibudilast attenuated the expression of cocaine-induced locomotor sensitization (Snider et al., 2013), and also attenuated morphine withdrawal-induced weight loss and somatic signs of withdrawal while enhancing the acute analgesic potency of morphine and oxycodone (Metz et al., 2017). Lastly, ibudilast and other PDE4 inhibitors reduce alcohol drinking in mice (Bell et al., 2015; Liu et al., 2017a) and humans (Grodin et al., 2021). However, it remains unknown if ibudilast affects cocaine self-administration and seeking. The present study will examine the effects of ibudilast pretreatments on cocaine self-administration, cocaine primed- and cue-induced reinstatement of cocaine seeking, then investigate the potential mechanisms involved.

PDEs are a family of enzymes (PDE1–11) that hydrolyze intracellular cAMP and cGMP (Conti et al., 2003). Ibudilast is a non-selective phosphodiesterase (PDE) inhibitor (PDE3, 4, 10, 11) with preferential inhibition of PDE4 (Kishi et al., 2001), an enzyme that selectively hydrolyzes cAMP (Conti et al., 2003). Dysregulation of cAMP signaling is linked to the development of dependency for many abused substances including cocaine (Chen et al., 2012; Raghavendra et al., 2004). PDE4 is expressed in mesolimbic dopamine system including the ventral tegmental area (VTA), prefrontal cortex (PFC), and nucleus accumbens (NAc) (Filiano et al., 2015). In previous studies, the mechanisms of modulating drug seeking behavior with ibudilast have been focused on its anti-inflammation activities (Beardsley and Hauser, 2014; Beardsley et al., 2010). As a PDE4 inhibitor, ibudilast is expected to increase cAMP levels in the brain (Huang et al., 2006). It has been shown that cAMP enhances neurotransmitter release and mediates long-term synaptic plasticity at many excitatory and inhibitory synapses (Capogna et al., 1995; Chavez-Noriega and Stevens, 1994; Chen and Regehr, 1997; Kaneko and Takahashi, 2004). Drugs of abuse induce neuroadaptations in the reward circuitry of the brain that might underlie the formation of addictive behavior (Dong et al., 2017; Hearing et al., 2018). We and others have shown that non-contingent cocaine exposure in vivo leads to a reduction of GABAergic inhibition and potentiation of glutamatergic excitation to VTA dopamine neurons (Bocklisch et al., 2013; Liu et al., 2005). In the present study, we determined whether the administration of ibudilast in vivo altered cocaine self-administration-induced inhibitory and excitatory synaptic plasticity in VTA dopamine neurons ex vivo. We found that cocaine self-administration led to a decrease in GABAergic inhibition and potentiation of glutamatergic excitation to VTA dopamine neurons, while ibudilast pretreatments prevented cocaine-induced reductions in GABAergic inhibition and did not further increase cocaine-induced potentiation of excitation, leading to a restoration of the balance between excitation and inhibition. These results reveal a potential mechanism for ibudilast-induced attenuation of cocaine self-administration.

2. Materials and methods

2.1. Animals

Male Long-Evans rats (8–10 weeks old) were purchased from Envigo (Indianapolis, IN). All animals were given ad libitum access to food and water in a room with controlled temperature (23 ± 1°C) and humidity (40–60%) on a reverse light-dark cycle. Rats were handled daily for 3–6 days prior to experiments. All behavioral experiments were completed during the dark cycle. Independent cohorts of rats were utilized for fixed ratio (FR), progressive ratio (PR), multiple-dose cocaine self-administration, electrophysiology recordings, cocaine prime-induced reinstatement, cue-induced reinstatement, and sucrose self-administration experiments. Rats were single-housed after intravenous catheterization to prevent cage-mates from damaging the implanted catheters. All animal maintenance and use were in accordance with protocols approved by the Institutional Animal Care and Use Committee of the Medical College of Wisconsin.

2.2. Chemical reagents

Cocaine HCl and ibudilast were provided by the NIDA Drug Supply Program. Cocaine was dissolved in sterile saline (Midwest Veterinary Supply, Sun Prairie, WI), diluted to unit doses appropriate for behavioral studies, and filtered through a 0.2 μm membrane prior to use. Ibudilast was first dissolved in PEG 200 (final concentration 35%, Electron Microscopy Sciences, Hatfield, PA), then further mixed with 10% Cremophore EL (Sigma-Aldrich), and 55% sterile saline with gentle heating (Poland et al., 2016). Picrotoxin and all other common chemicals were obtained from Sigma-Aldrich (St. Louis, MO). 6-cyano-7-nitroquinoxaline-2,3-dione disodium salt, (R)-CPP and H89 dihydrochloride were obtained from Tocris Bioscience (Ellisville, MO).

2.3. Jugular catheterization surgery

Rats were anesthetized with ketamine (90 mg/kg, i.p.) and xylazine (10 mg/kg, i.p.), and an intravenous catheter (MicroRenathane tubing with ID = 0.025 inches, OD = 0.040 inches; Braintree scientific, Inc, MA) was inserted into the right jugular vein (3.5 cm). The catheter was connected to a cannula assembly, consisting of a stainless-steel cannula (22-gauge; Plastics One, Roanoke, VA) mounted on a dental cement base made with nylon mesh, and implanted subcutaneously on the back of the rats. After the surgery, animals received a single subcutaneous injection of buprenorphine-SR (1 mg/kg), an analgesic. Catheters were flushed daily with 0.2 ml of heparinized saline (30 units/ml) and cefazolin (100 mg/ml). Rats were allowed to recover for 5–7 days prior to the start of self-administration experiments. Catheter patency was tested by intravenous infusion of 0.05 ml xylazine (20 mg/ml) every week after cocaine self-administration or when compromise of catheter patency was suspected (Wright et al., 2017).

2.4. Cocaine self-administration training

Cocaine self-administration was performed based on published procedures with minor modifications (Xi et al., 2008). Rats were first trained under a FR1 reinforcement schedule to facilitate the acquisition of cocaine self-administration (3-hour sessions). Each active lever press resulted in one cocaine infusion (FR1: 1 mg/kg/infusion; 40–50 μl over 2.3–2.8 sec based on the weight of the rats) and illumination of the cue light above the active lever for 5 sec, followed by a 10 sec timeout. Lever presses during timeout were counted but had no consequence. After 5 days of training, the rats were advanced to a FR2 reinforcement schedule (2 active lever presses resulted in one cocaine delivery, 0.5 mg/kg/infusion) until stable cocaine self-administration was established. Stable cocaine self-administration was defined as: ≥ 25 infusions and > 2:1 active/inactive response ratio for three consecutive days, and less than 20% variability in daily drug infusions across two consecutive sessions. The session ended early if 64 reinforcers were earned to avoid overdose. Animals that did not achieve the criteria after 10 days of FR2 training were excluded from further study. After stable cocaine self-administration was achieved, the effects of ibudilast on cocaine self-administration under FR2, PR schedules of reinforcement and multiple-dose cocaine self-administration were evaluated in different cohorts.

