Ethanol pre-exposure differentially impacts the rewarding and aversive effects of α-pyrrolidinopentiophenone (α-PVP): Implications for drug use and abuse

Abstract

Rationale:

Exposure to a drug can subsequently impact its own reactivity as well as that of other drugs. Given that synthetic cathinones, i.e., “bath salts”, users typically have extensive and varied drug histories, an understanding of the effects of drug history on its behavioral and physiological effects may be important to its abuse liability.

Objectives:

The goal of the current work was to assess the effects of an ethanol pre-exposure on the rewarding and aversive effects of α-PVP.

Methods:

Adult male Sprague Dawley rats were exposed to ethanol prior to combined conditioned taste avoidance/conditioned place preference training in which rats were injected with 1.5, 3 or 5 mg/kg of racemic α-PVP or vehicle. Following a 7-day washout period, rats were then tested for thermoregulatory effects of α-PVP using subcutaneous probes to measure body temperature changes over the course of 8 h. This was followed 10 days later by assessments for α-PVP-induced locomotor activity and stereotypies over a 1-h session.

Results:

α-PVP induced significant dose- and trial-dependent taste avoidance that was significantly attenuated by ethanol history and dose- and time-dependent increases in locomotor activity that were significantly increased by ethanol. α-PVP also induced place preferences and dose- and time-dependent increases in body temperature, but these measures were unaffected by ethanol history.

Conclusions:

α-PVP’s aversive effects (as measured by taste avoidance) were attenuated, while its rewarding effects (as indexed by place preference conditioning) were unaffected, by ethanol pre-exposure. Such a pattern may indicate increased α-PVP abuse liability, as changes in the balance of aversion and reward may impact overall drug effects and likelihood of drug intake. Future self-administration studies will be necessary to explore this possibility.

1. General Introduction

Synthetic cathinones (also known colloquially as “bath salts”) are a class of novel psychoactive substances that mimic the behavioral effects of psychostimulants such as amphetamine, methamphetamine, 3,4-methyl​enedioxy​methamphetamine (MDMA) and cocaine. One such compound, i.e., α-pyrrolidinopentiophenone (aka, α-PVP or “flakka”), became available following the scheduling of the first-generation cathinone 3,4-methylenedioxypyrovalerone (MDPV), a synthetic cathinone to which α-PVP is structurally and mechanistically similar (Baumann et al., 2016; Glennon and Young, 2016; Karila et al., 2018). Like MDPV, α-PVP is an uptake inhibitor of dopamine (DA) and norepinephrine (NE) at their respective transporters (DAT and NET) with little to no action at the serotonin (5-HT) transporter (SERT), leading to a buildup of DA and NE in the synaptic space (the IC₅₀ for uptake inhibition at DAT for α-PVP is 12 ±1 nM; the IC₅₀ for NET is 14 ±1 nM; for the IC₅₀ for SERT >10,000 nM; see Baumann et al., 2016; Eshleman et al., 2017; Glennon and Young 2016; Marusich et al. 2014; Meltzer et al., 2006). Similar to many psychostimulants (and related synthetic cathinones), α-PVP has rewarding (Gatch et al., 2015; Marusich et al., 2016; Nelson et al., 2017; Nelson et al., 2019b; Watterson et al., 2014) and aversive (Nelson et al., 2017; 2019a,b) effects, the balance of which is thought to be important to its self-administration (Cunningham, 1979; Lin et al., 2017; Stolerman and D’Mello, 1981; Verendeev and Riley, 2012; for examples of α-PVP’s reinforcing effects see Collins et al., 2019; Gannon et al., 2017; Gannon et al., 2018a,b; Schindler et al., 2019).

Although α-PVP has been investigated in relation to reward and aversion, factors that impact these effects have received little attention (for a review, see Riley, 2011; see also Baker and Cannon, 1982; Cunningham et al., 2008; Stolerman and D’Mello, 1981). One factor that has been reported to impact the rewarding and aversive effects of drugs in general is drug history (Albaugh et al., 2011; Davis and Riley, 2010; Gaiardi et al., 1991; Gauvin et al., 2000; Grakalic and Riley, 2002a,b; Lett, 1989; Shippenberg et al., 1996). The impact of a drug history is particularly relevant in this context as the vast majority of synthetic cathinone users engage in polydrug abuse (see Assi et al., 2017; Guirguis et al., 2017; James et al., 2010; López-Rodriguez and Viveros, 2019; Miotto et al., 2013; Prosser and Nelson, 2012; Romanek et al., 2017; Schifano et al., 2020; Schmoll et al., 2018).

Polydrug use has been defined as the use of multiple drugs in a single session (concurrent/simultaneous use) or the use of multiple drugs on separate occasions (sequential use) (for a recent review on polydrug use, see Crummy et al., 2020). In the greater synthetic cathinone-using population, concurrent polydrug use is prevalent. For example, the synthetic cathinones are popularly used by individuals who belong to certain subcultures, e.g., clubbers, music festival revelers, those who inject drugs, chem sex party attendees and abstinence treatment entrants (Busardo et al., 2020; Guirguis et al., 2017; Mohr et al., 2018; Romanek et al., 2017) known to engage in frequent recreational polydrug use (for more information see Calle et al., 2019; Castellanos et al., 2018; Donnadieu-Rigole, 2020; Gyarmathy et al., 2017). Interestingly, cathinone users regularly do not know the extent of their own polydrug use due to the fact that the cathinone products are often adulterated prior to sale to maintain their weight, enhance the dealer’s profits and to improve their intoxicating effects all without the user’s knowledge (Guirguis et al., 2017).

Even though it is well recognized that synthetic cathinone users take multiple drugs concurrently or are found to have more than one drug in their system at the same time (Brusin et al., 2017; Donnadieu-Rigole, 2020; Gyarmathy et al., 2017; Patocka et al., 2020), documentation of the unambiguous serial use of drugs involving the cathinones is limited as clinical surveys and case reports often do not document the temporal order in which users build their drug repertoires, their serial use of drugs or their general drug history (Crummy et al., 2020; Leri et al., 2003; López-Rodriguez and Viveros, 2019; Riley, 2011).

Although it is difficult to know the specific temporal order of what drugs are frequently used before the synthetic cathinones are ever encountered by an individual, it is possible to look at patterns of drug use in school-aged children (grades 8 – 12) and compare these patterns to what is seen in the synthetic cathinone user base (aged 18 – 35). According to the most recent Monitoring the Future (MTF): National Survey Results on Drug Use for the United States (Johnston et al., 2021), alcohol (ethanol) is the most widely used substance by teenagers with nearly two out of every three students in the year 2020 consuming more than just a few sips at some point in their lives and 26% doing so by the 8th grade. Interestingly, “bath salt” use in this population in 2018 was 0.9%, 0.5%, and 0.6% in 8th, 10th and 12th graders, respectively (data on the prevalence of the “bath salts” was not collected in 2019 or 2020 due to the Covid-19 pandemic). Given these patterns of drug intake, i.e., exposure to alcohol in young individuals and an older demographic for “bath salt” users, understanding the possible impact of early alcohol use on the synthetic cathinones may be important to predicting their abuse liability.

