Alteration of Ethanol Reward by Prior Mephedrone Exposure: The Role of Age and Matrix Metalloproteinase-9 (MMP-9)

Mephedrone, a synthetic cathinone, is widely abused by adolescents and young adults. The aim of this study was to determine: (i) whether prior mephedrone exposure would alter ethanol reward and (ii) whether age and matrix metalloproteinase-9 (MMP-9) are important in this regard. In our research, male Wistar rats at postnatal day 30 (PND30) received mephedrone at the dose of 10 mg/kg, i.p., 3 times a day for 7 days. To clarify the role of MMP-9 in the mephedrone effects, one mephedrone-treated group received minocycline, as an MMP-9 antagonist. Animals were then assigned to conditioned place preference (CPP) procedure at PND38 (adolescent) or at PND69 (adult). After the CPP test (PND48/79), expression of dopamine D1 receptors (D1R), Cav1.2 (a subtype of L-type calcium channels), and MMP-9 was quantified in the rat ventral striatum (vSTR). The influence of mephedrone administration on the N-methyl-D-aspartate glutamate receptors (NMDAR) subunits (GluN1, GluN2A, and GluN2B) was then assessed in the vSTR of adult rats (only). These results indicate that, in contrast with adolescent rats, adult rats with prior mephedrone administration appear to be more sensitive to the ethanol effect in the CPP test under the drug-free state. The mephedrone effect in adult rats was associated with upregulation of D1R, NMDAR/GluN2B, MMP-9, and Cav1.2 signaling. MMP-9 appears to contribute to these changes in proteins expression because minocycline pretreatment blocked mephedrone-evoked sensitivity to ethanol reward. Thus, our results suggest that prior mephedrone exposure differentially alters ethanol reward in adolescent and adult rats.


Introduction
Mephedrone (4-methylmethcathinone) is a synthetic cathinone derivative that is structurally and pharmacologically related to the psychostimulants (3,4-methylenedioxymethamphetamine (MDMA) and other amphetamines) [1] and has been classified as a new psychoactive substance (NPS) [2,3]. Mephedrone is abused by club drug users, predominantly adolescents and young adults [4,5], in a binge-like fashion ("stacking") [6]. Published data indicate that most mephedrone users are engaged in "heavy" alcohol use immediately prior to consuming mephedrone, and then they reduce alcohol consumption as the effects of mephedrone are experienced during the drug episode [7]. Another study shows that mephedrone is commonly used simultaneously with alcohol (ethanol) by polydrug users [8]. Such drug
Three-way ANOVA revealed no significant effect of mephedrone pretreatment (F (1, 79) = 3.225; p > 0.05) on the rewarding effect of ethanol at the dose of 0.3 and 1.0 g/kg (F (2, 79) = 0.8173; p > 0.05) in adolescent rats (PND 48) ( Figure 1C). A post hoc test did not show any differences between groups on the pre-test day. What is more, two-way ANOVA did not indicate significant effects of mephedrone pretreatment during adolescence (F (1, 42) = 0.005755; p > 0.05) and ethanol administration during conditioning on locomotor activity measured during the test (F (2, 42) = 0.3263; p > 0.05) (data not shown).

Influence of Minocycline Pretreatment on the Mephedrone Effect on the Ethanol CPP in Adult Rats
Minocycline, as an MMP-9 inhibitor, given before every mephedrone administration, attenuated the mephedrone induced sensitivity to ethanol rewarding effect in the CPP test in adult rats (PND79). Three-way ANOVA revealed the significant effect of minocycline/mephedrone pretreatment (F (3, 108) = 3.696; p < 0.05) and interaction between minocycline/mephedrone × ethanol (F (3, 108) = 3.813; p < 0.05) and minocycline/mephedrone × time (F (3, 108) = 3.941; p < 0.05). Post hoc analysis revealed that significant CPP was induced only in mephedrone but not minocycline/mephedrone-pretreated rats ( Figure 1D). A post hoc test did not show any differences between groups in the pre-test day according to the unbiased CPP method.
Post hoc analysis revealed that mephedrone (p < 0.001) but not ethanol (p > 0.05) increased the D1 receptor expression level in the vSTR of PND 79 rats. Moreover, ethanol but not saline administration during conditioning decreased D1 receptor upregulation in adult rats (PND 79) pretreated with mephedrone during adolescence (p < 0.05).  Post hoc analysis revealed that mephedrone (p < 0.001) but not ethanol (p > 0.05) increased the Cav1.2 expression level in the vSTR. Moreover, ethanol administration decreased Cav1.2 expression, compared with animals that also previously received mephedrone, but not in animals that received saline during the CPP (p < 0.01) of PND79 rats.
Post hoc analysis (Tukey's multiple comparisons test) revealed that mephedrone (p < 0.01) but not ethanol (p > 0.05) increased the MMP-9 expression level in the vSTR of PND 79 rats. There were no significant changes in adolescent (PND48) animals.
Post hoc analysis (Tukey's multiple comparisons test) revealed that mephedrone given during adolescence increased the MMP-9 expression level in the vSTR (p < 0.05) of adult rats. Minocycline given before mephedrone prevented this effect (p < 0.01) ( Figure S1).

