Slideshare Incubation of Cocaine Craving After Withdrawal a Review of Preclinical Data

New Results

Cannabidiol furnishings on cocaine-seeking behaviour and incubation of craving in mice

doi: https://doi.org/10.1101/2020.12.18.423391

Abstruse

Groundwork and Purpose To remain abstinent represents one of the major challenges for the treatment of cocaine utilize disorder. Cocaine seeking elicited by drug-associated cues progressively intensifies during abstinence in a process termed incubation of craving, representing an aggravating factor for relapse. Cannabidiol is a phytocannabinoid that exerts protecting furnishings upon cocaine-seeking behaviour, although its furnishings on cocaine-peckish incubation have never been elucidated.

Experimental Approach We adult a mouse model of behavioural economical assay of need curves and incubation of cue-induced cocaine craving. Changes in the protein expression of AMPAR subunits and ERK_1/2 phosphorylation were analysed. We too assessed the effects of cannabidiol (20 mg·kg^-1) administered either during conquering of cocaine self-assistants or abstinence.

Key Results Mice efficiently performed the demand task and incubation of cocaine peckish. Likewise, changes in GluA1 and GluA2 protein levels were institute along the abstinence in prelimbic cortex, ventral striatum and amygdala, as well as a decrease in ERK_1/2 phosphorylation in ventral striatum. Cannabidiol reduced ongoing cocaine intake when administered during the acquisition phase of the self-assistants, but failed to alter the subsequent need chore performance and incubation of cocaine craving. No effects were found when cannabidiol was administered during the forbearance menstruation.

Conclusion and Implications We provide here a novel model of behavioural economic analysis of demand curves and cue-induced incubation of cocaine-seeking behaviour for mice. Moreover, we prove that cannabidiol exerts differential effects on the current model depending on the self-administration stage in which it was administered.

What is already known

• Behavioural economics and incubation of cocaine craving are well-stablished paradigms to evaluate cocaine seeking in rats.

• CBD reduces cocaine-seeking and cocaine-taking behaviours.

What this study adds

• A mouse model of behavioural economic assay of demand curves and incubation of cue-induced cocaine craving.

• CBD reduces cocaine self-administration and has no effect over demand task and cocaine-craving incubation.

Clinical significance

• A new behavioural model for studying cocaine habit in mice.

• CBD exerts differential effects depending on when it was administered in the addictive procedure.

1. INTRODUCTION

Cocaine is one of the most used drugs worldwide, showing an increasing trend and doubling the prevalence in Europe compared to the global information (United nations publication, 2020). However, despite the extended use of cocaine among the population and the trouble that it represents for Public Health, the pharmacotherapeutic strategies used to treat cocaine employ disorder to engagement seem to have limited efficacy (Kampman, 2019).

The difficulty to stay abstemious and the take chances of relapse represent the major challenges for the treatment of cocaine use disorder. In this sense, cue-induced cocaine craving is one of the main factors contributing to relapse in abstinent addicts. Cocaine-seeking elicited past drug-associated cues progressively intensifies during abstinence in a process termed incubation of peckish (Grimm et al., 2001), firstly described in animal models. Clinical studies have already provided prove about the incubation of peckish for cocaine (Parvaz et al., 2016) and other drugs of corruption (Bedi et al., 2011; Wang et al., 2013; Li et al., 2015), although a mechanistic explanation for this phenomenon is nonetheless to be found.

In contrast to the scarce studies in humans, rodent models have been largely used to describe incubation of cocaine craving and to decipher the molecular and cellular mechanisms underlying this process (Lu et al., 2004). Incubation of cocaine craving requires from structural and functional changes in different brain areas, mainly located in the mesocorticolimbic organization (Pickens et al., 2011; Wolf, 2016). The accumulation of calcium-permeable α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acrid receptors (CP-AMPARs) defective GluA2 subunit in the nucleus accumbens (NAc) core is needed for the expression of cocaine-craving incubation after prolonged withdrawal (Conrad et al., 2008). These neuroplastic changes are specially significant since they functionally translate into increases in the number and strength of NAc core neurons responding to cocaine-associated cues (Hollander and Carelli, 2005; Purgianto et al., 2013). Besides, activation of glutamatergic signalling in the medial prefrontal cortex and amygdala are also required for the expression of cocaine-craving incubation. In particular, glutamatergic signalling within the prelimbic cortex (PL) is a key element for the incubation of cocaine-seeking behaviour during late withdrawal (West et al., 2014). More than precisely, the glutamatergic PL-NAc cadre pathway promotes incubation, while the infralimbic cortex (IL)-NAc shell signalling prevents information technology, based on a process of silent synapses remodelling (Ma et al., 2014). Similarly, distinct nuclei of the amygdala are also differentially implicated in the modulation of drug-seeking behaviour forth abstinence. Previous enquiry has demonstrated that both, central nucleus of the amygdala (CeA) and basolateral amygdala (BLA) participate in the first stages of cocaine withdrawal (Lu et al., 2005a). All the same, their function in prolonged withdrawal is still a matter of fence. Some studies evidence that ERK phosphorylation and glutamatergic activation of CeA, but not BLA, are required for the expression of cocaine-craving incubation during the tardily withdrawal (Lu et al., 2005b, 2007). Others propose that the glutamatergic BLA-NAc beat out pathway is essential for this process (Lee et al., 2013). Furthermore, a contempo work identifies the CeA as a common substrate of the incubation of drugs (including cocaine) and natural reinforcers (Roura-Martínez et al., 2019).