2.5. Examination of the effects of ibudilast on cocaine self-administration under FR2 reinforcement

The day following completion of the 10-day cocaine self-administration training, rats were injected with ibudilast (1, 3 or 10 mg/kg, i.p.) or vehicle (0 mg/kg) 30 minutes prior to a cocaine self-administration session under FR2 reinforcement in a Latin Square design. Following each drug treatment, rats were allowed to self-administer cocaine until responding restabilized as defined above, rats were then tested with subsequent doses of ibudilast in a within-subjects design.

2.6. Examination of the effects of ibudilast on cocaine self-administration under PR reinforcement

Rats were first trained to self-administer cocaine under FR1 and FR2 reinforcement schedules for 10 days, as outlined above. After stable cocaine self-administration was established, a PR reinforcement schedule was introduced under which the first active lever press resulted in a 0.5 mg/kg cocaine infusion but the number of lever pressed required for subsequent infusions successively increased according to the equation: Response ratio = [5e(injection number × 0.18)] – 5, following published procedure (Richardson and Roberts, 1996). The PR breakpoint was defined as the maximum number of lever presses completed for the last cocaine infusion prior to a 1-hour period in which no infusions were obtained. After the PR breakpoint was achieved and stabilized (defined as < 15% variability in the number of infusions over 3 days), rats were pretreated with ibudilast (3 or 10 mg/kg, i.p.) or vehicle (0 mg/kg). Cocaine self-administration under PR reinforcement was tested 30 min later, and subsequent doses were tested as described above.

2.7. Examination of the effects of ibudilast on multiple-dose cocaine self-administration

Rats were first trained to self-administer cocaine under FR1 and FR2 schedules as described in Methods. After criteria for stable cocaine-maintained responding were met, rats were switched to multiple-dose cocaine self-administration maintained by a full range of cocaine doses (0, 0.03125, 0.0625, 0.125, 0.25, 0.5, 1.0 mg/kg/infusion) in a single dose-response session under FR2 reinforcement schedule (Keck et al., 2014; Xi et al., 2017). Training sessions under the multiple-dose schedule began with a 25-min extinction trial (0 mg/kg/infusion of cocaine), then another six 25-min trials in which different doses of cocaine were presented in an ascending order; each dose trial was separated by a 5-min intertrial time-out period. Cocaine doses were determined by adjusting cocaine concentrations (0.5 mg/ml and 4 mg/ml) and the infusion durations (i.e., volume). For lower cocaine doses (0, 0.03125, 0.0625, 0.125 mg/kg/infusion), the drug solution consisted of 0.5 mg/ml cocaine, 0, 25, 50, and 100 μl injections were delivered in 0, 0.72, 1.44 and 2.88 sec, respectively. For higher cocaine doses (0.25, 0.5, 1.0 mg/kg/infusion), the drug solution consisted of 4 mg/ml cocaine so that 25, 50, and 100 μl injections were delivered in 0.72, 1.44 and 2.88 sec, respectively. Ibudilast pretreatment testing began once cocaine self-administration behavior stabilized, and stability was defined as (1) a minimum of 5 mg/kg cocaine intake within a session and less than 20% variation in total number of cocaine infusions for 2 consecutive test sessions; (2) the dose of cocaine that maintained maximal response rates varied by no more than one-half log unit over two consecutive test sessions (Keck et al., 2014; Xi et al., 2017). Rats received vehicle (0 mg/kg) or ibudilast (1, 3 mg/kg, i.p.) 30 minutes prior to the test session, the order of testing for the various doses of ibudilast or vehicle was counterbalanced. Following each vehicle or ibudilast treatment, rats were allowed to self-administer under the multiple-dose cocaine schedule until responding restabilized as defined above before subsequent doses of ibudilast or vehicle were tested.

2.8. Slice electrophysiology

Four groups of rats received saline and cocaine self-administration (FR1/FR2) for 10 days as described above. On the 11th day, rats received i.p. injection of either vehicle (0 mg/kg) or ibudilast (10 mg/kg) in a 2 × 2 factorial design and were tested under FR2 reinforcement schedule. The next day, rats were anesthetized by isoflurane inhalation and perfused through the aorta with artificial cerebrospinal fluid (ACSF, 4–6°C) containing (in mM): 119 NaCl, 2.5 KCl, 2.5 CaCl2, 1 MgCl2, 1.25 NaH2PO4, 26 NaHCO3, and 10 glucose. The brain was removed, trimmed, and embedded in low-gelling-point agarose, and horizontal slices (200 μm thick) containing the VTA were cut using a vibrating slicer (Leica VT1200s, Nussloch, Germany), as described in our recent studies (Vickstrom et al., 2020). Slices were prepared in a cutting solution containing the following (in mM): 110 choline chloride, 2.5 KCl, 1.25 NaH2PO4, 0.5 CaCl2, 7 MgSO4, 26 NaHCO3, 25 glucose, 11.6 sodium ascorbate, and 3.1 sodium pyruvate. The VTA slices were cut at the midline to produce two individual slices from each section. After slice cutting, ACSF was progressively spiked into the choline solution every 5 min for 20 min at room temperature to gradually reintroduce Na+, similar to a previous method (Ting et al., 2018). The slices were allowed to recover for at least an additional 30 min in ACSF prior to recording. All solutions were saturated with 95% O2 and 5% CO2.