Effect of Mephedrone Treatment on the Expression of NMDAR Subunits (GluN1, GluN2A, and GluN2B) in the vSTR
Statistical analysis revealed that mephedrone administration during adolescence induced significant changes only in the GluN2B subunit of NMDAR in the vSTR of adult rats (p < 0.01 by Student's t-test; p = 0.0031) ( Figure 2D).

Discussion
The current study provides evidence, for the first time, that adult rats with prior mephedrone experience express a more robust ethanol-induced CPP than their adolescent counterparts. The mephedrone effect in adult rats was not observed when minocycline, as an MMP-9 antagonist, was given before mephedrone administration. Along with these behavioral changes, we observed a substantial upregulation of D1R, Cav1.2, and MMP-9 expression in the vSTR in adult, but not adolescent rats with prior mephedrone administration. Moreover, upregulation of the GluN2B subunit of NMDAR was noted in adult rats with prior mephedrone administration. Ethanol administration during the conditioning sessions did not affect the MMP-9 level but reduced the upregulation of D1R and Cav1.2 in adult rats that had received mephedrone in adolescence. Together, these results suggest that prior mephedrone treatment differentially affected the rewarding action of ethanol in adolescent and adult rats.
Prior works have found differences in behavioral and neurochemical changes induced by amphetamine-type stimulants between adolescent and adult rodents [49][50][51][52]. Consistent with these studies, we found behavioral changes between adolescent and adult rats following ethanol conditioning with prior (binge) mephedrone administration during adolescence. Treated adolescent rats did not exhibit CPP preference following ethanol conditioning at the dose that induced robust CPP in adult rats ( Figure 1). Moreover, no differences were observed in the locomotor activity of animals. Herein, the CPP response in adolescent but not in adult rats contrasts with a previous study showing that past stimulant users appear to be more sensitive to ethanol rewarding properties [10]. Unfortunately, we did not perform a dose-effect testing for ethanol CPP in adolescent rats. However, we assumed that adolescents show a shifted dose-effect curve. Thus, our experiment suggests that adolescent but not adult rats previously treated with mephedrone might need higher doses of ethanol (will drink more) to achieve the same response. Extending these results to humans, it is conceivable that adolescent mephedrone users may be at a greater risk of developing ethanol addictive behaviors than adults, similar to MDMA adolescent users [53]. Furthermore, due to enhanced reward experience, adults most likely chase the drug and develop addictive behaviors faster than adolescents.
The CPP response under a drug-free state (CPP expression) represents the motivational effects of the contextual cues that gain saliency following pairing with addictive drugs [54,55]. It appears that prior mephedrone exposure has no impact on the motivational valence of the context (associated with ethanol pairing) in adolescents but that it increases it in adult rats, as we observed a robust CPP in adult rats with prior mephedrone experience but not in adolescent rats. The CPP expression, on the other hand, is a representative of the memory retrieval of the conditioned response that is acquired due to pairing of the subjective effects of the drug and the context during conditioning [54,55]. This response is more robust in adult compared with adolescent rats with prior mephedrone administration. Although we expected to observe a greater response in adolescent rats, it appears that prior mephedrone exposure may differentially bring about molecular changes, thereby leading to these behavioral changes. These results suggest that mephedrone exposure during adolescence, similar to amphetamines exposure [56], may promote long-lasting neurophysiological alterations (remodeling) that influence their future behaviors, including increased reward sensitivity in adult but not adolescent rats.
Release of DA in the NAc is a key process related with the reinforcing and rewarding properties of a drug [57]. Because the NAc D1R is mainly involved in the acquisition of ethanol induced CPP [58], we evaluated the expression of this receptor in the vSTR in the adolescent and adult ethanol conditioned rats with/without prior mephedrone administration. We found that ethanol alone did not have significant impact on the expression of D1R and L-type Cav1.2 channels expression in the vSTR of adolescent or adult rats in the drug-free state during the CPP test (24 h after the last ethanol conditioning session). However, there was a significant upregulation of D1R in adult rats with prior mephedrone treatment. This mephedrone effect was accompanied by an upregulation of L-type Cav1.2 channels expression. Such effects were not observed in adolescent animals-(probably) because mephedrone-induced neurophysiological alterations were not sufficiently developed. Furthermore, ethanol administration during conditioning in adults increases burst firing of DA neurons in the NAc, see [19]; therefore, ethanol conditioning reduced the upregulation of D1R and Cav1.2 in the mephedrone-treated animals. These findings suggest that the dopamine system in adults with prior mephedrone administration was more vulnerable than adolescents to the rewarding effect of ethanol. Thus, even a minor ethanol-induced change/increase in extracellular dopamine was able to reduce the upregulation of D1R and Cav1.2 expression in the animals with prior mephedrone administration. The mechanism underlying the resistance to the effects of ethanol in adolescent rodents is not well understood. However, published data show that compared with adult rodents, adolescent rodents show reduced dopamine level in the NAc and PFC [52]. These findings provide evidence that dopamine responses to ethanol exposure during adolescence are/can be less intense than that induced by ethanol exposure in adults.