Interestingly, prior drug exposure has been reported to alter both the rewarding and aversive effects of drugs (see above). In this context, Simpson and Riley (2005) reported that Sprague Dawley (SD) rats exposed to morphine prior to a combined conditioned taste avoidance/conditioned place preference (CTA/CPP) procedure with morphine displayed increased place preferences and attenuated taste avoidance (see Gaiardi et al., 1991; Lett, 1989; Shippenberg et al., 1996; see also Martin et al.,1998 where morphine pre-exposure had no effect on morphine-induced CPP). Hutchison and Riley (2012) demonstrated that adolescent and adult male SD rats exposed to ethanol prior to CPP training displayed preferences at a lower dose than non-pre-exposed animals and a weaker ethanol-induced taste avoidance (Hutchison et al., 2010; although see Busse et al., 2005 where an ethanol pre-exposure had no effect on the rewarding effects of cocaine or a cocaine + ethanol combination in the CPP design). Increased reward (see Horger et al., 1992; Lett, 1989; Kalivas et al., 1998; Schenk and Izenwasser, 2002) and attenuated avoidance (see Barker and Johns, 1978; Batson and Best, 1979; Braveman, 1975; Cappell and LeBlanc, 1975; Randich and LoLordo, 1979; Riley and Simpson, 2001; Risinger and Cunningham, 1995) following drug history have been reported for a variety of drugs and in a number of behavioral procedures.

In one of the few studies to assess the effect of a drug history with the synthetic cathinones, López-Arnau et al. (2017) showed that MDPV pre-exposure during adolescence increased breakpoints for cocaine intravenous self-administration (IVSA), cocaine-induced reinstatement and cocaine-induced place preferences (for other work assessing serial drug exposure with cathinones, see Creehan et al., 2015; see also Listos et al., 2017 for a demonstration of the effects of mephedrone pre-exposure in adolescent rats on the rewarding effects of morphine in adults). Conversely, Woloshchuk et al. (2016) found that MDPV pre-exposure attenuated taste avoidance induced by MDPV and cocaine, but not LiCl. Similar to effects reported with other compounds, drug history significantly impacts the rewarding and aversive effects of synthetic cathinones.

Given the relatively limited information regarding the effect of serial drug interactions involving the synthetic cathinones in general and no published work assessing this effect with α-PVP, the goal of the current work was to determine the effects of drug pre-exposure on the rewarding (CPP) and aversive (CTA) effects of α-PVP. Specifically, animals were exposed to ethanol prior to taste avoidance and place preference conditioning with α-PVP. Ethanol was chosen for these assessments given that human cathinone users will likely have engaged in ethanol use prior to their first encounter with α-PVP (see above discussion of Monitoring the Future; Johnston et al., 2021). In the context of previous findings assessing the effects of an ethanol pre-exposure on cocaine’s rewarding (as assessed by a CPP design; see Hutchison et al., 2012) and aversive (as assessed by CTA; see Grakalic and Riley 2002a,b; Hutchison et al., 2010) effects and the similar mechanisms of action of cocaine and α-PVP (Baumann et al., 2016; Eshleman et al., 2017; Marusich et al., 2014; Meltzer et al., 2006), an ethanol history should attenuate the aversive (and increase the rewarding) effects of α-PVP. Given earlier clinical reports of elevated body temperature and locomotor agitation (adverse reactions related to psychostimulant use and abuse) and their contribution to α-PVP overdose (see Castellanos et al., 2018; Cherry and Rodriguez, 2017; Grapp et al., 2016; for a review of clinical data with MDPV and α-PVP, see Karila et al., 2018), these endpoints were also assessed for the impact of ethanol pre-exposure.

2. General Methods

2.1. Subjects

The subjects were 96 experimentally naïve, adult male SD rats. Subjects were trained in two replicates where each replicate contained 48 animals and included all tested groups (see below). Animals were either bred within the American University animal research facility and were allowed to mature undisturbed until the start of testing (Replicate 1) or they entered the animal research facility at American University on post-natal day (PND) 74 and allowed to acclimate until the start of testing (Replicate 2; Envigo, Indianapolis, IN). Beginning on approximately PND 80, all animals were weighed daily for 8 days to index health status and to reduce handling stress during experimental procedures. At the start of experimental procedures (approximately PND 90), subjects weighed between 288 and 490 grams and throughout testing were socially housed in OptiRat Plus cages (38.9 × 56.9 × 26.2 cm; 1181 cm²). The room in which the cages were located was maintained on a 12-h light/dark cycle (08.00 – 20.00h) at 23 °C. Training and testing took place during the lights-on phase of the light cycle, and unless otherwise stated, food and water were available ad libitum. All procedures adhered to the Guidelines for the Care and Use of Laboratory Animals (National Research Council, 2011) and the Guidelines for the Care and Use of Mammals in Neuroscience and Behavioral Research (National Research Council, 2003) and were approved by the Institutional Animal Care and Use Committee at American University.

2.2. Drugs and Solutions

Ethanol was prepared as a concentration of 15% by being diluted with isotonic saline (0.9%) from a 95% stock solution and was administered intraperitoneally (IP) at a dose of 2 g/kg. This dose of ethanol was based on previous work from our laboratory and others (Berman and Cannon, 1974; Grakalic and Riley, 2002b; Hutchison and Riley, 2012; Hutchison et al., 2010; Kulkosky et al., 1980; Miceli et al., 1980) reporting taste avoidance with ethanol and its attenuation with ethanol history. Racemic α-pyrrolidinopentiophenone HBr (α-PVP) (synthesized and generously provided by the Drug Design and Synthesis Section, MTMDB, NIDA and NIAAA) was dissolved in isotonic saline and injected IP at 1.5, 3 or 5 mg/kg. These doses were based on previous work in our laboratory in which racemic α-PVP at 3 mg/kg induced a significant, but intermediate, suppression of saccharin consumption and 6 mg/kg produced almost complete suppression (Nelson et al., 2017; 2019a). These doses have also been examined across a range of behavioral and physiological indices (Aarde et al., 2015; Gatch et al., 2015; Marusich et al., 2014; Schindler et al., 2019; Watterson et al., 2014) and are encompassed in the range of doses from clinical reports (from 1 to < 20 mg nasally; from 1 to < 25 mg orally; Zawilska and Wojcieszak, 2017). Each drug (and vehicle) solution was prepared daily and passed through a 0.2-μm filter prior to injection to remove any potential particulates. For α-PVP, concentration was held constant across doses and the injection volume varied with the animal’s weight. Equivolume isotonic saline (vehicle) was administered to controls. Saccharin (Sodium Saccharin, Acros Organics) was prepared as a 1 g/l (0.1%) solution in tap water.