Whole-cell recordings were performed from VTA dopamine neurons, which were identified by a broad triphasic extracellular action potential with a width > 2 ms and a relatively slow firing rate (<10 Hz) mode, and the presence of Ih current (Chieng et al., 2011; Johnson and North, 1992; Jones and Kauer, 1999; Liu et al., 2005; Melis et al., 2013; Melis et al., 2009). Spontaneous inhibitory postsynaptic currents (sIPSCs), evoked IPSCs, AMPAR/NMDAR ratio, and excitation/inhibition (E/I) ratio were measured based on published studies (Liu et al., 2016; Liu et al., 2017b). Whole-cell and cell-attached patch clamp recordings were made from VTA dopamine neurons using patch-clamp amplifiers (Multiclamp 700B) under infrared differential interference contrast (DIC) microscopy. Data acquisition and analysis were performed using DigiData 1440A and 1550B digitizers and the analysis software pClamp 10.7 (Molecular Devices). Signals were filtered at 2 kHz and sampled at 10 kHz. For recording spontaneous inhibitory postsynaptic currents (sIPSCs, Fig. 2) and evoked IPSCs (Fig. 3), glutamate receptor antagonists 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and (R)-CPP (10 μM) were present in the ACSF throughout the experiments and dopamine neurons were voltage-clamped at −70 mV unless otherwise specified. Glass pipettes (3–5 MΩ) were filled with an internal solution containing (in mM): 90 K-gluconate, 50 KCl, 10 HEPES, 0.2 EGTA, 2 MgCl2, 4 Mg-ATP, 0.3 Na2GTP, and 10 Na2-phosphocreatine, pH 7.2 with KOH. For recording of evoked excitatory postsynaptic currents (EPSCs) or IPSCs, electrical stimulation was delivered by a bipolar tungsten stimulation electrode (WPI) that was placed at fixed distance (~150 μm) rostral to the soma of the recorded dopamine neuron. The stimulation strength was set to 30–40 μA, which yielded EPSCs or IPSCs of 20–30% maximum amplitude. AMPA/NMDA receptor (AMPAR/NMDAR) ratio was measured based on published studies (Liu et al., 2016). GABAA receptor antagonist picrotoxin (100 μM) was present in the ACSF. Neurons were voltage-clamped at +40 mV to record dual component EPSCs containing both AMPAR- and NMDAR-EPSCs that were isolated pharmacologically. After a stable baseline recording of total EPSCs, NMDAR antagonist (R)-CPP (10 μM) was applied in the bath for 6–10 min to isolate fast AMPAR-EPSCs. NMDAR-EPSCs were calculated as the subtraction of AMPAR-EPSCs from the total EPSCs from the same neuron. An average of 10–20 consecutive EPSCs was collected for each type of EPSCs. AMPAR/NMDAR ratio was calculated by dividing the peak of the AMPAR-EPSCs by the peak of the NMDAR-EPSCs. For recording of EPSCs/IPSCs (E/I) ratio, NMDAR antagonist (R)-CPP (10 μM) was present throughout the experiment. VTA dopamine neurons were voltage-clamped at the reversal potential for IPSCs (−60 mV) and EPSCs (0 mV) to isolate EPSCs and IPSCs, respectively. The E/I ratios were calculated by dividing the amplitude of EPSCs by the amplitude of IPSCs. For recordings of AMPAR/NMDAR ratio and E/I ratio, glass pipettes (3–5 MΩ) were filled with an internal solution containing (in mM): 130 Cs-methanesulfonate, 10 CsCl, 10 HEPES, 1.1 EGTA, 2 MgCl2, 4 MgATP, 0.3 Na2GTP, and 10 Na2-phosphocreatine, pH 7.2 with CsOH. Series resistance (10–20 MΩ) was monitored throughout all recordings, and data were discarded if the resistance changed by more than 20%. All recordings were performed at 32 ± 1°C by using an automatic temperature controller (Warner Instruments, Inc.).

Fig. 2: Ibudilast pretreatments blocked the cocaine self-administration induced reduction of GABAergic inhibition in dopamine neurons.

Fig. 2:

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A Timeline of self-administration training, vehicle or ibudilast treatment and electrophysiology. B Representative sIPSCs recorded from VTA dopamine neurons in slices prepared from saline- or cocaine-self-administration rats pre-treated with vehicle or ibudilast. C,D The averaged frequency (C) and amplitude (D) of sIPSCs in VTA dopamine neurons in these four groups of rats. The mean frequency and amplitude of sIPSCs were significantly decreased in cocaine self-administration vehicle-treated rats (**p < 0.01, n = 13), and this decrease was blocked by ibudilast pretreatments (**p < 0.01, n = 13–14). E,F The cumulative probability plots indicated that cocaine self-administration led to shifts in the distribution of the inter-event intervals (E) and amplitude (F) in vehicle-treated rats; these shifts were blocked by ibudilast pretreatments (p < 0.001, n = 13–14). Each data set was obtained from 3–4 rats.

Fig. 3. Ibudilast potentiated IPSCs by enhancing cAMP/PKA signaling.

Fig. 3.

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A Timeline of self-administration training and electrophysiology. B Bath application of ibudilast (10 μM) increased the amplitude of evoked IPSCs in slices from rats that had undergone saline and cocaine self-administration (p < 0.001, n = 7), there was no significant difference between these two groups (p > 0.05, n = 7). C The ibudilast-induced potentiation was blocked by the PKA inhibitor H89 (1 μM; p < 0.001 vs. Ibudilast alone, n = 6–7) in either group. Each data set was obtained from 2–3 rats.

2.9. Examination of the effects of ibudilast on sucrose self-administration

The procedures for oral sucrose self-administration testing were identical to the procedure used for cocaine self-administration, except for the following: (1) no surgery was carried out in this experiment; and (2) active lever presses led to delivery of a reward of sucrose pellets (40 mg). Briefly, rats underwent daily sessions of 3-hour training in which they learned to lever press to earn a reward of sucrose pellets (40 mg) under a FR1 reinforcement schedule. Active lever presses resulted in delivery of a sucrose pellet and illumination of 5 sec light cue and 10 sec reward timeouts. All inactive lever presses were recorded but had no programmed consequence. Training sessions were terminated if 64 reinforcers were obtained. After 5 days of training, rats advanced to a FR2 reinforcement schedule. Stable sucrose self-administration was defined as the following: ≥ 25 intakes and > 2:1 active/inactive response ratio for three consecutive days, and less than 20% variability in daily drug infusions across two consecutive sessions. All rats reached the criteria after 10 days of training. On subsequent days, rats were injected with vehicle (0 mg/kg, i.p.) or ibudilast (1, 3 or 10 mg/kg, i.p.) 30 min prior to a test session under FR2 reinforcement in a Latin Square design. Following drug treatment, rats self-administered sucrose as before until responding returned to stable levels, using the above criteria. Subsequent doses of ibudilast or vehicle were then tested in a within-subjects design.

2.10. Examination of the effects of ibudilast on cocaine prime-induced reinstatement of cocaine seeking

To facilitate the acquisition of cocaine self-administration, rats were first trained to lever-press for sucrose pellets under FR1 reinforcement schedule over the course of 5 consecutive days. Rats were switched to cocaine self-administration under FR1 reinforcement schedule (0.5 mg/kg/infusion) for 5 days and FR2 reinforcement schedule (0.5 mg/kg/infusion) for 5 days. Rats that established stable cocaine self-administration underwent extinction training in which lever presses (without limitation) were recorded but cocaine delivery and cue light activation were disabled. Each extinction session lasted for 3 hours. After 7–10 days of extinction training, drug-seeking behavior was extinguished, which was defined as: ≤ 20 active lever presses during each 3-hour session for at least 3 consecutive days. One day after achieving extinction criteria, the effects of ibudilast pretreatments on reinstatement of drug-seeking behavior triggered by cocaine prime was examined. Rats with extinguished drug-seeking were pretreated with ibudilast (10 mg/kg, i.p.) or vehicle (0 mg/kg, i.p.) 30 min prior to a cocaine prime-induced reinstatement trial. Before the start of the test, rats were injected with cocaine (10 mg/kg, i.p) and placed into the same operant chambers, and drug-seeking behavior was tested for 3 hours. As in extinction sessions, active lever presses were recorded without drug infusions or accompanying cues.