Despite the vast research on cocaine-peckish incubation in preclinical studies, about of the records to date have been extracted from rat models and only a scattering of articles have been published addressing this issue in mouse models (Terrier et al., 2015; Nugent et al., 2017). Besides, long-admission cocky-administration (6h/day) has been the preferred protocol to induce incubation of cocaine craving in both mice and rats (Conrad et al., 2008; Terrier et al., 2015; Calipari et al., 2019).

In the last years, cannabidiol (CBD) has emerged as a new potential treatment for drug abuse (Chye et al., 2019), including cocaine (Calpe-López et al., 2019; Rodrigues et al., 2020). CBD's multi-target nature (Izzo et al., 2009) seems to contribute to its effectiveness in treating dissimilar psychiatric disorders, such as drug addiction. However, it is maybe also the reason why the molecular mechanisms underlying its effects are so circuitous and, frequently, poorly understood. Although unlike preclinical studies have evaluated the furnishings of CBD on acquisition of cocaine cocky-administration (Mahmud et al., 2016; Luján et al., 2018), extinction (Lujan et al., 2020), reinstatement (Mahmud et al., 2016; Gonzalez-Cuevas et al., 2018; Luján et al., 2018; Lujan et al., 2020), and spontaneous cocaine withdrawal (Gasparyan et al., 2020), its effects on forbearance and cocaine-craving incubation accept non yet been confirmed. Of note, CBD or CBD-containing drugs have been clinically tested for the handling of tobacco (Hindocha et al., 2018) and cannabis (Crippa et al., 2013; Allsop et al., 2014; Trigo et al., 2018) withdrawal, but non for cocaine. Moreover, none of these studies specifically assessed incubation of craving.

In the present study, nosotros implemented a demand task analysed under the behavioural economics paradigm in CD1 male mice exposed to a cocaine-induced cocky-administration procedure. This strategy allowed us to evaluate relevant parameters related to cocaine-seeking and -taking behaviours. And then, we set a new procedure for incubation of cue-induced craving in mice during cocaine withdrawal from brusk-access cocaine self-assistants (2h·twenty-four hour period^-1). AMPAR subunit composition and extracellular indicate-regulated kinase i and 2 (ERK1/2) phosphorylation were determined in PL, ventral striatum (vSTR) and amygdala during the incubation of craving. Finally, we examined the effects of CBD on cocaine-induced conquering of cocky-assistants demand job and incubation of cocaine craving.

2. METHODS

two.1. Animals

Male CD1 mice (postnatal day 56) were purchased from Charles River (Barcelona, Spain) and transported to our animal facility (UBIOMEX, PRBB). Animals were maintained in a 12-hour light-nighttime cycle (lights on 7:thirty-xix:30). Mice were housed at a stable temperature (22 °C ± two) and humidity (55% ± 10%), with food and water advertising libitum, and they were allowed to acclimatize to the new environmental conditions for at least five days prior to surgery. Once mice were recovered from surgery, they were moved to the experimental room in an inversed 12-60 minutes light-night wheel (lights on xix:30-7:thirty). All creature care and experimental protocols were approved by the UPF/PRBB Animate being Ethics Committee (CEEA-PRBB-UPF), in accordance with European Community Council guidelines (2016/63/Eu).

2.ii. Experimental design

Experiment i: Cocaine cocky-administration, demand job and incubation of craving

Mice were start trained to self-administrate cocaine in a stock-still ratio (FR) 1 and FR3 schedules of reinforcement, and 24 hours later underwent the need job. Afterwards, mice started the forbearance period in their home cages and were tested for cue-induced seeking on withdrawal days (WD) one, fifteen, xxx, and 45 (Figure 1A). Besides, brains were extracted immediately after the cue-induced seeking tests on WD1, WD15 and WD30 for Western blot assay (Figure 2a).

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Figure 1.

Behavioural economic analysis of demand curve and incubation of cocaine peckish paradigms for mice. (A) Schematic timeline of the experiment 1. (B) Active and inactive nosepokes during FR1 and FR3 (n=22) in the self-administration paradigm. Tukey's test, **p<0.01 and ***p<0.001, active vs inactive nosepokes (C) Infusions during FR1 and FR3. (D) Intrasession active nosepokes during FR1 and FR3. 2-way ANOVA, ***p<0.001, FR1 vs FR3. (East) Exponential need bend where consumption plotted as a office of price (n=19). (F) Consumption at a minimally constraining cost or Q₀. (1000) Demand elasticity or α. (H) Point of unit elasticity or P_max. (I) Maximum number of responses at P_max or O_max. (J) Active and inactive nosepokes during cue-induced seeking tests (northward=19). Two-way ANOVA, ***p<0.001 active vs inactive hole; Tukey'south test, *p<0.01, baseline vs WD15 and WD30. (K) Percentage of active nosepokes change from WD1. (L) Per centum of mice that reached incubation criteria. (Chiliad) Active nosepokes of incubated (n=8) and non-incubated (northward=11) mice. Sidak's test, **p<0.01 and ***p<0.001, vs WD1; #p<0.05, vs not-incubated mice. (N) Percentage of active nosepokes change from WD1 of incubated and non-incubated mice. Sidak's test, ***p<0.001, vs WD1; #p<0.05, vs non-incubated mice.