2.3. Apparatus

During taste avoidance conditioning, subjects were placed in individual hanging, stainless-steel wire-mesh test cages (24.3 × 19 × 18 cm) on the front of which graduated Nalgene tubes could be placed for fluid access. For place preference testing, animals were placed in one of eight identical testing apparatuses. These measured 68.5 × 21 × 34.5 cm (San Diego Instruments Place Preference System, San Diego, CA) and were divided into three separate areas, each equipped with a 16 × 4 photo beam array near the floor of the apparatus to record location and time spent in specific locations within each chamber. The left side chamber of the apparatus (28 × 21 × 34.5 cm) had white walls and white aluminum diamond-plate flooring, and the right side (28 × 21 × 34.5 cm) had black walls and black plastic haircell textured flooring. The middle connecting chamber (14 × 21 × 34.5 cm), which was not used for conditioning, had grey walls and metal grid flooring consisting of stainless-steel rods spaced approximately 1 cm apart. The place preference chambers (as well as the room in which the chambers were located) were unlit in order to create an unbiased apparatus (see Clasen et al., 2020; Cunningham et al., 2003; Nelson et al., 2019b; Roma and Riley, 2005). A white noise generator was used to mask background noise.

For subsequent activity assessments, animals were placed in the same testing apparatuses as during CPP assessments, but each apparatus was converted so that they no longer had the appearance of clearly defined areas (see Hutchison et al., 2010). The walls of each apparatus were clear Plexiglas with a grey plastic box placed around the outside to give the effect of opaque grey walls, and the floor was covered with a single 68.5 × 21 cm sheet of haircell textured grey Kydex plastic. The 16 × 4 photo beam array described previously was used in this phase to record both locomotor activity (consecutive beam breaks) as well as stereotypies (repeated breaking of the same beam) (San Diego Instruments Place Preference System, San Diego, CA). Each apparatus had four white LED lights set to maximum brightness within the otherwise unlit room, and a white noise generator was again used to mask background noise.

2.4. Procedure

2.4.1. Ethanol Pre-exposure

At the outset of experimental procedures, rats were deprived of water for 24 hours before being transferred to the stainless-steel hanging test cages and given 20-min access to tap water delivered in graduated 50-ml Nalgene tubes. Fluid intake was calculated by measuring the difference between pre- and post-consumption volumes. Following fluid access, the animals were returned to their home cages. The rats were given 6 days to habituate to this limited fluid-access procedure. On the 7^th day of limited fluid access, rats were divided into two groups based on water consumption such that the average fluid intake was equivalent between groups. Five hours after the fluid intake period, rats were removed from their home cages and taken to an adjacent room where they were injected with either ethanol (2 g/kg, IP) or equivolume isotonic saline (the vehicle, IP) before being returned to their home cages. For the following 3 days, rats were given 20-min access to water but were not injected. This procedure of ethanol exposure followed by 3 recovery days was repeated for a total of five cycles (over 26 days, see Figure 1).

Figure 1.

An illustrated timeline of the behavioral procedures. Created with BioRender.com

2.4.2. Combined Conditioned Taste Avoidance and Conditioned Place Preference

Most drugs of abuse have both rewarding and aversive effects which impact its abuse liability (Cunningham, 1979; Lin et al., 2017; Riley, 2011; Stolerman and D’Mello, 1981; Verendeev and Riley, 2012). Specifically, the drug’s rewarding effects establish its use, whereas the drug’s aversive effects are a consequence of this use and serve to limit drug intake. The balance of these two affective properties of a drug impacts the likelihood of its subsequent intake. Furthermore, these affective properties can be dissociated and impacted differently by a host of physiological, behavioral and pharmacological manipulations (for a discussion, see Cunningham et al., 2008; Verendeev and Riley, 2012). The combined CTA/CPP design is a concurrent measure of both drug reward and aversion in which animals are exposed to a novel taste, subsequently injected with a dose of drug and then placed on one side of a place preference chamber. Avoidance of the drug-paired taste and preference for the drug-paired side are reflective of the aversive and rewarding effects of the drug, respectively (see Brockwell et al., 1991; Clasen et al., 2020; Hempel et al., 2016; King et al., 2015b; Reicher and Holman, 1977; Sherman et al., 1980; Simpson and Riley, 2005; for a general review of the combined CTA/CPP design, see Davis, 2013).

2.4.2.1. Pre-Test

On Day 1 of this phase, subjects were given 20-min access to water in the test cages before being placed into the middle grey chamber of the place preference apparatus and allowed to freely explore all chambers for 15 min, at which point they were returned to their home cages. Time spent in each area was recorded via photobeam breaks. Each apparatus was wiped down with a cleaning solution (Sani-Plex 128M, 1-step disinfectant germicidal detergent) between animals. For each replicate, a paired sample t-test of absolute time spent on the white side vs. absolute time spent on the black side during the 15-min Pre-Test indicated an unbiased apparatus (both ps > 0.05). Time spent in the middle chamber was not used for conditioning or used in the calculation of side preferences.

2.4.2.2. Conditioning

On the day following the place preference Pre-Test, all subjects were placed in the test cages and given 20-min access to a novel saccharin solution. Subjects from both pre-exposure groups (ethanol and vehicle) were run in each replicate and matched on saccharin consumption within each group. Approximately 15-min following saccharin access, subjects in each of the two pre-exposure groups were then assigned to one of four drug groups and injected (IP) with either the saline vehicle or 1.5, 3 or 5 mg/kg of racemic α-PVP. This resulted in a total of eight experimental groups, i.e., Groups VV, V1.5, V3, V5, EV, E1.5, E3 and E5 where the first letter indicates the pre-exposure condition (vehicle or ethanol) and the second letter or number indicates vehicle or the dose of α-PVP (1.5, 3 or 5 mg/kg) (n = 12 per experimental group). Immediately following the injections, subjects were placed on one side of the place preference apparatus in a counterbalanced fashion such that half the animals in a given group were placed on their preferred side (defined as the side on which they spent the most time during the Pre-Test) and the remaining half were placed on their non-preferred side for 30 min. Subjects were then returned to their home cages, and the testing chambers were sanitized prior to the introduction of the next set of animals. On the next day (Day 2), subjects were given 20-min access to water in the test cages, injected with vehicle and placed on the opposite side of the place preference chamber for 30 min. This 2-day cycle was repeated for a total of four cycles.

2.4.2.3. Post-Test

On Day 10, animals were given 20-min access to tap water, placed in the center gray middle area of the place preference apparatus and once again given 15 min to investigate all three chambers (Post-Test). Time spent in each area was recorded via photobeam breaks, and the time spent in the two conditioning chambers was evaluated for any changes in side-preference relative to the amount of time spent in the chambers on the Pre-Test.

2.4.3. Temperature

Following a 7-day wash-out period subsequent to combined CTA/CPP testing (see above), temperature probes (Bio Medic Data Systems, Seaford, DE; Model #IPTT-300) were implanted subcutaneously between the shoulder blades of each animal. This procedure took place while the animals were under inhaled isoflurane anesthesia, and the surgical site was first sterilized with 70% ethanol before implantation occurred. Subjects were allowed to recover undisturbed for 1 day. On the following 3 days they were weighed to check for health status and the temperature transponders were scanned to check for proper functioning. After these preliminary checks, all subjects were again scanned for temperature, weighed and then injected IP with saline for 3 days. The initial scans taken on these days were not used in any statistical assessments as they were used only for habituating the animals to the procedure and confirming the proper functioning of the equipment. For baseline measures, rats were then weighed, scanned and injected with 0.3 ml saline and additional temperature scans were taken 30-min, 1-h and again every hour up until 8-h post injection. The final procedure was similar to the day before with the exception that subjects were randomized into new experimental groups before being injected with either the saline vehicle or 1.5, 3 or 5 mg/kg racemic α-PVP, resulting in new Groups VV, V1.5, V3, V5, EV, E1.5, E3 and E5 (n = 12 per group). For each temperature recording, the probe was scanned three times and the three measurements averaged. Temperature measurements at any given time point for any specific animal typically varied by only ± 0.1 degree. All temperature assessments took place in the animal colony during the lights-on phase. Rats remained in their home cages except for when they were removed for weighing, injections and scanning.