2.11. Examination of the effects of ibudilast on cue-induced reinstatement of cocaine seeking.

Rats were first trained to acquire and then extinguish cocaine self-administration behavior, as described above for cocaine prime-induced reinstatement, but in an independent cohort. Rats with extinguished drug-seeking were pretreated with ibudilast (10 mg/kg, i.p.) or vehicle (0 mg/kg, i.p.) 30 min before a cue-induced reinstatement trial performed in the same operant chambers. The cue-induced drug seeking session began with a 5 s presentation of the cocaine-conditioned light cue, and active lever presses resulted in further light cues but no cocaine infusions.

2.12. Statistics

Data are presented as the mean ± SEM. Data sets were compared with either Student’s t-test, one-way or two-way ANOVA followed by Tukey’s post hoc analysis, or two-way repeated measures ANOVA. Post hoc analyses were performed only when ANOVA yielded a significant main effect or a significant interaction between the two factors. As the analysis of breakpoints for the progressive ratio violates the assumption of homogeneity of variance(Richardson and Roberts, 1996), we used Kruskal-Wallis one-way ANOVA on ranks to compare the effects of vehicle and different doses of ibudilast on breakpoints, followed by Dunn’s post hoc analysis for pair-wise comparisons. The cumulative distributions for inter-event intervals and amplitude of sIPSCs from different treatment groups were compared with Kolmogorov-Smirnov test (K-S test). Results were considered to be significant at p < 0.05.

3. Results

3.1. Ibudilast dose-dependently decreased cocaine intake and motivation to self-administer cocaine

We first sought to determine the effects of ibudilast on cocaine self-administration under FR2 reinforcement schedule in rats. To facilitate acquisition of cocaine self-administration, rats were trained to self-administer 1 mg/kg/infusion cocaine under FR1 reinforcement schedule for 5 days, followed by 0.5 mg/kg/infusion under FR2 reinforcement schedule for an additional 5 days (see Materials and Methods). Three rats were removed from the study due to loss of catheter patency and failure to acquire stable cocaine self-administration. The remaining rats rapidly learned to discriminate the active lever from the inactive lever and acquired stable cocaine self-administration within the 10-day training period (Fig.1A). On subsequent days, the effects of ibudilast were tested under a FR2 reinforcement schedule. The rats were injected with ibudilast (1, 3 or 10 mg/kg, i.p.) or vehicle (0 mg/kg, i.p.) in a Latin Square design, 30 min prior to self-administration testing. Ibudilast doses and pretreatment times were based on previous studies (Poland et al., 2016) and our own pilot experiments. Ibudilast pretreatments produced a significant and dose-dependent suppression of active lever presses (F3,28 = 14.0, p < 0.001; Fig. 1B) and cocaine infusions (F3,28 = 12.0, p < 0.001; Fig. 1D) but had no significant effect on inactive lever presses (F3,28 = 0.08, p = 0.970; Fig. 1C). Tukey’s post hoc tests revealed that ibudilast pretreatments dose-dependently reduced active lever presses (p < 0.001; Fig. 1B) and cocaine infusions (p < 0.01; Fig. 1D) at both 3 mg/kg and 10 mg/kg doses compared with vehicle pretreatment.

Fig. 1: Ibudilast dose-dependently decreased cocaine intake and motivation to self-administer cocaine and shifted cocaine dose-response curve downward.

Fig. 1:

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A The number of active lever presses, inactive lever presses and cocaine infusions during FR1 and FR2 training. B,C,D Ibudilast pretreatments produced a significant and dose-dependent suppression of active lever presses (B; ***p < 0.001, n = 6–10 rats) and cocaine infusions (D; **p = 0.001, ***p < 0.001, n = 6–10 rats), but did not alter the number of inactive lever presses (C; p > 0.05, n = 6–10 rats). E Ibudilast pretreatments significantly attenuated the number of cocaine infusions under PR reinforcement (*p = 0.048, ***p < 0.001, n = 7–9 rats). F Ibudilast pretreatments significantly attenuated the PR breakpoint in rats (***p < 0.001, n = 7–9 rats). G Ibudilast pretreatments produced a significant downward shift in the cocaine dose-response curve (3 mg/kg vs 0 mg/kg, **p < 0.01, ***p < 0.001; 1 mg/kg vs 0 mg/kg, #p < 0.05, ##p < 0.01, ###p < 0.001; n = 8 rats).

We next examined whether ibudilast affected cocaine self-administration under a PR schedule, in which rats were required to make an increasing number of lever presses for each successive reward (cocaine infusion) until a breakpoint level is achieved. Rats underwent 10-day cocaine self-administration training under FR1/FR2 reinforcement schedules as described above, then were switched to a PR reinforcement schedule at 0.5 mg/kg/infusion cocaine. When PR breakpoints were stabilized for 3 consecutive days, the effects of ibudilast (3 or 10 mg/kg, i.p.) or vehicle (0 mg/kg, i.p.) on cocaine infusions and breakpoints were examined. One-way ANOVA indicated significant effects of ibudilast on cocaine infusions (F2, 23 = 12.4, p < 0.001). Tukey’s post hoc tests revealed that ibudilast pretreatments significantly reduced cocaine infusions (3 mg/kg, p = 0.048; 10 mg/kg, p < 0.001; Fig. 1E) compared with vehicle pretreatments. As breakpoint data failed the Brown-Forsythe equal variance test (p < 0.05), we performed non-parametric Kruskal-Wallis one-way ANOVA on ranks and the results showed that ibudilast decreased the breakpoints for cocaine in a dose-dependent manner (H = 14.248, p < 0.001; Fig. 1F). Dunn’s post hoc tests revealed that breakpoints were reduced by ibudilast pretreatments at 10 mg/kg (p < 0.001) but not at 3 mg/kg (p = 0.250) compared with vehicle pretreatments. There was no significant difference on breakpoints between 3 and 10 mg/kg ibudilast doses (p = 0.126). Thus, ibudilast also reduces cocaine self-administration when the workload (active lever presses) for obtaining cocaine rewards is progressively increased.

3.2. Ibudilast produced a downward shift in the cocaine dose-response curve in rats

To test the hypothesis that ibudilast depresses the reinforcing effects of cocaine, we examined the effects of ibudilast on multiple-dose cocaine self-administration. Rats were trained to self-administer cocaine for 10 days under FR1/FR2 reinforcement, as described above. After stable self-administration was achieved, the rats were switched to multiple-dose cocaine self-administration maintained by a full range of cocaine doses in a single session (see Materials and Methods). The dose-effect curve for cocaine had an inverted-U shape and maximal infusions occurred at the 0.125 mg/kg/infusion dose (Fig. 1G). Ibudilast (1 or 3 mg/kg, i.p.) or vehicle was injected 30 min prior to multiple dose cocaine self-administration. We found that ibudilast produced dose-dependent downward shifts in cocaine dose-response functions under a FR2 schedule. A two-way repeated-measures ANOVA over cocaine dose revealed a significant treatment (vehicle vs ibudilast) main effect (F2,21 = 42.7, p < 0.001), a significant cocaine dose main effect (F6,126 = 11.0, p < 0.001) and a significant treatment × cocaine dose interaction (F12,126 = 3.9, p < 0.001) (Fig. 1G). Taken together, these results suggest that ibudilast reduces cocaine’s reinforcing strength and/or motivation for drug-taking behavior.