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Figure 2.

AMPAR subunit limerick and ERK phosphorylation changes along abstinence in PL, vSTR and amygdala (n=six-8). (A) Schematic timeline of the experiment 1. GluA1/2 and pERK/ERK changes on WD1, WD15 and WD30 in: (B) PL; Tukey's test; *p<0.05, vs WD1; ##p<0.01, vs WD15; (C) vSTR; Tukey'southward test, *p<0.05, vs WD1; (d) Amygdala; Tukey'southward exam, *p<0.05, vs WD1; #p<0.05, vs WD15.

Experiment 2: Effects of CBD administered during conquering of cocaine self-administration

Mice followed the aforementioned protocol as in Experiment 1, except that vehicle or CBD were administered only before entering the operant conditioning cages during the conquering of cocaine self-administration and too immediately prior to the need task. Therefore, as the length of the acquisition phase depends on how quick animals learn the task, the number of days and the number of CBD injections varied between nine and 16. Mice that succeeded to acquire self-administration behaviour underwent cue-induced seeking tests on WD1 and WD15 (Figure 3a).

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Effigy three.

CBD decreases cocaine seeking and taking when administered during conquering, but such effects are non maintained during abstinence. (A) Schematical timeline of the experiment two. (B) Active and inactive nosepokes during FR1 and FR3 (Vehicle due north=eighteen; CBD due north=17). (C) Infusions during FR1 and FR3. Two-way ANOVA, *p<0.05, vehicle- vs CBD-treated group. (D) Full cocaine intake during acquisition of self-administration. Pupil'southward t test, *p<0.05. (Eastward) Exponential demand curve where consumption is plotted as a function of toll for vehicle- (n=eleven) and (F) CBD-treated group (due north=eight). (M) Consumption at a minimally constraining price or Q₀. (H) Need elasticity or α. (I) Signal of unit of measurement elasticity or P_max. (J) Maximum number of responses at P_max or O_max. (K) Active and inactive nosepokes during cue-induced seeking tests (Vehicle n=8; CBD n=7). Tukey's test, *p<0.05, baseline vs WD15. (L) Percentage of active nosepokes change from WD1. (M) Pct of incubation. (N) Active nosepokes for vehicle-treated (incubated mice n=iv; non-incubated mice north=4) and (O) CBD-treated (incubated mice n=i; mon-incubated mice n=6) groups. (P) Percentage of change from WD1 for vehicle-treated and (Q) CBD-treated groups. Sidak's test, **p<0.01, vs WD1; #p<0.05 and ##p<0.01, vs non-incubated mice.

Experiment three: Effects of CBD administered during incubation of cocaine craving

Mice followed the aforementioned protocol equally in Experiment 1, except that CBD (20 mg·kg^-1) was administered once a day from WD1 to WD15. Mice underwent cue-induced seeking tests on WD1, WD15 and WD30 (Figure 4a).

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Figure 4.

CBD has no effects on cocaine-craving incubation when administered during forbearance. (A) Schematical timeline of the experiment 2. (B) Active and inactive nosepokes during FR1 and FR3 (northward=30). Tukey's test, ***p<0.001 vs inactive hole. (C) Infusions during FR1 and FR3. (D) Exponential demand curve where consumption is plotted every bit a function of price (n=27). (Eastward) Agile and inactive nosepokes during cue-induced seeking tests (Vehicle north=fourteen; CBD n=14). Tukey's test, *p<0.05, baseline vs WD15. (F) Per centum of active nosepokes change from WD1. Tukey's exam, *p<0.05, WD1 vs WD15. (G) Percentage of mice that reached incubation criteria. (H) Active nosepokes for vehicle-treated (incubated mice north=7; not-incubated mice n=7) and (I) CBD-treated (incubated mice due north=five; not-incubated mice n=9) groups. Sidak's test, ***p<0.001, vs WD1. (J) Percentage of change for vehicle-treated and (K) CBD-treated groups. Sidak's examination, ***p<0.001, vs WD1; Sidak's test, **p<0.01, vs WD1; ###p<0.001, vs not-incubated mice.

two.three. Surgery

The surgical process was conducted as previously described (Soria et al., 2005; Lujan et al., 2020) with pocket-size changes. Surgical implantation of the catheter into the jugular vein was performed following anaesthetization with a mixture of ketamine hydrochloride (75 mg·kg^-1; Imalgène1000, Lyon, French republic) and medetomidine hydrochloride (1 mg·kg^-1; Medeson®, Barcelona, Kingdom of spain). The anaesthetic solution was injected in a volume of 0.15 mL·10 g^-1 trunk weight, i.p. Briefly, a vi cm length of silastic tubing (0.iii mm inner diameter, 0.half-dozen mm outer bore) (silastic, Dow Corning, Houdeng-Goegnies, Belgium) was fitted to a 22-gauge steel cannula (Semat, Herts, England) that was bent at a right angle and then embedded in a cement disk (Dentalon Plus, Heraeus Kulzer, Germany) with an underlying nylon mesh. The catheter tubing was inserted one.3 cm into the correct jugular vein and anchored with a suture. The remaining tubing ran subcutaneously to the cannula, which exited at the mid-scapular region.