Published data suggest that MMP-2 and MMP-9 are involved in the regulation of methamphetamine (METH)-induced changes in DA release and uptake in the NAc [34,59]. Additionally, METH-induced behavioral sensitization and reward were markedly attenuated in MMP-2-and MMP-9-deficient mice, compared with those in wild-type [32]. The current study confirms the role of MMP-9 in the ethanol-induced reward in adult rats with prior mephedrone administration. Minocycline is a commonly used semi-synthetic tetracycline with anti-inflammatory and anti-apoptotic properties [60,61]. Minocycline interferes with MMP activity [62,63] and has been shown to be neuroprotective in cerebral ischemia [64] and in other models of brain injury [65,66]. Published data showed that minocycline inhibits enzymatic activity of gelatin protease, especially of MMP-9 [45][46][47]. Our results support its inhibitory effect on MMP-9 expression in the vSTR ( Figure S1). Herein, minocycline given before mephedrone administration in adolescence reduced the rewarding effect of ethanol in adult rats. Furthermore, our findings indicate that binge mephedrone administration during adolescence induced upregulation of MMP-9 in the vSTR in adult but not in adolescent rats, which was abolished by minocycline pretreatment (see Figure S1). Considering these data, we hypothesized that mephedrone treatment during adolescence differentially affected the expression of MMP-9 in adolescent vs. adult rats.
Published data confirm that treatment with drugs of abuse increases MMPs activity in various brain structures [28,32,36,67]. However, data concerning ethanol are controversial. Some of the research has demonstrated that both acute and chronic intermittent ethanol exposure increased [68], but others [69] found decreased MMP-9 expression in various brain structures after ethanol injections. However, MMP-9 activity is increased in the brain of alcoholics, suggesting that the timing of measuring MMP activity after ethanol exposure may yield different results [70].
On the other hand, the acquisition of CPP for abuse drugs is a learning process [71]. Still, while MMP-9 is increased during drug-induced learning processes, when learning/plasticity is posited to have been completed, MMP-9 expression returns to pharmacological control levels in some brain structures, including the NAc [35,69,[72][73][74]. Thus, we hypothesize that transient changes in MMP-9 could have occurred in the vSTR during the active phase (rising phase of the acquisition curve when performance is improving) of learning and memory consolidation of ethanol-induced CPP in rats. However, there were no changes in the MMP-9 level on the test day when this task had been established. These results suggest that on the test day the synaptic changes had been completed and that MMP-9 levels had returned to normal.
In contrast with ethanol effects, our findings indicate that binge mephedrone administration during adolescence induced marked changes in MMP-9 level in adult (PND79), but not adolescent (PND48) rats. These results confirm our previous findings [30], when significant upregulation of MMP-9 expression was noticed after a delay of 14 days and lasted up to 38 days following the last mephedrone administration, suggesting that MMP-9 might contribute to later occurring processes. Other researchers found that dopaminergic and glutamate transmission can affect the ECM that surrounds and stabilizes synapses [75][76][77][78]. Interestingly, prime candidates for ECM remodeling are extracellular proteases, including MMP-9 [79]. Published data reported that neuromodulation via D1-type dopamine receptor can induce ECM proteolysis specifically via NMDAR/NR2B activation and intracellular calcium signaling [80,81], hence taking part in synaptic plasticity (in striatal medial spiny neurons and frontal cortex neurons). Thus, we hypothesize that such a mechanism could be involved in our study in adult rats with prior mephedrone administration. However, future studies will be needed to explain the influence of ethanol CPP on the NMDAR subunits expression in prior mephedrone-exposed rats.
In summary, our results suggest that binge-like mephedrone administration during adolescence sensitized adult but not adolescent rats to the rewarding effects of ethanol. In adult rats, these changes are associated with the upregulation of D1R and NMDAR/Glu2B, as well as upregulation of L-type of calcium channel and MMP-9 expression in the vSTR. It seems that MMP-9 activation was responsible for these neuroplastic changes in reward pathways because the blockade of its activity by minocycline inhibits vulnerability to ethanol reward. The decrease in the ethanol rewarding action in adolescents, compared with adult rats, may suggest that this may be one cause of the dose escalation in chasing pleasure in this population. In turn, adults with prior mephedrone exposure more rapidly develop addictive behavior than adolescents.