3.2. Ibudilast pretreatments prevented the reduction of GABAergic inhibition induced by cocaine self-administration

Repeated non-contingent cocaine exposure in vivo leads to a decrease in IPSCs in VTA dopamine neurons. This reduction of GABAergic inhibition facilitates long-term synaptic plasticity at glutamatergic inputs to VTA dopamine neurons (Bocklisch et al., 2013; Liu et al., 2005). However, it remained unknown whether cocaine self-administration was associated with changes in the strength of GABAergic inputs to VTA dopamine neurons. If so, ibudilast pretreatments could affect the cocaine self-administration-induced change in GABAergic inhibition. Rats received cocaine or saline self-administration under FR1/FR2 reinforcement schedules as described in Methods. On the 11th day, the effects of ibudilast on cocaine or saline self-administration were examined (Fig. 2A, Fig. S1A). Two-way ANOVA showed that cocaine self-administration and ibudilast pretreatments had significant effects on the active lever presses (cocaine: F1,30 = 192.5, p < 0.001; Ibudilast: F1,30 = 66.4, p < 0.001; cocaine x ibudilast interaction: F1,23 = 77.4, p < 0.001; Fig. S1B) and number of infusions (cocaine: F1,30 = 175.2, p < 0.001; Ibudilast: F1,30 = 60.4, p < 0.001; cocaine x ibudilast interaction: F1,30 = 70.7, p < 0.001; Fig. S1C). Tukey’s post hoc tests indicated that cocaine self-administration led to significant decreases in the active lever presses (p < 0.001; Fig. S1B) and number of infusions (p < 0.001; Fig. S1C). The cocaine self-administration-induced increase in active lever presses (p < 0.001; Fig. S1B) and number of infusions (p < 0.001; Fig. S1C) were prevented in rats that received ibudilast pretreatments. Ex vivo midbrain slices were prepared from the four groups of rats 1 day after behavioral tests (Fig. 2A, Fig. S1A). The self-administration experiments were staggered to start and end on different days to coordinate data acquisition for electrophysiology experiments across groups. Spontaneous IPSCs (sIPSCs) were recorded from VTA dopamine neurons. Two-way ANOVA showed that cocaine self-administration and ibudilast pretreatments had significant effects on the mean frequency of sIPSCs (cocaine: F1,52 = 5.7, p = 0.021; Ibudilast: F1,52 = 7.8, p = 0.007; cocaine x ibudilast interaction: F1,52 = 6.4, p = 0.014; Fig. 2B,C), and the mean amplitude of sIPSCs (cocaine: F1,52 = 5.2, p = 0.027; Ibudilast: F1,52 = 9.1, p = 0.004; cocaine x ibudilast interaction: F1,52 = 5.5, p = 0.023; Fig. 2B,D). Tukey’s post hoc tests indicated that cocaine self-administration led to significant decreases in the mean frequency (p = 0.001; Fig. 2C) and amplitude (p = 0.002; Fig. 2D) of sIPSCs. The cocaine self-administration-induced decreases in the frequency (p < 0.001; Fig. 2C) and amplitude (p < 0.001; Fig. 2D) of sIPSCs were prevented in slices from rats that received ibudilast pretreatments. The cumulative distribution for inter-event intervals of sIPSCs was shifted to the right in the vehicle pretreatment/cocaine self-administration group compared to that in the vehicle pretreatment/saline self-administration group (K-S test, p < 0.001), and this shift was blocked by ibudilast pretreatments (K-S test, p < 0.001; Fig. 2E). The cumulative distribution for the amplitude of sIPSCs was shifted to the left in the vehicle pretreatment/cocaine group (K-S test, p = 0.013), and this shift was blocked by ibudilast pretreatments (K-S test, p = 0.013; Fig. 2F). Together, these results indicate that cocaine self-administration led to decreases in sIPSC frequency and amplitude, and these decreases were prevented by ibudilast pretreatments.

How might ibudilast affect IPSCs? We examined the effects of bath application of ibudilast on basal IPSCs in VTA dopamine neurons in slices prepared from rats that underwent saline and cocaine self-administration (Fig. 3A, Fig. S1A). We found that bath application of ibudilast (10 μM) caused significant increases in the amplitude of evoked IPSCs in both the saline group (147.7 ± 6.1% of baseline, paired t-test, t6 = 7.0, p < 0.001 vs. baseline) and the cocaine group (145.2 ± 5.0% of baseline, paired t-test, t6 = 7.4, p < 0.001 vs. baseline), the magnitude of ibudilast-induced potentiation of IPSCs was not significantly different between the two groups (t-test, t12 = 0.3, p = 0.753; Fig. 3B,C). Ibudilast-induced inhibition of PDE4 is known to increase intracellular cAMP, while cAMP enhances neurotransmitter release at many excitatory and inhibitory synapses via the activation of protein kinase A (PKA) (Chavez-Noriega and Stevens, 1994; Kaneko and Takahashi, 2004). We found that the effect of ibudilast on IPSCs was prevented in the presence of PKA inhibitor H89 (1 μM) in both saline (t-test, t11 = 5.6, p < 0.001) and cocaine groups (t-test, t12 = 10.4, p < 0.001; Fig. 3B,C). Thus, ibudilast enhances IPSCs by increasing cAMP/PKA activity.

3.4. Effects of cocaine self-administration and ibudilast pretreatments on the AMPAR/NMDAR Ratio in VTA dopamine neurons

We next determined whether cocaine self-administration and ibudilast pretreatments altered AMPAR/NMDAR ratio in VTA dopamine neurons, using rats described in Fig. S1A. The experiment timeline was similar to those described in Fig. 2 above except the AMPAR/NMDAR ratio was recorded (Fig. 4A). Cocaine self-administration and ibudilast pretreatments altered the AMPAR/NMDAR ratio (cocaine: F1,45 = 10.3, p = 0.003; ibudilast: F1,45 = 11.2, p = 0.002; cocaine x ibudilast interaction: F1,45 = 6.9, p = 0.012; Fig. 4B,C). Tukey’s post hoc tests indicated that cocaine self-administration resulted in a significant increase in the AMPAR/NMDAR ratio (p < 0.001; Fig. 4C) in vehicle-pretreated rats. Interestingly, compared with the vehicle pretreatment group, ibudilast pretreatments alone increased the AMPAR/NMDAR ratio in saline self-administration rats (p < 0.001) but did not cause any further increase in the AMPAR/NMDAR ratio in cocaine self-administration rats (p = 0.614; Fig. 4C). Thus, cocaine self-administration led to the increased AMPAR/NMDAR ratio ex vivo, suggesting that cocaine self-administration led to potentiation of excitatory synaptic transmission to VTA dopamine neurons. On the other hand, ibudilast pretreatments increased the AMPAR/NMDAR ratio in saline self-administration rats but did not produce further increase in the AMPAR/NMDAR ratio in cocaine self-administration rats.