Meloxicam (0.v mg·kg^-one s.c.; Metacam®, Barcelona, Spain), enrofloxacin (7.5 mg·kg^-1 i.p.; Baytril® 2.v%; Barcelona, Spain), atipamezole hydrochloride (0.five mg·kg^-1 i.p.; Revertor®, Barcelona, Espana), and 1 ml glucose 5% solution were injected later the surgery. Home cages were placed on thermal blankets to avoid mail-anaesthesia hypothermia. Mice were daily monitored for their weight and treated with meloxicam for 48 h and were immune to recover for four days before the conquering phase of the cocky-administration procedure began.

2.four. Acquisition of cocaine cocky-assistants

Cocaine operant self-administration was adjusted from Soria et al. (2005) and Luján et al. (2018). The self-administration experiments took place in operant conditioning chambers (Model ENV-307A-CT, Med Associates, Inc. Cibertec. Madrid. Espana) with two nosepoke holes. Animals were connected to a microinfusion pump (PHM-100A, Med-Assembly, Georgia, VT, Usa). Active and inactive nosepokes were selected randomly. Mice were first trained to self-administer cocaine in a FR1 schedule, where a nosepoke in the active pigsty resulted in the delivery of a twenty μl cocaine infusion over 2 seconds (0.75 mg·kg^-1·inf^-1) and a stimulus light for iv seconds. Each infusion was followed by a 15-second time-out menstruation. All sessions started with a priming infusion and lasted two hours. Mice switched to FR3 schedule for 5 days when the following criteria were met on three sequent days: a minimum of 5 infusions and 65% of responses received at the active nosepoke. No fourth dimension-out catamenia was applied in FR3. If animals did not meet the criteria within 10 days under FR1 schedule, they were removed from the study. Mice that failed to increase nosepokes >30% from baseline (three last days of FR1) during iii out of the 5 days of FR3 were also excluded from the study. No functioning-based exclusion criteria were applied in Experiment 2, since the treatments were administered during the acquisition and the fact that some animals failed in acquiring the self-administration behaviour either in FR1 or FR3 was considered an effect of the treatment and, therefore, subject to assay.

2.5. Demand task and behavioural economical assay

Demand job sessions consisted of a single 2-hour inside-session process adjusted from Bentzley et al. (2013). Acquired mice were placed on the operant cages and received a cocaine priming infusion (0.75 mg·kg^-1·inf^-1). The session was divided in 12 ten-minute bins, where the requirement for earning an infusion followed an ascending trend on the active nosepoke: 1, two, 4, 6, 8, xi, 14, 18, 21, 28, 35, 42. Inactive nosepokes had no consequences. The first bin of the session was considered a loading bin where the animate being would response in gild to achieve the preferred level of intoxication and not in a cost-dependent way. Therefore, this first bin was excluded from the analysis. Mice that had no active responses along the session (excluding the loading bin) were excluded from the analysis, since they failed on learning the job.

For the representation and the analysis of Q0 and α, we used a GraphPad template, kindly provided by the Institutes for Behavior Resource (IRB; Baltimore, Maryland, United states). Demand curve was analysed post-obit the exponential model (Hursh and Silberberg, 2008): logQ = logQ₀ + thou (e ^−αQ0C − 1). In the exponential demand bend graphs, consumption is plotted as a office of the price (responses·mg^-1), so when the price increases, the consumption decreases. The value of the constant chiliad was automatically calculated for each experiment and set up equal for all animals. Q₀ represents the consumption at a minimally constraining toll, meaning the preferred level of consumption with a minimum price. α is a measure of behavioural elasticity, which determines the sensitivity of need to changes in article price (Hursh, 1980). On general terms, a low α value is indicative of niggling changes in cocaine consumption in response to requirement increases. For the analysis of P_max and O_max, nosotros used the Excel figurer provided by Kaplan and Reed (2014). P_max or point of unit elasticity is considered a metric of motivation, and it refers to the maximal toll the creature is willing to pay to maintain the level of consumption. O_max is the number of responses that occur at P_max.

ii.half-dozen. Cue-induced seeking tests

The procedure for cue-induced seeking tests was adapted from Terrier et al. (2015). Mice were placed on the operant cages for two hours. An active nosepoke resulted in the presentation of a stimulus light, merely no cocaine infusion. Inactive nosepokes had no consequences. An animal was considered to limited incubation of craving when the increase in active nosepokes on WD15 was >50% from WD1 and/or the increase on WD15 and 30 was >thirty% from WD1. On Experiment 2, since cue-induced seeking tests were merely undertaken on WD1 and 15, an animal was considered to limited incubation of peckish when the increase of active nosepokes on WD15 was >50% from WD1.

2.7. Western blot

Animals were euthanized by cervical dislocation and PL, vSTR and amygdala were dissected and stored at -80°C. The tissue was homogenized in lysis buffer [0.15M NaCl, i% TX-100, x% glycerol, 1mM EDTA, 50mM TRIS pH=7.4 and a phosphatase and protease inhibitor cocktail (Roche, Basel, Switzerland)]. Equal amounts of poly peptide (sixteen μg) for each sample were mixed with loading buffer (153 mM TRIS pH = 6.8, vii.5% SDS, 40% glycerol, 5 mM EDTA, 12.5% 2-β-mercaptoethanol, and 0.025% bromophenol bluish), loaded onto ten% polyacrylamide gels, and then transferred to PVDF sheets (Immobilion-P, MERCK, Burlington, USA). Membranes were blocked for xxx minutes with five% bovine serum albumin at room temperature and so immunoblotted overnight at 4 °C using chief antibodies. On the side by side day, membranes were incubated for 1 h with their corresponding secondary fluorescent antibodies. Poly peptide expression was quantified using an Amersham™ Typhoon™ scanner and quantified using Image Studio Lite software v5.2 (LICOR, U.s.a.). Protein expression signals were normalized to the detection of housekeeping command protein in the same samples and expressed in terms of fold-change with respect to WD1 values.