Animals
Male Wistar rats (OMD, Lublin, Poland) weighing 100-135 g at the initial phase of study were used in our experiments, as this experiment is a follow-up to a previous study [30] in which only males were included. The results obtained from female rats may [82] or may not be [83] different because of estrous cycle stage. The experiments began at PND 30. The 234 animals (n = 7-8/group) were kept double-housed in cages (55 cm × 33 cm × 20 cm) under standard laboratory conditions such as a constant temperature of 22 ± 1 • C, controlled humidity within 55 ± 10%, natural day-night cycle (12/12 h), and free access to drinking water and standard laboratory chow (Sniff Spezialdiäten GmbH, Soest, Germany). Before the behavioral experiments, rats were handled for 5 min per day for 5 consecutive days. Experiments were conducted between 8:00 a.m. and 7:00 p.m. All procedures were approved by the Local Ethic Committee (No. 67/2019) and were carried out according to the National Institute of Health Guidelines for the Care and Use of Laboratory Animals and the European Community Council Directive of November 2010.

CPP Apparatus
In the experiment, six identical rectangular boxes (54 cm × 35 cm × 49 cm) were used. Each was composed of two compartments with the same dimensions but differing in color of walls and texture of the floor. Between the compartments, there was a square passage closed with a guillotine door allowing, when opened, free movement between apparatus compartments. The walls of one were uniformly black, while the other was black and white vertically striped. The experimental room's luminance was adjusted so that the environmental (visual and tactile) cues did not produce a significant baseline preference for one of both compartments. Between each paradigm procedure, the whole apparatus was cleaned with 10% ethanol solution to neutralize the odor cues. The apparatus was kept in a soundproof room with neutral noise masking and dim illumination (40 lx). The CPP performance was measured by computerized video tracking (VideoMot, TSE-Systems, Bad Homburg, Germany). Time spent in the compartment associated with ethanol was measured pre-test and test as measure of the degree of conditioning induced by the drug.