Fig. 4. Effects of cocaine self-administration and ibudilast pretreatments on AMPAR/NMDAR ratio.

Fig. 4.

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A Timeline of self-administration training, vehicle or ibudilast treatment and electrophysiology. B Representative AMPAR- and NMDAR-mediated EPSCs recorded from VTA dopamine neurons in rats. C In saline self-administration rats, ibudilast pretreatments significantly increased AMPAR/NMDAR ratio in dopamine neurons compared with vehicle treatment (***p < 0.001, n = 11–12). Ibudilast pretreatments did not further increase the AMPAR/NMDAR ratio in slices prepared from cocaine self-administration rats (p = 0.614; ns, not significant; n = 11–12). Each data set was obtained from 3–4 rats.

3.5. Ibudilast pretreatments prevented cocaine-induced disruption of excitatory/inhibitory balance

We determined whether cocaine self-administration and ibudilast pretreatments altered the balance of excitatory and inhibitory input to VTA dopamine neurons. We recorded evoked EPSCs and IPSCs sequentially from the same VTA dopamine neurons in slices prepared from the four groups of rats described above (Fig. S1A, Fig. 5A). The NMDAR antagonist R-CPP (10 μM) was present throughout the experiment. VTA dopamine neurons were voltage-clamped alternatively at the reversal potential for IPSCs (−60 mV) and EPSCs (0 mV) to isolate EPSCs and IPSCs, respectively. Indeed, IPSCs recorded at 0 mV were abolished by the GABAA receptor blocker picrotoxin (50 μM), while EPSCs recorded at −60 mV were abolished by the AMPAR antagonist CNQX (20 μM) (Fig. 5B). We next determined whether cocaine self-administration and ibudilast treatments altered the EPSCs/IPSCs (E/I) ratio in slices from these four groups of rats. Two-way ANOVA indicated that cocaine self-administration and ibudilast pretreatments had significant effects on the E/I ratio (cocaine: F1,42 = 36.9, p < 0.001; Ibudilast: F1,42 = 8.7, p = 0.005; cocaine x ibudilast interaction: F1,42 = 12.7, p < 0.001; Fig. 5C,D). Tukey’s post hoc tests indicated that the E/I ratio was significantly increased in the vehicle/cocaine group compared to the vehicle/saline group (p < 0.001; Fig. 5D).

Fig. 5: Ibudilast pretreatments partially restored the cocaine-induced imbalance of excitation and inhibition in the VTA.

Fig. 5:

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A Timeline of self-administration training, vehicle or ibudilast treatment and electrophysiology. B EPSCs and IPSCs were isolated by voltage-clamping VTA dopamine neurons at the reversal potentials of IPSCs (−60 mV) and EPSCs (0 mV), respectively. EPSCs were blocked by the AMPAR antagonist CNQX (n = 6), while IPSCs were blocked by the GABAA receptor blocker picrotoxin (n= 6). C Sample EPSCs and IPSCs recorded in slices from saline- or cocaine self-administration rats that received vehicle or ibudilast pretreatments. D Cocaine self-administration led to an increase in the E/I ratio (***p < 0.001, n = 10–11), which was blocked by ibudilast pretreatments (***p < 0.001, n = 11). Ibudilast pretreatments did not have significant effect on E/I ratio in slices prepared from cocaine self-administration rats (p = 0.080; ns, not significant; n = 10–11). Each data set was obtained from 3–4 rats.

The increase in the E/I ratio induced by cocaine self-administration was blocked by ibudilast pretreatments (p < 0.001; Fig. 5D). Together, these results suggest that ibudilast restores the imbalance between excitation and inhibition in VTA dopamine neurons induced by cocaine self-administration.

3.6. Effects of ibudilast on oral sucrose self-administration

To determine whether ibudilast affects non-drug reinforcement, we evaluated the effect of ibudilast on oral sucrose self-administration under FR2 reinforcement. Rats established stable sucrose self-administration during the 10-day training period which emulated the procedure for cocaine self-administration training, but with sucrose pellet rewards rather than cocaine (Fig. 6A). On subsequent days, the effects of ibudilast (0, 1, 3 or 10 mg/kg, i.p.) on sucrose self-administration were tested using a Latin Square design. One-way ANOVA found that ibudilast pretreatments had significant effect on active lever presses (F3,28 = 3.3, p = 0.036; Fig. 6B) and sucrose intake (F3,28 = 3.2, p = 0.041; Fig. 6D), but has no significant effect on inactive lever presses (F3,28 = 1.2, p = 0.346; Fig. 6C). Tukey’s post hoc tests indicated that ibudilast at 10 mg/kg dose significantly decreased active lever presses (p = 0.040; Fig. 6B) and sucrose intake (p = 0.044; Fig. 6D). These results suggested that ibudilast pretreatments at a higher dose significantly decreased oral sucrose self-administration.

Fig. 6: The effects of ibudilast pretreatments on oral sucrose self-administration.

Fig. 6:

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A The number of active lever presses, inactive lever presses and sucrose intake during FR1 and FR2 training. B,C,D Ibudilast pretreatments at a higher dose (10 mg/kg, i.p.) produced a significant suppression of active lever presses (B; *p = 0.040, n = 8 rats) and sucrose intakes (D; *p = 0.044, n = 8 rats), but did not alter the number of inactive lever presses (C; p = 0.514, n = 8 rats).

3.7. Effects of ibudilast on cocaine prime- and cue-induced reinstatement of cocaine seeking

To investigate whether ibudilast is effective in preventing relapse to drug seeking, we examined the effects of ibudilast on cocaine prime-induced reinstatement of cocaine seeking. Rats were first trained to lever-press to obtain sucrose pellets followed by 10 days of cocaine self-administration training under FR1 and FR2 reinforcement schedules (0.5 mg/kg/infusion). After rats acquired stable cocaine self-administration as shown by robust active lever pressing, they began extinction training until extinction criteria (≤ 20 active lever presses during each 3 hours session for at least 3 consecutive days) was attained. The next day, the two groups of rats received vehicle or ibudilast pretreatments (10 mg/kg, i.p.) 30 min before a priming dose of cocaine (10 mg/kg, i.p.). The timeline of these experiments is described in Fig. 7A. Different cohorts of rats were used for cocaine prime- and cue-induced reinstatement experiments. In rats pretreated with vehicle, cocaine priming induced robust reinstatement of active lever pressing, while ibudilast pretreatments attenuated cocaine prime-induced active lever responding (t16 = 3.8, p = 0.002; Fig. 7B) but had no significant effect on inactive lever responses (t16 = 0.1, p = 0.895; Fig. 7C). Thus, ibudilast pretreatment reduces cocaine prime-induced reinstatement of cocaine seeking.