2.8. Data and statistical analysis

The information and statistical analysis comply with the recommendations of the British Journal of Pharmacology on experimental design and analysis in pharmacology (Curtis et al., 2018). Except for demand curves, data are presented as hateful ± SEM. For statistical analysis nosotros used GraphPad Prism 8.0. software. When one, two or three factors were analysed, we used one, two or three-way ANOVA, respectively. When an experimental condition followed a within-subject design, repeated measures ANOVA was used. The factors used in this study were schedule (FR1/FR3), time (days or minutes), pigsty (active/inactive), treatment (CBD/vehicle) and incubation (incubated/non-incubated). The statistical threshold for significance was gear up at p<0.05. When F achieved significance and there was no significant variance in homogeneity, Tukey'south or Sidak's post hoc (when most appropriate) tests were run. We analysed the results of single factor with two levels, parametric measures with unpaired Student's t tests. Percentage of incubation was analysed using Fisher'due south exact exam.

2.ix. Materials

Cocaine HCl (0.75 mg·kg^-1·inf^-1) was purchased in Alcaliber S.A. (Madrid, Spain), and was dissolved in 0.9% NaCl. CBD (20 mg·kg^-1 i.p.) was provided by courtesy of Phytoplant Inquiry S.50., (Córdoba, Spain) and was dissolved in ethanol, cremophor E.Fifty. and 0.9% NaCl (0.5:ane:eighteen.5).

For Western blot, the primary antibodies used were anti-ERK_one/2 (i:thousand; Abcam; #ab54230; RRID:AB_2139967), anti-pERK_one/2 (1:2500; Abcam; #ab50011; RRID:AB_1603684), anti-GluA1 (1:one thousand; Abcam; ABN241; RRID:AB_2721164), anti-GluA2 (one:1000; Abcam; AB1768-I; RRID:AB_2313802) and anti-beta Three Tubulin (one:2500; Abcam; ab18207; RRID:AB_444319). The fluorescent secondary antibodies used were anti-mouse (ane:2500; Abcam; #ab216772; RRID:AB_2857338) and anti-rabbit (ane:2500; Rockland; 611-144-002-0.5; RRID:AB_11182462).

RESULTS

Experiment ane: Mice efficiently perform the demand task and incubation of cocaine craving paradigms

In order to validate the behavioural paradigm under our experimental conditions, a naive group of mice acquired cocaine self-administration, and so underwent need task and cue-induced seeking tests, equally shown on Figure 1A. Regarding the acquisition phase, the factors time, hole and their interaction reached significance indicating how mice learned to discriminate between active and inactive hole along the days, showing even a higher number of active responses when they were moved to FR3 (Figure 1B) in society to maintain the number of infusions (Figure 1C). Likewise, the intrasession data showed a significant effect for time and schedule, only not an interaction, indicating that mice on FR3 performed more active responses than on FR1 throughout the session (Figure 1D).

On need chore, 3 animals were excluded from the assay, since they failed to reach any infusion. The exponential model showed a R^two = 0.88 (Figure 1E), suggesting a expert fitting of the analysed data. The consumption at a minimally constraining price (Q₀) was 0.61 mg·kg^-one (Figure 1F), demand elasticity (α) was 0.017 (Figure 1F), animals displayed a maximal price (P_max) to maintain the consumption at 80.65 responses·mg^-1 (Figure 1H), and the number of responses at that toll (O_max) was viii.81 responses (Figure 1I).

Regarding the cue-induced seeking behaviour, the factors day and hole, just not their interaction, reached significance, indicating both, discrimination between agile and inactive holes and differential responding along withdrawal days (Figure 1J). More precisely, Tukey's mail service hoc analysis showed that on WD15 and WD30 mice displayed more than nosepokes than on baseline. When data were shown as the per centum of agile nosepokes change from WD1, no effect was found (Figure 1K).

Finally, mice were divided into two groups depending on whether they exhibited or non peckish incubation, according to their performance on the cue-induced seeking tests. When expressing the data every bit agile nosepokes (Figure 1M), no consequence was establish for incubation or mean solar day, just it was for their interaction. In particular, Sidak'south postal service hoc analysis revealed that incubated mice had more active responses on WD15 and WD30 compared to WD1, and the grouping that did not express incubation had fewer active responses on WD45 compared to WD1. Besides, incubated mice significantly had more than active responses than non-incubated mice on WD30. Yet, when expressing information as per centum of alter from WD1 (Figure 1N), the factors incubation, solar day and their interaction became pregnant. Sidak's post hoc analysis showed that the group showing incubation increased the percentage of responding on WD15, WD30 and WD45 compared to WD1, while the group that did not incubate showed no differences along days. Also, the percentage of change on WD30 was significantly higher on incubated mice compared to those that did non incubate.