2.4.4. Activity and Stereotypies

Following an additional 10-day wash-out period, animals were weighed and handled for 3 consecutive days. Following this handling period, they were weighed and then injected IP with 0.3 ml saline (vehicle). Immediately following the injection, animals were taken to an adjacent room and placed into the activity testing apparatuses. Counts of gross activity (consecutive beam breaks) and stereotypies (repetitive beam breaks) were recorded for each animal over a 1-h session that was broken up into twelve 5-min bins. Activity is assessed by consecutive beam breaks which assay lateral movement within the chamber. Stereotypies are fine motor movements such as repetitive head motions, sniffing the same locations repeatedly, repetitive circular motions and repetitive grooming behaviors, all of which result in repetitive breaks of the same photobeam. The 1-h assessment of activity/stereotypies is similar to that often used in such studies (see Baumann et al., 2013; Berquist et al., 2016; Nelson et al., 2019b) and is sensitive to the behavioral differences in activity induced by synthetic cathinones. On the next day of testing, the procedure was identical to the previous day, with the exception that subjects were randomized into their third and final experimental groups before being injected with either saline or 1.5, 3 or 5 mg/kg racemic α-PVP, again resulting in Groups VV, V1.5, V3, V5, EV, E1.5, E3 and E5 (n = 12 per group). Eight activity chambers were used, and each animal was placed in the same chamber on each testing day. Chambers were sanitized (Sani-Plex 128M, 1-step disinfectant germicidal detergent) between animals.

2.5. Statistical Analysis

Data from each of these assessments were analyzed using SPSS statistical software (IBM SPSS Statistics) in separate mixed model ANOVAs where the between subject factors in each was Dose [vehicle, 1.5, 3 and 5 mg/kg] and Pre-exposure [vehicle or ethanol]. The within-subjects factor(s) varied depending on the assessment (for taste avoidance [Trials 1 – 4]; for place preference [Pre-Test and Post-Test]; for temperature [Time (pre-injection, 30-min, and 1- to 8-h post-injection)]; for motor activity and stereotypies [Time (5-min intervals over the span of 1 h)]. In the instance of a significant interaction, simple effects of Dose and Pre-exposure at each within-subjects factor was assessed (multivariate analysis) followed by Bonferroni-adjusted multiple comparisons.

Statistical significance was set to p ≤ 0.05.

3. Results

The outcomes of the analysis of each assessment are presented in Tables 1 – 5 and in Figures 1 – 5. Results of each analysis are described below.

Table 1.

Results of statistical analyses used to analyze saccharin consumption between ethanol or vehicle pre-exposed subjects which were then injected with 0, 1.5, 3, or 5 mg/kg of α-PVP

Factor	DF	F Statistic	P
Trial	3, 264	31.529	< 0.0001
Pre-exposure	1, 88	46.390	< 0.0001
Dose	3, 88	54.089	< 0.0001
Pre-exposure × Dose	3, 88	5.052	0.003
Trial × Pre-exposure	3, 264	18.631	< 0.0001
Trial × Dose	9, 264	29.062	< 0.0001
Trial × Pre-exposure × Dose	9, 264	2.369	0.014

The 4 × 2 × 4 mixed model ANOVA on saccharin consumption showed a significant main effect of Trial [F(3, 264)= 31.529, p < 0.0001], a significant main effect of Pre-exposure [F(1, 88)= 46.390, p < 0.0001], a significant main effect of Dose [F(3, 88)= 54.089, p < 0.0001], a significant interaction of Pre-exposure × Dose [F(3, 88)= 5.052, p= 0.003], Trial × Pre-exposure [F(3, 264)= 18.631, p < 0.0001], Trial × Dose [F(9, 264)= 29.062, p < 0.0001] and a significant three-way interaction of Trial × Pre-exposure × Dose [F(9, 264)= 2.369, p= 0.014].

Table 5.

Results of Statistical Analyses Used to Analyze Stereotypic Effects Between Ethanol or Vehicle Pre-Exposed Subjects Which Were Then Injected with 0, 1.5, 3, or 5 mg/kg of α-PVP

Factor	DF	F Statistic	P
Timepoint	11, 968	0.961	0.481
Pre-exposure	1, 88	0.146	0.703
Dose	3, 88	32.363	< 0.0001
Timepoint × Pre-exposure	11, 968	0.955	0.486
Timepoint × Dose	33, 968	8.952	< 0.0001
Pre-exposure × Dose	3, 88	0.786	0.505
Timepoint × Pre-exposure × Dose	33, 968	1.465	0.045

The 12 × 2 × 4 mixed model a showed significant main effect of Dose [F(3, 88)= 32.363, p < 0.0001]. There was no significant main effect of Pre-exposure [F(1, 88)= 0.146, p= 0.703] or Timepoint [F(11, 968)= 0.961, p= 0.481], and no significant interaction of Timepoint × Pre-exposure [F(11, 968)= 0.955, p= 0.486], or Pre-exposure × Dose [F(3, 88)= 0.786, p= 0.505]. There was a significant interaction of Timepoint × Dose [F(33, 968)= 8.952, p < 0.0001] and a significant three-way interaction of Timepoint × Pre-exposure × Dose [F(33, 968)= 1.465, p= 0.045].

Figure 5.

Mean (+/−SEM) consecutive beam breaks for groups pre-exposed to either vehicle (saline) or ethanol (2 g/kg) then injected with α-PVP at 0 (vehicle), 1.5 mg/kg (panel A), 3 mg/kg (panel B) or 5 mg/kg (panel C) in 5-min intervals for 1-h. ^#pre-exposure conditions (vehicle or ethanol) significantly differ. *ethanol pre-exposed subjects that were injected with α-PVP significantly different from ethanol pre-exposed subjects injected with vehicle. ⁺vehicle pre-exposed subjects that were injected with α-PVP significantly different from vehicle pre-exposed subjects injected with vehicle.

3.1. Conditioned Taste Avoidance

Subjects from both pre-exposure conditions displayed significant dose- and time-dependent taste avoidance, and ethanol pre-exposure significantly attenuated the aversive effects of α-PVP at all tested doses. On Trial 1, subjects from all experimental conditions were matched for saccharin consumption such that it was comparable across groups. Animals pre-exposed to vehicle and then injected with vehicle during conditioning (Group VV) increased their average consumption of saccharin from approximately 10 ml on Trial 1 to 17 ml on Trial 2. Saccharin consumption remained at these high levels on Trials 3 and 4. Subjects that were pre-exposed to ethanol and then injected with vehicle during conditioning (Group EV) displayed the same level of saccharin consumption as Group VV and did not differ statistically from this group at any point in conditioning. On Trials 2 – 4, vehicle pre-exposed subjects injected with α-PVP (Groups V1.5, V3 and V5) drank significantly less saccharin than both Groups VV and EV. Subjects pre-exposed to ethanol and injected with 1.5 mg/kg α-PVP (in Group E1.5) never significantly differed from their controls (Group EV), while subjects pre-exposed to ethanol and conditioned with 3 or 5 mg/kg α-PVP (Groups E3 and E5) consumed significantly less saccharin than their controls over trials. On Trials 3 and 4, subjects in Group E3 showed significant attenuation of saccharin consumption compared to Group V3. This attenuation was evident in subjects in Group E5 (relative to those in Group V5) throughout conditioning (see Table 1 and Figure 2).