Fig. 7: Ibudilast attenuated cocaine prime- and cue-induced reinstatement of cocaine seeking.

Fig. 7:

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A Timeline of self-administration (SA) training, extinction training and reinstatement testing. Separate groups of rats were used for cocaine prime- and cue-induced reinstatement experiments. B Cocaine prime induced robust reinstatement of active lever presses, but this increase was attenuated by ibudilast pretreatments (**p = 0.002, n = 9 rats). C Both vehicle and ibudilast pretreatments did not affect the inactive lever presses during cocaine prime-induced reinstatement (p = 0.895, n = 9 rats). D Ibudilast pretreatments induced a significant reduction in active lever presses in rats under cue-induced reinstatement compared with vehicle pretreatments (*p = 0.014, n = 7–9 rats). E Both vehicle and ibudilast pretreatments did not significantly change inactive lever responses in cue-induced reinstatement (p = 0.222, n = 7–9 rats).

We further evaluated whether ibudilast affected cue-induced reinstatement of cocaine seeking in another cohort of rats. The experiments resembled those described above except the cocaine prime injection was replaced with drug-associated light cues to induce reinstatement of cocaine seeking. After cocaine operant responding stabilized, rats underwent extinction in which no cues and drug were delivered in response to lever presses. Prior to testing cue-induced reinstatement, rats received vehicle or ibudilast pretreatments (10 mg/kg, i.p.). The presence of light cues previously paired with cocaine availability markedly reinstated active lever responding in rats that received vehicle pretreatments. Ibudilast pretreatments induced a significant reduction in active lever-presses (t14 = 2.8, p = 0.014; Fig. 7D) but did not significantly change inactive lever responses (t14 = 1.3, p = 0.222; Fig. 7E). Together, these data demonstrated that ibudilast pretreatments significantly attenuated cocaine prime- and cue-induced reinstatement of cocaine seeking.

4. Discussion

Here we have demonstrated that the PDE4 inhibitor and glial activity modulator ibudilast (Huang et al., 2006; Schwenkgrub et al., 2017) produced a significant and dose-dependent reduction of intravenous cocaine self-administration in rats under an array of reinforcement schedules. Ibudilast prevented cocaine self-administration-induced changes in inhibitory and excitatory synaptic plasticity and restored the balance between excitation and inhibition in VTA dopamine neurons. Finally, ibudilast significantly reduced both cocaine prime- and cue-induced reinstatement of cocaine-seeking. Our findings suggest that ibudilast may serve as a potential treatment for cocaine use disorder.

We found that ibudilast (3 and 10 mg/kg) reduced cocaine self-administration under FR2 reinforcement schedule. In contrast, it significantly inhibited sucrose self-administration under FR2 reinforcement only at the high dose (10 mg/kg). Thus, ibudilast shows relative selectivity in reducing cocaine self-administration. In addition, ibudilast (3 and 10 mg/kg) produced a dose-dependent reduction in the number of PR cocaine infusions earned. FR and PR schedules of drug self-administration measure different aspects of drug reinforcement, with FR schedules more sensitive to satiety factors and the PR schedule being a more direct measure of the motivational and reinforcing efficacy of cocaine (Arnold and Roberts, 1997; Hodos, 1961; Richardson and Roberts, 1996). A decrease in PR cocaine infusions is often interpreted as a reduction in drug reward strength and in the motivation for obtaining drug reward (Arnold and Roberts, 1997; Hodos, 1961; Richardson and Roberts, 1996). Finally, ibudilast at doses of 1 mg/kg and 3 mg/kg produced downward shifts of dose-response curve and reduced the number of cocaine infusions earned across a wide range of cocaine doses. Taken together, the robust attenuation of cocaine self-administration under FR, PR and multiple cocaine-dose reinforcement schedules indicates that ibudilast pretreatments suppress the reinforcing and motivating effects of cocaine self-administration.

However, one important caveat needs to be considered when interpreting the above results. It has been shown that i.p. injections of ibudilast to drug naïve rats at doses from 2.5 to 10 mg/kg led to a decrease in locomotor activity in an open field during the first 20–30 min but had little or no significant effects after the initial habituation (Lilius et al., 2009; Poland et al., 2016). Ibudilast-induced reduction of locomotor activity would be expected to reduce operant responding. Of note, the cocaine and sucrose self-administration experiments were started 30 min after i.p. injections of ibudilast. In addition, ibudilast significantly inhibited sucrose self-administration only at the 10 mg/kg dose but not at the 1 and 3 mg/kg doses. Even at 10 mg/kg ibudilast, the rats still had 70.4 ± 10.4 (n = 8 rats) active lever presses during the 3-hour period of sucrose self-administration. Finally, ibudilast did not significantly alter inactive lever responding during cocaine or sucrose self-administration. Thus, ibudilast-induced reduction of cocaine taking and seeking cannot be entirely explained by suppression of locomotor activity, although we could not exclude the possibility that the suppression of locomotor activity may partially contribute to the suppression of operant responding, particularly at higher doses such as 10 mg/kg.

We examined the potential mechanisms that might underline ibudilast-induced reduction of cocaine self-administration. VTA dopamine neurons regulate reward, reinforcement, incentive salience, and decision making (Lammel et al., 2012; Morales and Margolis, 2017; Zweifel et al., 2011), and their firing is governed by inhibitory and excitatory synaptic inputs (Polter et al., 2018). Proper function of VTA dopamine neurons depends on the balance between excitation and inhibition. We found that cocaine self-administration led to a decrease in GABAergic inhibition to VTA dopamine neurons, while ibudilast pretreatment prevented the cocaine self-administration-induced decrease in GABA inhibition. Cocaine enables long-term depression at inhibitory synapses (I-LTD) (Pan et al., 2008b), but this I-LTD can be blocked by PKA inhibitors, suggesting a cAMP-dependent mechanism (Pan et al., 2008a). Ibudilast is a PDE4 inhibitor that increases cAMP levels by blocking its degradation (Huang et al., 2006; Schwenkgrub et al., 2017). Indeed, we found that ibudilast potentiated evoked IPSCs in VTA dopamine neurons in slices prepared from rats that received saline and cocaine self-administration, and these effects were blocked by the PKA inhibitor H-89. It is thus likely that ibudilast reversed cocaine-induced I-LTD-like synaptic modification in vivo (Pan et al., 2008a), which may explain why ibudilast prevented cocaine self-administration-induced GABAergic inhibition.