Experiment ane: GluA1/GluA2 and pERK/ERK proteins levels are modulated during cocaine-craving incubation

To analyse the protein levels of GluA1, GluA2, ERK_one/2 and pERK_ane/2 during abstinence, mice were euthanised on WD1, WD15 and WD30 immadiately afterward the cue-induced seeking tests (Figure 2A). Regarding the results obtained in PL (Figure 2B), GluA1 protein expression was increased in WD30 compared to WD1, while GluA2 was increased on WD30 compared to WD15. However, no significant changes on the ratio GluA1/2 were observed. By contrast, both ERK_1/2 and pERK_1/2 remained unaltered during the forbearance.

On vSTR (Figure 1C), GluA1 protein levels were increased on WD30 compared to WD1, while no changes in GluA2 or GluA1/2 were observed. Besides, nosotros also found a decrease in the ratio pERK_1/2 /ERK_1/2 on WD30 compared to WD1.

Finally, the results obtained in amygdala (Effigy second) showed a decreased in GluA1/2 ratio on WD15 compared to WD1 and WD30, merely no changes in ERK_ane/2 phosphorylation.

Experiment 2: CBD decreases cocaine seeking and taking when administered during acquisition, just such effects are non maintained during abstinence

CBD treatment was administered just earlier the acquisition and demand task sessions (Effigy 3A). The nosepokes curve shows significant effects for time, hole and treatment, too as the interaction between time and pigsty (Effigy 3B). Regarding infusions, CBD-treated mice significantly self-administered fewer infusions forth the acquisition (Figure 3C), as it is too reflected in the total intake (Figure 3D).

For the demand task, mice that failed to encounter the acquisition criteria on FR1 and FR3 were excluded from the analyses as they could not encounter the requirements of the task. Besides, 3 and 4 mice in the vehicle- and CBD-treated groups, respectively, were as well excluded from the analysis as they failed to reach whatever infusion. The behavioural economic assay of the exponential demand curves for vehicle- (Effigy 3E) and CBD-treated groups (Effigy 3F) did not show significant differences amongst the different parameters (Figure 3G-J).

In this experiment, we also analysed the effects of CBD handling administered during the acquisition on the posterior incubation of cocaine craving on WD1 and WD15. The performance during the cue-induced seeking tests revealed a significant effect of time and hole, but non treatment or any interaction, indicating that animals incubated their response regardless of the treatment (Figure 3K). Specifically, we found an increase in the responding on WD15 compared to the baseline. When expressing active nosepokes every bit per centum of modify from WD1, no significant effects were institute (Figure 3L). Fisher's test for percent of incubation showed no significant differences (Figure 3M).

Finally, vehicle- and CBD-treated mice were divided into incubated or non-incubated groups depending on whether agile nosepokes on WD15 showed an increment of >50% from WD1. For the vehicle-treated group, the 2-manner ANOVA for active nosepokes revealed a significant interaction between time and incubation, but Sidak's post hoc showed no significant furnishings (Figure 3N). However, when expressed equally percentage of change, incubated mice showed a meaning increase in the percentage of change on WD15 while non-incubated group did not (Figure 3P). Besides, Sidak's post hoc assay besides showed that both groups of mice were significantly different on both days. For the CBD-treated grouping, no significant furnishings were found when data was expressed as active nosepokes (Figure 3O) or when expressed as per centum of change (Effigy 3Q), due to the fact that only one mouse met the criteria for incubation.

Experiment 3: CBD has no effects on cocaine-craving incubation when administered during abstinence

After mice underwent conquering for cocky-administration (Figure 4B, C) and demand task (Figure 4D), they were treated with CBD or vehicle for fifteen days and tested for cue-induced seeking on WD1, WD15 and WD30 (Effigy 4A). Three-way ANOVA for nosepokes during the cue-induced seeking behaviours revealed a significant upshot for time and hole, merely not for handling or any interaction (Figure 4E). More than precisely, Tukey's post hoc showed that mice conducted more responses on WD15 compared to the baseline. When data was expressed as per centum of alter from WD1, time, but not handling nor their interaction, revealed a significant outcome, being the per centum of change during WD15 increased compared to WD1 (Figure 4F). Fisher's exam for percent of incubation showed no pregnant differences (Figure 4G).

Mice were then divided into two groups depending on their craving-incubation exhibition. For the vehicle-treated group, the two-way ANOVA revealed a significant interaction of time and incubation for both active nosepokes (Figure 4H) and percentage of change (Figure 4J). For active nosepokes, Sidak's post hoc analysis showed that incubated mice conducted more agile responses on WD15 compared to WD1. When expressed every bit percentage of modify, nosotros observed that mice that showed incubation too conducted more agile responses on WD15 compared to WD1 and compared to the mice that did not evidence incubation. For the CBD-treated group, two-style ANOVA for active nosepokes revealed a significant upshot for fourth dimension and the interaction of time and incubation (Figure 4I). Sidak's post hoc analysis showed that incubated mice performed more active nosepokes on WD15 compared to WD1. When data were expressed as percentage of change from WD1, two-way ANOVA showed a significant effect for fourth dimension, incubation and the interaction betwixt factors (Effigy 4K). The post hoc assay revealed that incubated mice increased the per centum of change on WD15 compared to WD1 and to mice that did non show incubation.