Figure 2.

Mean (±SEM) saccharin consumption (ml) over Trials 1 – 4 for groups pre-exposed to either vehicle (saline) or ethanol (2 g/kg) then injected with α-PVP at 0 (vehicle), 1.5 mg/kg (panel A), 3 mg/kg (panel B) or 5 mg/kg (panel C). ^#pre-exposure conditions (vehicle or ethanol) significantly differ. *ethanol pre-exposed subjects that were injected with α-PVP significantly different from ethanol pre-exposed subjects injected with vehicle. ⁺vehicle pre-exposed subjects that were injected with α-PVP significantly different from vehicle pre-exposed subjects injected with vehicle.

3.2. Conditioned Place Preference

Although the testing apparatus was unbiased, a number of individual animals spent more than 65% of the 15-min testing time on one side of the testing apparatus during the Pre-Test (indicative of a strong natural bias for one side of the testing chamber) and were excluded from subsequent statistical analysis (although still run in all behavioral assessments). The resulting group sizes (Groups VV: n = 8; V1.5: n = 4; V3: n = 7; V5: n = 8; EV: n = 6; E1.5: n = 9; E3: n = 10; E5: n = 9) reflect this exclusion. There was a significant main effect of Test [F(1, 53)= 38.457, p < 0.0001], but there were no significant main effects of Pre-exposure (p= 0.781) or Dose (p= 0.306). There were also no significant Test × Pre-exposure (p= 0.409), Test × Dose (p= 0.080) or Test × Pre-exposure × Dose (p= 0.561) interactions.

When the data were subsequently collapsed across Dose (producing new Groups: Veh-Veh: n = 8, Veh-α-PVP: n = 19; EtOH-Veh: n = 6; EtOH-α-PVP: n = 28; where the Veh or EtOH signifies Pre-exposure and the Veh or α-PVP denotes Conditioning) and rerun as a separate 2 × 2 × 2 mixed model ANOVA (results presented in Table 2), the analysis revealed a significant main effect of Test (p < 0.0001), no main effect of Pre-exposure (p= 0.592) and no main effect of Conditioning (p= 0.053). There was a significant interaction of Test × Conditioning (p= 0.019), but there was no significant interaction of Pre-exposure × Conditioning (p= 0.553) or of Test × Pre-exposure × Conditioning (p= 0.449). In both the vehicle and ethanol control groups (Groups Veh-Veh and EtOH-Veh; see Figure 3), there was no significant increase in time spent on the DPS on the Post-Test compared to the time spent on the DPS during the Pre-Test. In contrast, there was a significant increase in time spent on the DPS on the Post-Test compared to the Pre-Test for Groups Veh-α-PVP and EtOH-α-PVP (see Table 2 and Figure 3).

Table 2.

Results of Statistical Analyses Used to Analyze Conditioned Place Preferences Between Ethanol or Vehicle Pre-Exposed Subjects Which Were Then Injected with Vehicle or α-PVP

Factor	DF	F Statistic	P
Test	1, 57	15.901	< 0.0001
Pre-exposure	1, 57	0.290	0.592
Conditioning	1, 57	3.905	0.053
Test × Conditioning	1, 57	5.873	0.019
Pre-exposure × Conditioning	1, 57	0.356	0.553
Test × Pre-exposure × Conditioning	1, 57	0.581	0.449

The 2 × 2 × 2 mixed model ANOVA showed a significant main effect of Test [F(1, 57)= 15.901, p < 0.0001], no main effect of Pre-exposure [F(1, 57)= 0.290, p= 0.592], and no main effect of Conditioning [F(1, 57)= 3.905, p= 0.053]. There was a significant interaction of Text × Conditioning [F(1, 57)= 5.873, p= 0.019], but there was no significant interaction of Pre-exposure × Conditioning [F(1, 57)= 0.356, p= 0.553], or of Test × Pre-exposure × Conditioning [F(1, 57)= 0.581, p= 0.449].

Figure 3.

Mean (+/− SEM) time spent on the drug-paired side (DPS) for each group at Pre-Test (white bars) and Post-Test (black bars) for groups pre-exposed either vehicle or ethanol that were later conditioned with either α-PVP or vehicle. *significantly different between Pre-Test and Post-Test.

3.3. Temperature

Due to chip malfunction, three animals were excluded from analysis, one each from Groups E3, E5 and V5. The resulting group sizes for temperature analysis (Groups VV: n = 12, V1.5: n = 12, V3: n = 12, V5: n = 11, EV: n = 12, E1.5: n = 12, E3: n = 11, E5: n = 11) reflect this exclusion.

α-PVP produced dose- and time-dependent increases in body temperature that were not affected by pre-exposure condition (see Table 3). The average temperature of the subjects in the control groups (VV and EV) did not significantly differ from each other at any time, with both groups showing a gradual decline in body temperature throughout the 8-h observations. From the pre-injection scan to the 1-h sampling period, there were no significant differences in body temperature between any groups. At the 2-h sampling period, Groups V1.5 and V3 showed significantly increased body temperatures compared with Group VV. This effect continued until the 4-h sampling period after which neither dose group differed significantly from vehicle controls (Group VV) (see Figure 4, panels A: 1.5 mg/kg and B: 3 mg/kg). From 3-h until the end of testing, subjects in Group V5 showed a significantly increased body temperature compared to Group VV (Figure 4, panel C). Subjects in Group E1.5 never significantly differed from ethanol controls (Group EV) (Figure 4, panel A). At the 4-h sampling period, subjects in Groups E3 and E5 displayed significantly elevated body temperatures compared to Group EV. This effect was no longer evident after the 5-h timepoint for animals who received the 3 mg/kg dose (Group E3) but continued until the 7-h timepoint for animals who received the 5 mg/kg dose (Group E5).

Table 3.

Results of Statistical Analyses Used to Analyze Body Temperature Changes Between Ethanol or Vehicle Pre-Exposed Subjects Which Were Then Injected with 0, 1.5, 3, or 5 mg/kg of α-PVP

Factor	DF	F Statistic	P
Timepoint	9, 765	65.950	< 0.0001
Pre-exposure	1, 85	0.100	0.753
Dose	3, 85	6.877	< 0.0001
Timepoint × Pre-exposure	9, 765	0.615	0.785
Timepoint × Dose	27, 765	8.231	< 0.0001
Pre-exposure × Dose	3, 85	0.329	0.805
Timepoint × Pre-exposure × Dose	27, 765	0.572	0.961

The 10 × 2 × 4 mixed model showed a significant main effect of Timepoint [F(9, 765)= 65.950, p < 0.0001], a significant main effect of Dose [F(3, 85)= 6.877, p < 0.0001] as well as a significant interaction of Timepoint × Dose [F(27, 765)= 8.231, p < 0.0001]. There was no significant main effect of Pre-exposure [F(1, 85)= 0.100, p= 0.753], no significant interaction of Timepoint × Pre-exposure [F(9, 765)= 0.615, p= 0.785], Pre-exposure × Dose [F(3, 85)= 0.329, p= 0.805] and no significant interaction of Timepoint × Pre-exposure × Dose [F(27, 765)= 0.572, p= 0.961].