GABAergic inhibition puts a brake on excitatory synaptic plasticity, and the reduced inhibition facilitates the induction of long-term potentiation (LTP) (Huang et al., 1999; Wigstrom and Gustafsson, 1983). Non-contingent cocaine exposure induces potentiation of excitatory synaptic inputs to VTA dopamine neurons, as manifest by an increase in the AMPAR/NMDAR ratio ex vivo (Argilli et al., 2008; Bellone and Luscher, 2006; Borgland et al., 2004; Liu et al., 2005; Mameli et al., 2007; Ungless et al., 2001). We extended these findings by showing that cocaine self-administration led to an increase in the AMPAR/NMDAR ratio ex vivo, and that ibudilast pretreatments alone increased the AMPAR/NMDAR ratio in the saline self-administration group but did not further increase the AMPAR/NMDAR ratio in the cocaine self-administration group. PKA activation is thought to underlie the increase in the AMPAR/NMDAR ratio in VTA dopamine neurons induced by cocaine exposure (Argilli et al., 2008; Brown et al., 2010; Liu et al., 2016). PKA phosphorylates GluA1 at S845, increasing the conductance and open probability of the AMPAR channel (Banke et al., 2000). It is likely that ibudilast and cocaine potentiate AMPAR-EPSCs via PKA-dependent mechanisms, which might explain why ibudilast did not further increase the AMPAR/NMDAR ratio. Cocaine self-administration induces a reduction of inhibition and potentiation of excitation which shifted the E/I balance towards over-excitation. Ibudilast pretreatments enhanced inhibition without significantly altering excitation and restored the E/I balance. These mechanisms might contribute to the effects of ibudilast on cocaine self-administration.

We acknowledge the limitations of the present study. First, we have examined the effects of acute, but not chronic, ibudilast treatments on cocaine self-administration. Treatments for substance use disorders have historically been chronically administered (Mello and Negus, 1996). In rat cocaine self-administration models, chronic drug treatment may diminish or even reverse the effects seen after acute administration (Haney and Spealman, 2008; Thomsen et al., 2017). Second, our experiments were carried out only in male rats. These two concerns could perhaps be lessened by human clinical trial studies showing efficacious therapeutic effects for the treatments of alcohol (Grodin et al., 2021; Ray et al., 2017) and methamphetamine use disorders (DeYoung et al., 2016; Li et al., 2020) and opioid withdrawal (Cooper et al., 2016) in both males and females after chronic ibudilast administration. Third, our electrophysiological experiments have been carried out in brain slices ex vivo. While brain slices offer more accessibility and facilitate pharmacological studies, slice cutting severs afferent and efferent projections. In vivo electrophysiology (Peyrache and Destexhe, 2019) or recording neuronal activity via fluorescent reporters in behaving animals (Vickstrom et al., 2021) may provide further insight into cocaine-induced neuroadaptations. Finally, it was thought that ibudilast attenuates methamphetamine hyperlocomotion and sensitization by inhibiting glial proinflammatory activity (Snider et al., 2012), our experiments have not directly addressed whether the effects of ibudilast on cocaine self-administration result from anti-neuroinflammatory or glial cell modulation activity. Future studies should determine whether anti-inflammatory mechanisms are involved in cocaine-induced synaptic plasticity and behavioral effects.

Relapse to drug-taking behavior can be precipitated by re-exposure to the drug itself (i.e., administering a drug prime) and drug-associated cues. We examined the effects of ibudilast pretreatments on reinstatement of cocaine-seeking behavior induced by systemic injections of a relatively low cocaine dose (10 mg/kg) or cocaine-associated cues. Cocaine prime and cue-induced restatement of cocaine seeking are well-validated models for studying relapse to drug seeking (Beardsley and Shelton, 2012). We found that ibudilast pretreatment remarkably suppressed cocaine seeking induced by either cocaine prime or drug-associated cues. We speculate that enhancing cAMP-mediated neuroadaptations may contribute to its inhibitory behavioral effects on cocaine prime and cue-induced cocaine seeking.

5. Conclusion

Ibudilast pretreatments selectively and dose-dependently attenuated cocaine-self administration under a variety of reinforcement schedules, shifting the cocaine dose-response curve downward. This downward shift suggests that the inhibitory effects of ibudilast on cocaine taking cannot be overcome by increased cocaine availability. We propose that ibudilast-induced enhancement of ex vivo GABA-IPSCs would counter the cocaine-induced reductions in GABAergic inhibition and prevent disruption of E/I balance in VTA dopamine neurons. We report that ibudilast increased AMPAR/NMDAR ratio independently of cocaine exposure, but AMPAR/NMDAR ratio was not further elevated by pretreatment in the cocaine self-administration groups. Ibudilast was also found to attenuate both cue and cocaine-prime induced reinstatement of cocaine seeking, models of relapse. Together, these studies suggest that ibudilast reduces the motivational and reinforcing efficacy of cocaine and could be effective in reducing relapse to cocaine seeking. Ibudilast readily crosses the blood-brain-barrier (Cooper et al., 2016) and is safe and well-tolerated in humans (Cooper et al., 2016). As such, ibudilast may serve as a promising candidate for pharmacological treatment of cocaine use disorder.

Supplementary Material

1. Fig. S1. Cocaine self-administration behavioral data for ex vivo electrophysiology presented in Figs. 2, 4, and 5.

A Timeline of saline and cocaine self-administration training, vehicle or ibudilast treatment and electrophysiology. B Ibudilast pretreatments (10 mg/kg, i.p.) produced a significant suppression of active lever presses (B; ***p < 0.001, n = 7–8 rats) and cocaine infusions (C; ***p < 0.001, n = 7–8 rats). The self-administration experiments were staggered to start and end on different days to coordinate data acquisition for electrophysiology experiments across groups.

Highlights.

  • Ibudilast dose-dependently decreased cocaine self-administration under both fixed ratio and progressive ratio reinforcement schedules.

  • Ibudilast shifted the dose-response curve of cocaine downward.

  • Cocaine self-administration led to a decrease in GABAergic inhibition and an enhancement of glutamatergic excitation to ventral tegmental area (VTA) dopamine neurons.

  • Ibudilast enhanced GABAergic inhibition and restored the balance between excitation and inhibition to VTA dopamine neurons.

  • Ibudilast decreased cocaine prime- and cue-induced reinstatement of cocaine seeking.

Acknowledgments

Funding and disclosure: The authors declare no competing financial interests. This work was supported by NIH Grants DA035217 (QSL) and DA047269 (QSL). It was also partially funded through the Research and Education Initiative Fund, a component of the Advancing a Healthier Wisconsin endowment at the Medical College of Wisconsin.

Footnotes

Declaration of competing interest

The authors declare that they have no conflicts of interest.

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Associated Data

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Supplementary Materials

1. Fig. S1. Cocaine self-administration behavioral data for ex vivo electrophysiology presented in Figs. 2, 4, and 5.

A Timeline of saline and cocaine self-administration training, vehicle or ibudilast treatment and electrophysiology. B Ibudilast pretreatments (10 mg/kg, i.p.) produced a significant suppression of active lever presses (B; ***p < 0.001, n = 7–8 rats) and cocaine infusions (C; ***p < 0.001, n = 7–8 rats). The self-administration experiments were staggered to start and end on different days to coordinate data acquisition for electrophysiology experiments across groups.

NIHMS1749369-supplement-1.pptx (47.2KB, pptx)

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