DISCUSION

Incubation of cocaine craving is an of import process contributing to the high risk of relapse in both humans and rodents. Mainly, rat models of cue-induced cocaine craving take been used to study the neurobiological components underlying such process. In the nowadays study, we adult a mouse model for behavioural economic analysis of demand curves and incubation of cocaine craving in mice. The molecular analysis indicated changes in GluA1 and GluA2 protein levels along forbearance in PL, vSTR and amygdala, likewise as a subtract in ERK_1/2 phosphorylation in vSTR. Besides, nosotros observed a differential effect of CBD depending on the handling regime. Thus, CBD was able to reduce cocaine seeking in the self-administration paradigm when administered during the acquisition, but failed to modify the subsequent incubation of cocaine craving. Similarly, CBD administered during the cocaine forbearance period did not exert whatever effect upon incubation.

Behavioural economic analysis of demand curves has been lately applied to rat models of drug consumption (Bentzley et al., 2014; Calipari et al., 2018). However, this analysis has never been employed in mice despite their extended apply in the field of addiction research. Based on previous research (Bentzley et al., 2013), we used a single within-session procedure with ascending unit prices. The increase of the prices in every bin was adapted to brand it more suitable for mouse demands, that is, lower increment in each bin compared to rat models. In Experiments 1 and 3, the exponential need curves had a reasonable fit to the mouse performance on the chore (r^two 0.88 and 0.94, respectively) and most of the animals were able to complete it consuming at any bin. For Experiment 2, the fit of the model was not as good as expected. This may be due to unlike reasons. Outset, since mice were divided into two dissimilar groups (vehicle- and CBD-treated mice), the sample size was smaller compared to the previous experiment, complicating the extraction of an accurate curve. Second, this kind of task requires from animals considerably engaged with drug-seeking behaviour. Since assistants of CBD during acquisition macerated this behaviour, CBD-treated mice consumed less during the need task. Thus, the exponential model is provided with more express information, resulting in a poorer fitting of the data. Keeping these limitations in mind, behavioural assay of need curves is a highly valuable tool in addiction research equally information technology offers a wide range of measures, such equally consummatory behaviour, motivation and behavioural elasticity (Hursh and Silberberg, 2008; Bentzley et al., 2013), which is at present prepared to be applied in mouse models of cocaine self-administration.

We also presented a mouse model for incubation of cocaine craving later on short-admission cocaine cocky-administration. To our knowledge, simply two studies take previously utilized mice as experimental subjects (Terrier et al., 2015; Nugent et al., 2017). Our findings signal that WD15 represented the peak of incubation, followed by WD30, whereas on WD45 responding levels were already lower. Similarly, Nugent et al. (2017) found the maximal responding on WD28 also after a short-access cocaine self-administration. Although, in this example, the cocaine dose was lower (0.v mg·kg^-1·inf^-ane) than in our study (0.75 mg·kg^-1·inf^-1). Furthermore, Terrier et al. (2015) plant no craving incubation when applying a short-admission self-assistants with the aforementioned dose used in our written report (0.75 mg·kg^-1·inf^-1). Notwithstanding, since we found the maximal response on WD15 and they only tested cocaine craving on WD1 and WD30, information technology is possible that the incubation did in fact occur, but they did non observe information technology.

Regarding the molecular results, we found an increase in GluA1 subunit on WD30 within the vSTR, although the GluA1/ii ratio remained unaltered. Since NAc core and beat take opposite roles on either promoting or inhibiting incubation of cocaine peckish, respectively, and nosotros analysed the whole vSTR, it is possible that detached changes in any of these areas were mutually curtained. Otherwise, larger expositions to the drug (i.e., long admission) would possibly cause more evident changes in the glutamatergic system (Purgianto et al., 2013). Surprisingly, Western absorb analysis revealed a subtract in ERK_1/2 phosphorylation on WD30 coinciding with the GluA1 subunit increase. Since ERK_one/ii phosphorylation is related to neural activeness and plasticity, these results could be somehow conflicting. Yet, a written report plant that ERK₁ knockout mice presented an enhanced synaptic plasticity in the STR (Mazzucchelli et al., 2002). In the same line, Sun et al. (2015) reported a decrease of pERK_1/2 in the NAc associated with the incubation of heroin seeking. Otherwise, our results show an increase of both GluA1 and GluA2 poly peptide levels on WD30 in PL, but no differences on the ratio. In accord, Ma et al. (2014) demonstrated that PL neurons projecting to NAc mature during withdrawal past recruiting non-CP-AMPARs, which could explain the lack of increase in GluA1/2 ratio. Alternatively, albeit both GluA1 and GluA2 subunits are increased on WD30, they could differ in the number of subunits that are either internalised or expressed on the jail cell surface (Conrad et al., 2008). Besides, we found no changes on ERK_1/two phosphorylation along abstinence. Accordingly, Whitfield et al. (2011) besides reported no differences in ERK_1/two phosphorylation on WD7 in the dorsomedial PFC in rats. Finally, we found a decrease of GluA1/two ratio on WD15 compared to WD1 and WD30 in the amygdala, although nosotros cannot distinguish whether this finding is due to changes in the CeA or in the BLA, which could imply unlike functional changes (Wolf, 2016). Furthermore, despite the fact that ERK activation in CeA has been shown to be critical for the incubation of cocaine craving (Lu et al., 2005b), we establish no differences throughout forbearance.