Figure 4.

Mean (+/−SEM) temperature in °C for groups pre-exposed to either vehicle (saline) or ethanol (2 g/kg) then injected with α-PVP at 0 (vehicle), 1.5 mg/kg (panel A), 3 mg/kg (panel B) or 5 mg/kg (panel C) at Pre-Injection, and 30-min, 1-h and every hour up until 8-hrs post-injection. *ethanol pre-exposed subjects that were injected with α-PVP significantly different from ethanol pre-exposed subjects injected with vehicle.

⁺vehicle pre-exposed subjects that were injected with α-PVP significantly different from vehicle pre-exposed subjects injected with vehicle.

3.4. Motor Activity and Stereotypies

α-PVP produced time- and dose-dependent increases in locomotor activity that were impacted by ethanol pre-exposure at the 3 mg/kg dose (Group E3). Subjects in the vehicle and ethanol control groups (Groups VV and EV) did not significantly differ from each other throughout the 1-h observation window, with both groups showing a decline in locomotor activity from the 5-min sampling period to the 20-min sampling period where the average number of consecutive beam breaks remained consistent (approximately 200 beam breaks or less) until the end of testing. Subjects injected with α-PVP (Groups V1.5, V3, V5, E1.5, E3 and E5) displayed significant increases in consecutive photobeam breaks compared to their respective control groups (see Figure 5, panels A: 1.5 mg/kg; B: 3 mg/kg and C: 5 mg/kg). Subjects in Group E3 displayed significantly greater activity compared to their vehicle pre-exposed counterparts (Group V3) beginning at the start of observation (5-min) to the 40-min sampling period.

α-PVP produced significant stereotypies in both vehicle and ethanol pre-exposed subjects at each tested dose of α-PVP (Groups V1.5, V3, V5, E1.5, E3 and E5) (see Table 5 and Figure 6). There were no significant differences among groups from the 5-min sampling period to the 15-min sampling period. Vehicle and ethanol control groups (Groups VV and EV) did not significantly differ from each other at any point throughout the 1-h observation window, showing a gradual decline in repetitive beam breaks from 10-min to 25-min where the average number of breaks remained consistent (approximately 20 – 30 breaks) until the end of testing. There was only one timepoint (50-min) and one dose (5 mg/kg) where the pre-exposure conditions significantly differed (see Figure 6, panel C). Specifically, subjects in Group E5 displayed significantly more stereotypies than vehicle pre-exposed subjects at this same dose (Group V5).

Figure 6.

Mean (+/−SEM) Repeated Beam Breaks (stereotypy) for Groups Pre-Exposed to Either Vehicle (saline) or Ethanol (2 g/kg) Then Injected with α-PVP at 0 (vehicle), 1.5 mg/kg (panel A), 3 mg/kg (panel B) or 5 mg/kg (panel C) in 5-min Intervals for 1-h. ^#pre-exposure conditions (vehicle or ethanol) significantly differ. *ethanol pre-exposed subjects that were injected with α-PVP significantly different from ethanol pre-exposed subjects injected with vehicle. ⁺vehicle pre-exposed subjects that were injected with α-PVP significantly different from vehicle pre-exposed subjects injected with vehicle.

4. Discussion

Experiential factors such as a drug history can impact the overall balance of the aversive and rewarding effects of drugs which may alter drug acceptability and the probability of drug taking (see Gaiardi et al., 1991; Gauvin et al., 2000; Stolerman and D’Mello, 1981). To date, no studies have assessed the impact of a drug history on α-PVP’s rewarding and aversive effects. To address this, ethanol pre-exposure was used to determine its effect on α-PVP’s affective properties as indexed by a combined CTA/CPP design and followed by measures of body temperature and locomotion/stereotypies.

Although animals from both pre-exposure conditions displayed significant dose- and trial-dependent α-PVP-induced CTA, rats with an ethanol history had significantly attenuated aversions that were evident at all doses of α-PVP. These results show for the first time that an ethanol history significantly and dose-dependently attenuates the aversive effects of α-PVP, with no detectable aversive effects at the lowest tested dose and aversive effects effectively halved at the highest. These results are consistent with our predictions and with previous work (Grakalic and Riley, 2002a) in which ethanol pre-exposure significantly attenuated the aversive effects of ethanol, cocaine and a cocaine + ethanol combination (see also Grakalic and Riley, 2002b; Hutchison et al., 2010; Kunin et al., 1999). These findings are also similar to those reported by Woloshchuk and colleagues (2016) who found that MDPV pre-exposure significantly attenuated the avoidance of saccharin induced by both MDPV and cocaine. Considering that α-PVP and MDPV are highly similar structurally (the only difference being the methylenedioxy moiety that is present on the phenyl ring in MDPV and absent in α-PVP) with similar mechanisms of action as monoamine uptake inhibitors (Baumann et al., 2016; see also Eshleman et al., 2017; Glennon and Dukat, 2017; Glennon and Young, 2016; Marusich et al., 2014; Meltzer et al., 2006; Rickli et al., 2015), it is tempting to predict that an α-PVP pre-exposure would have similar attenuating effects on cocaine and MDPV in a taste avoidance design. Future studies investigating the effects of α-PVP pre-exposure on the affective properties of MDPV and cocaine (or synthetic cathinones with different mechanisms of action, e.g., methylone, mephedrone) would provide an interesting opportunity to study structure/function relationships in the context of drug-induced aversive effects.

α-PVP also conditioned place preferences, although this was only evident after groups were collapsed across the factor of dose (α-PVP vs. vehicle). Specifically, animals that were conditioned with α-PVP spent significantly more time on the DPS than animals injected with vehicle. This generally weak effect in response to α-PVP has been found in previous work from our laboratory assessing α-PVP-induced CPP. Specifically, Nelson et al. (2017) utilized a biased CPP design (that did not involve exclusion criteria) and found significant place preferences were conditioned by α-PVP, although again only when the data were collapsed across the factor of dose (for assessments of α-PVP’s rewarding effects using a biased CPP design in male mice, see Gatch et al., 2015; see also Marusich et al., 2016 where an unbiased apparatus was utilized to test for the rewarding effects of inhaled α-PVP vapor in male mice; for an assessment of α-PVP’s rewarding effects using an unbiased CPP design in male, but not female, SD rats, see Nelson et al., 2019b). Although relatively weak in the present assessment, place preferences induced by α-PVP were unaffected by ethanol history. This result was unexpected as previous work has shown that ethanol pre-exposure (at 2 g/kg, IP) in either adolescent or adult male SD rats significantly increased the rewarding effects of cocaine (Hutchison and Riley, 2012). Specifically, Hutchison and colleagues found subjects with an ethanol history acquired cocaine-induced place preferences at half the dose of that for animals pre-exposed to vehicle, an indication of cocaine’s increased rewarding effects (see also Hutchison et al., 2010; Mateos-Garcia et al., 2015; Molet et al., 2013 for other work in which an ethanol pre-exposure increased cocaine reward; though see Le Pen et al., 1998; Fredriksson et al., 2017; Busse et al., 2005; see also Busse and Riley 2002; Busse et al., 2004; Lewis and June, 1994; McCance-Katz et al., 1998 for assessments of the modulatory effects of acutely administered ethanol on cocaine’s rewarding effects; see Duart-Castells et al., 2020 for cross reinstatement between MDPV and cocaine using CPP).

α-PVP also produced significant dose- and time-dependent increases in body temperature that were not affected by an ethanol history. This may suggest that α-PVP toxicity is not significantly impacted by previous exposure to ethanol, although additional studies will be required to explore this interaction in order to confirm this. Given that drug-induced temperature effects are not often reported in response to a drug history in general and information regarding this with the synthetic cathinones is especially lacking, it is difficult to compare our findings to previous work. That said, some studies done with methylone and other drugs do address this. For example, Piper et al. (2005) found that adolescent exposure to MDMA in male SD rats reduced the subsequent effects of MDMA on body temperature compared to vehicle pre-exposed subjects (an effect that diminished with repeated testing, see Piper et al., 2005; see also Clemens et al., 2007; Rodsiri et al., 2011). Although not a study of drug pre-exposure, Shortall et al. (2015) examined body temperature changes in response to three short-term repeated mephedrone injections spaced 2 h apart in adult male Lister hooded rats and found that the hypothermic effects of mephedrone were attenuated with repeated dosing (see also Baumann et al., 2012 for hypothermic effects following repeated mephedrone administration and hyperthermic effects with repeated methylone and MDMA injections). Finally, in one of the few studies to assess the thermoregulatory effects of a chronic exposure to a synthetic cathinone, Goldsmith and colleagues (2019) found that after administering weekly injections of methylone to adult male and female SD rats for 6 weeks, females developed a tolerance to methylone’s ability to induce hyperthermia (before changing to a hypothermic response) more rapidly than males. There is a general lack of published research on the relationship of a drug history on the effects of the synthetic cathinones on temperature, and further research in this area is needed.

In the current work, α-PVP produced dose- and time-dependent increases in locomotor activity that were significantly impacted by ethanol pre-exposure at the intermediate (3 mg/kg) dose, but not at the lower (1.5 mg/kg) or higher (5 mg/kg) doses of α-PVP. Specifically, counts of consecutive beam breaks were significantly increased at the 3 mg/kg dose for the first 40 min of testing in animals with a history of ethanol. α-PVP also produced significant stereotypies in both vehicle and ethanol pre-exposed subjects at each tested dose of α-PVP, but stereotypies were generally unaffected by an ethanol history. There was only a single timepoint (50-min post-injection) and at one dose (5 mg/kg) where an ethanol pre-exposure produced significantly more stereotypies than vehicle-pre-exposed subjects that received the same dose. The reason why increased locomotor effects are apparent at the intermediate (3 mg/kg) dose following ethanol pre-exposure, but not at the higher (5 mg/kg) dose, is unknown, as one might expect locomotor activity to increase with dose. Although the basis for this pattern of locomotor effects is unknown, increased locomotor activity has been seen with a history of other drugs. For example, animals with an amphetamine history show significantly increased locomotor behavior when subsequently re-exposed to amphetamine (Vezina et al., 1999; see also Pierre and Vezina, 1997; Schenk et al., 1991; for cross-drug effects on increased locomotor effects, see Achat-Mendes et al., 2003; Babbini and Davis, 1972; Cunningham et al., 2002; Hutchison et al., 2010; Kalivas et al., 1998; Pomfrey et al., 2015; Vandershuren et al., 1999). Interestingly, differential drug pre-exposure effects have also been reported. For instance, an amphetamine history enhances the locomotor effects of subsequent cocaine exposures, but a nicotine history does not (Schenk et al., 1991; see also Horger et al., 1992). Further, THC pre-exposure produces cross-tolerance to the motor-depressant effects of heroin but has no effect on the locomotor effects produced by cocaine (Panlilio et al., 2007; see also Hempel et al., 2020 where germline THC exposure did not alter nicotine locomotor sensitization). Given that these effects appear to be drug-dependent, it will be important to assess the impact of other drugs on the locomotor and stereotypic effects of α-PVP.

It is important to note that the current examination of α-PVP’s temperature and activity effects were made in animals that had prior experience with α-PVP as part of the initial examination of conditioned taste avoidance/conditioned place preference training and, thus, the animals not only had an ethanol or vehicle pre-exposure but also had a prior history with α-PVP which may have impacted the temperature and locomotor results obtained. While possible, animals experienced wash-out periods between testing phases and were sorted into new treatment groups to limit the impact of an α-PVP history. In this context, Giannotti and colleagues (2017) reported that after a 1 mg/kg injection of α-PVP (IP), a return to baseline levels of immediate early gene expression in the mouse frontal lobe and striatum was obtained following a 2-h washout period (effects that were maintained at the 24-h time point; see also Hambuchen et al., 2017; McClenahan et al., 2019). Furthermore, the current data parallel previous work from our laboratory where the wash-out periods were longer at 21 and 24 days (see Nelson et al., 2017 and Nelson et al., 2019b). This suggests that the length of the washout periods between assessments (7 and 10 days, respectively) was sufficient, both at the behavioral and biochemical levels.

The mechanism underlying the effects reported here were not experimentally assessed. One possibility is that the ethanol pre-exposure phase alone may have impacted anxiety and locomotor activity levels, thus impacting the results seen with α-PVP later in the experiment. For a variety of measures in the present design, e.g., changes in saccharin preference, initial and final side preferences, temperature changes and activity/stereotypies, a control group was included that was pre-exposed to ethanol and treated with vehicle (Group EV) to determine if these animals differed relative to another set of control subjects (Group VV) that were given vehicle exposure and subsequently treated with vehicle. As demonstrated, there was no evidence that an ethanol history alone impacted any of these indices as behavior in the ethanol pre-exposed controls did not significantly differ on any assessment from animals with the vehicle history. It is also possible that while there were no residual effects of ethanol on these indices, an ethanol history could change α-PVP’s effects on activity and anxiety. While possible, it is important to note that neither activity nor anxiety is consistently associated with the acquisition of taste avoidance or place preferences. For example, aversions and preferences are induced by drugs that both increase and decrease activity (e.g., cocaine and morphine, respectively; for cocaine see: Clasen et al., 2020; Mayer and Parker, 1993; Pomfrey et al., 2015; for morphine see: King and Riley, 2013; Loney et al., 2021; Simpson and Riley, 2005; Verendeev and Riley, 2011) and increase and decrease anxiety (e.g., “bath salts” and diazepam, respectively; for “bath salts” see: King et al., 2015b; Nelson et al., 2017; Nelson et al., 2019b; for diazepam see: Gray et al., 1999; Stephens and Dunworth, 2000). As such, it is hard to speculate on whether any effects of ethanol on these indices would necessarily be associated with increases or decreases in CTA and CPP.