Previous work in our laboratory has already provided evidence supporting the beneficial role of CBD in the handling of cocaine-seeking behaviour. In particular, Luján et al. (2018) demonstrated that CBD (twenty mg·kg^-ane), when administered during the acquisition phase (FR1) of cocaine self-administration, is able to reduce cocaine-seeking and -taking behaviours, an effect that is dependent on developed hippocampal neurogenesis (Luján et al., 2019). Similarly, we besides establish a decrease in cocaine self-administration when CBD was concomitantly administered during the acquisition process. Indeed, such effect was likewise maintained when the requirements for each infusion became more exigent (FR3). Nonetheless, CBD-induced subtract in cocaine-seeking behaviour faded during cue-induced cocaine-seeking tests, and both vehicle- and CBD-treated mice incubated their responsiveness over the withdrawal period.

When CBD was administered in the abstinence period, no effect was found during acute (WD1) or prolonged (WD15) treatment, or later (WD30). In a previous study (Gonzalez-Cuevas et al., 2018), CBD was able to reduce drug-seeking behaviour induced by stress or context exposure, although no assay for incubation of cocaine peckish was performed. Moreover, various methodological differences might hinder the comparisons, including the experimental designs, creature species (rat vs mouse), doses of cocaine (0.25 vs 0.75 mg·kg^-1·inf^-1) and CBD (15 vs xx mg·kg^-ane) or routes of assistants (transdermal vs intraperitoneal). A more recent report by Gasparyan et al. (2020) demonstrated that CBD normalized motor and somatic disturbances in a spontaneous cocaine withdrawal model in CD1 mice. In this instance, CBD would play a beneficial role in the management of concrete symptoms associated with cocaine withdrawal, an approach which is substantially different from ours.

In the nowadays study, nosotros applied a criterion for determining whether mice had incubated their seeking behaviour due to the exposition to cocaine-associated cues. Therefore, we can observe considerably unlike curves for mice that express cocaine-craving incubation and mice that do not. This observation is helpful at the fourth dimension of validating the following findings: (I) the existence of two different populations of mice regarding their expression of craving incubation, and (Ii) the lack of CBD effectivity on the incubation of cue-induced cocaine-seeking behaviours, since the curves of both vehicle- and CBD-treated groups resemble the same behaviour.

Altogether, we suggest that CBD only exerts its protecting effects on cocaine-seeking behaviour while learning processes are taking place. Thus, CBD was able to reduce drug-seeking behaviour, but could not influence the formation of long-lasting memories associated to the operant task. Hence, once the treatment ceased, the furnishings of CBD faded and CBD-treated animals were able to incubate cue-induced seeking behaviour equally much as control animals. On the same note, CBD administered during the withdrawal menses (in one case the acquisition of self-administration had finished) was not able to revert the seeking behaviour that had already been established in these animals. Therefore, according to this hypothesis, CBD's lack of furnishings could be due to the fact that it was not being concomitantly administered when the operant learning was taking place.

In conclusion, we provide a novel model of behavioural economic analysis of demand curves and cue-induced incubation of cocaine-seeking behaviour in mice. Furthermore, nosotros also provide bear witness supporting that CBD is able to reduce cocaine consumption when administered during the acquisition of self-administration but information technology shows no effects over incubation of cocaine craving, suggesting that CBD is but effective when administered while learning of seeking and taking behaviours are taking place.

AUTHOR CONTRIBUTIONS

L.A.Z. and O.5. were responsible for the study concept and design. Fifty.A.Z., L.C, A.Grand.Southward. and A.G.B. carried out the experimental studies. L.A.Z. and Yard.A.L. programmed the demand task code and analysis. L.A.Z and O.V. drafted the manuscript. All authors critically reviewed the content and approved the terminal version for publication.

Conflict OF INTEREST

The authors declare no conflicts of involvement

Announcement OF TRANSPARENCY AND SCIENTIFIC RIGOUR

This Declaration acknowledges that this paper adheres to the principles for transparent reporting and scientific rigour of preclinical enquiry as stated in the BJP guidelines for Blueprint & Analysis, Immunoblotting and Immunochemistry, and Animal Experimentation, and equally recommended by funding agencies, publishers, and other organizations engaged with supporting research.

ACKNOWLEDGEMENTS

This study was supported past the Ministerio de Economia y Competitividad (grant number PID2019-104077RB-100), Ministerio de Sanidad (Retic-ISCIII, RD16/017/010 and Plan Nacional sobre Drogas 2018/007). 50.A.Z received a FPI grant (BES-2017-080066) from Ministerio de Economia y Competitividad. A.G.B received a FI-AGAUR grant from the Generalitat de Catalunya (2019FI_B0081). The Department of Experimental and Health Sciences (UPF) is a "Unidad de Excelencia María de Maeztu" funded past the AEI (CEX2018-000792-One thousand).

ABBREVIATIONS

BLA

Basolateral amygdala

CBD

Cannabidiol

CeA

Primal nucleus of the amygdala

CP-AMPAR

Calcium-permeable α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor

ERK_one/ii

Extracellular signal-regulated kinase 1 and 2

R

Fixed ratio

IL

Infralimbic cortex

NAc

Nucleus accumbens

pERK_1/2

Phosphorylated extracellular betoken-regulated kinase 1 and ii phosphorylation

PL

Prelimbic cortex

vSTR

Ventral striatum

WD

Withdrawal day

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Source: https://www.biorxiv.org/content/10.1101/2020.12.18.423391v1.full

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