Given that CTA typically involves water deprivation, there may be concern that any resulting stress as a consequence of this deprivation may have some impact on the results obtained during CPP. While little has been done, there are a few studies that have examined facets of this issue. In one of these studies (Verendeev and Riley, 2011), the relationship between the acquisition of taste aversions and place preferences induced by morphine or amphetamine was examined in the combined CTA/CPP design. Verendeev and Riley found no consistent relationship between the acquisition of these two behaviors, i.e., animals that formed a strong aversion were just as likely to show a weak or a strong place preference; conversely animals that formed a weak taste aversion were just as likely to show a weak or strong CPP. Further, all animals were exposed to the same CTA conditions concurrent with a CPP assessment so the variability in the display of the CPP appeared to be unrelated to the design (as all animals were exposed to the same concurrent procedure).

The only study that has directly assessed whether taste aversion conditioning impacts place preference conditioning is that by King and Riley (2013). In that study, animals were given taste aversion training with morphine (or control injections) and then immediately assessed for place preference conditioning with morphine (morphine or vehicle). Interestingly, there was no differences in place preferences between groups with the aversion history vs. those without the history, i.e., control subjects with no pairings of the taste with morphine. Interestingly, Spear and her colleagues have examined the combined CTA/CPP procedure in nondeprived animals (to induce drinking, these nondeprived animals were given a highly palatable sucrose/saccharin solution to drink). Similar to other reports using the combined procedure, nicotine-induced aversions and place preferences were evident and the effects reported paralleled those in independent assessments of each. Specifically, adolescents were both more sensitive to the rewarding effects and less sensitive to the aversive effects of nicotine as compared to adults (Dannenhoffer and Spear, 2016; see also Shram et al., 2006 who assessed these same endpoints using CTA and CPP performed separately in water-deprived rats and found that periadolescent rats experienced more reward and less aversive effects compared to their adult counterparts).

The authors know of no study that has directly compared the acquisition of place preferences in animals that are given concurrent CTA/CPP training with those that only have CPP training. Comparisons across studies suggest that place preference conditioning is similar between the two designs and that common parametric manipulations on the two designs affect CPP similarly (see Simpson and Riley 2005; Yu et al., 2021 for assessments of the combined CTA/CPP assay with morphine compared to Lett, 1989 and Shippenberg et al.,1996 for assessments of morphine using only CPP; for a similar comparison with MDPV see King et al., 2015b as compared to Risca et al., 2020; for α-PVP see Nelson et al., 2017 and Nelson et al., 2019b as compared to Gatch et al., 2015 and Marusich et al., 2016; and for caffeine see Brockwell et al., 1991 as compared to Bedingfield et al., 1998). While there could be an impact of the aversion (or the training conditions under which the aversions are acquired), the little data that have been reported do not support this position.

Sex as a biological variable is a vital component for discovering potential risk factors involved in drug use and abuse (see Klein et al., 2015; Miller et al., 2017; Wetherington, 2007, 2010). Limited studies involving the “bath salts” have been done assessing the effects of sex, although some work has been done in this area in recent years (see: Alsufyani and Docherty, 2017; Daniel and Hughes, 2016; Hambuchen et al., 2017; Javadi-Paydar, 2018; King et al., 2015b; Marusich et al., 2016). That being said, only male subjects were run in the current studies largely because the majority of the work on α-PVP to date has been done using male subjects. As such, this choice allows for comparisons across studies regarding the effects of this compound. While sex comparisons are limited, we have recently examined each of the behavioral and physiological indices reported here and have observed sex differences. In this work (Nelson et al., 2019b), a significant conditioned place preference was not seen in adult female SD rats treated with α-PVP. Given that the current work was focused on demonstrating an effect of an ethanol history on several affective properties of α-PVP (including aversion and reward), it was important that the initial demonstration was completed with male subjects known to display both of these effects. Given the findings with males, similar assessments should be made with females in future studies.

In conclusion, ethanol pre-exposure reduced α-PVP’s aversive effects as measured by a robust attenuation of CTAs. Furthermore, ethanol pre-exposure significantly increased α-PVP’s locomotor effects (at the 3 mg/kg dose). On the other hand, ethanol history had no impact on α-PVP’s rewarding effects as measured by CPP or α-PVP’s effects on temperature or stereotypies. Although the basis for the specific interactions of ethanol and α-PVP is unknown, that ethanol pre-exposure had a different influence on the various effects of α-PVP may be an indication that these effects function through separate mechanisms that are differentially impacted by an ethanol history. Exploring the basis of ethanol’s effects on α-PVP for each interaction could provide insight into these differential effects of ethanol history on their display. The aversive effects α-PVP (as measured by CTA) were attenuated, while its rewarding effects (as measured by CPP) were unaffected, by an ethanol pre-exposure. This pattern of effects may indicate increased α-PVP abuse liability as changes in the balance of aversion and reward could impact overall drug effects and likelihood of drug intake. Future IVSA studies will be necessary to explore this possibility. This is an especially important factor to recognize as the majority of synthetic cathinone users participate in polydrug use although the concurrent and/or serial nature of this polydrug use is unclear. In addition to assessing the impact of a drug history in combination with other subject and experiential factors, e.g., sex, age, concurrent drug combinations, etc., it will be important to extend these findings by assessing the impact of a history of other drugs such as THC, cocaine, MDMA and nicotine commonly used by this population. Furthermore, given the lasting impact of an individual’s drug history, clinical reports and surveys would be greatly improved by probing deeper into participants’ past drug use and inquiring about any serial drug use practices.

Table 4.

Results of Statistical Analyses Used to Analyze Locomotor Activity Effects Between Ethanol or Vehicle Pre-Exposed Subjects Which Were Then Injected with 0, 1.5, 3, or 5 mg/kg of α-PVP

Factor	DF	F Statistic	P
Timepoint	11, 968	112.919	< 0.0001
Pre-exposure	1, 88	1.878	0.174
Dose	3, 88	42.208	< 0.0001
Timepoint × Pre-exposure	11, 968	2.518	0.004
Timepoint × Dose	33, 968	7.188	< 0.0001
Pre-exposure × Dose	3, 88	1.624	0.190
Timepoint × Pre-exposure × Dose	33, 968	2.017	0.001

The 12 × 2 × 4 mixed model a showed significant main effect of Timepoint [F(11, 968)= 112.919, p < 0.0001], a significant main effect of Dose [F(3, 88)= 42.208, p < 0.0001] as well as a significant interaction of Timepoint × Pre-exposure [F(11, 968)= 2.518, p= 0.004] and of Timepoint × Dose [F(33, 968)= 7.188, p < 0.0001]. There was no significant main effect of Pre-exposure [F(1, 88)= 1.878, p= 0.174] and no significant interaction of Pre-exposure × Dose [F(3, 88)= 1.624, p= 0.190]. There was a significant three-way interaction of Timepoint × Pre-exposure × Dose [F(33, 968)= 2.017, p= 0.001].