CPP Procedure
The CPP unbiased paradigm was performed according to the method described earlier [87] with minor modifications. It consisted of four phases that lasted for 11 consecutive days: habituation (1 day), pre-test (1 day), conditioning (8 days), test (1 day). Time spent in each compartment was measured during the pre-test phase to separate rats into groups with approximately equal biases for each compartment. An appropriate control group (0.9% NaCl-treated during all phases of experiments) was employed. This underwent the same CPP procedure as the drug-treated rats.
Habituation: On the first day, rats were placed in the random compartment for 15 min with free access to both rooms and could freely explore the apparatus.
Pre-test: On the second day, the procedure was the same, but computerized video tracking measured the time the rats spent in each apparatus compartment. The analysis showed no preferences. No drugs were administered during the first and the second day of this phase.
Conditioning: The animals were conditioned twice daily (morning and afternoon sessions) for eight consecutive days with at least a 3 h rest period between sessions. In the morning, the animals received ethanol (0.3, 1.0, or 1.5 g/kg, 15% v/v, i.p.), or equivalent volume of 0.9% NaCl, and were injected immediately prior to the conditioning session and then placed for 30 min into the drug-paired compartments with the guillotine doors closed. In the afternoon, all animals were injected with saline and placed into the drug-free compartment for 30 min.
Test (CPP expression): The rats were confined individually in the apparatus and left for 15 min, having free access to both compartments. The animals were not injected during this phase. The amount of time spent by the animals in each compartment was measured and recorded. The locomotor activity of individual rats was measured as the distance traveled within 15 min of the test phase (CPP test).

The Effect of Mephedrone Pretreatment on the Ethanol CPP
At PND30, the rats were injected with saline or mephedrone (10 mg/kg), three times a day for 7 consecutive days. Next, the animals were assigned into two cohorts. The first cohort was subjected to CPP procedure at PND38 (habituation (1 day, PND38), pre-test (1 day, PND39), conditioning (8 days,, and test (1 day, PND48)).
After the completion of the ethanol CPP test, the animals were decapitated, and the brain structures were removed and frozen (at −80 • C) for further neurochemical analyzes.

The Influence of Minocycline Pretreatment on the Mephedrone Effect on the Ethanol CPP
This part of the study was performed to indicate whether MMP-9 is involved in mephedrone-induced effects. For this purpose, minocycline, an inhibitor of MMP-9 [48], was given at the dose of 45 mg/kg [75] during mephedrone administration (n = 7-8/group), every day before first drug injection (PND30-36). Next, the animals (PND69) were subjected to the CPP procedure according to the method described above, with the exception that only one dose of ethanol (1.0 g/kg) was given during conditioning. After CPP procedure, one group of animals was decapitated, and the dissected brain structures were subjected to biochemical experiments to evaluate the influence of minocycline and mephedrone on MMP-9 expression in the vSTR.

The Ethanol CPP
A separate group of rats was subjected to the CPP procedure (PND69) with saline pretreatment (PND30-36). Ethanol was given at the dose of 0.3, 1.0, or 1.5 g/kg, 15% v/v, i.p. during conditioning to indicate whether ethanol alone induced the rewarding effect in CPP.

ELISA Assay
Quantitative measurement of MMP-9, D1R, and Cav1.2 in such rat brain structures as the vSTR was performed using a Rat MMP-9 ELISA Kit (Reddot Biotech, Kelowna, BC, Canada), a Rat Dopamine Receptor D1 (DRD1) ELISA kit (Reddot Biotech, Kelowna, BC, Canada), and Rat Voltage-dependent L-type Calcium Channel Subunit Alpha-1C ELISA Kit (Bioassay Technology Laboratory, Shanghai, China), respectively, following manufacturers' protocols. Firstly, frozen brain structures were homogenized in cold buffer at pH 7.4 (0.32 M sucrose, 1 mM HEPES, 1 mM MgCl 2 , 1 mM NaHCO 3 , and 0.1 mM PMSF) containing cocktails of protease and phosphatase inhibitors (Sigma-Aldrich, Saint Louis, MO, USA), using a homogenizer ball (Bioprep-24, Allsheng, China) (10 s at 10,000 rpm). Then, homogenates were centrifuged for 5 min at 5000× g, the supernates were immediately removed, and protein concentration in the supernates was measured using a bicinchoninic acid assay (BCA) protein assay kit (Serva, Heidelberg, Germany). From each sample, 100 µg of protein was used in each ELISA assay. All data are expressed in ng/mL.

Statistical Analysis
The data obtained in the CPP procedure are expressed as mean ± SEM of time spent in drug-paired compartment. The CPP results were evaluated by a three-way analysis of variance (ANOVA), followed by Tukey's multiple comparisons test in order to compare differences between groups. Two-way analysis of variance (ANOVA) with the Tukey's post hoc test was used to analyze the effect of mephedrone pretreatment, ethanol treatment, and age of rats on MMP-9, D1R, and Cav1.2 expression in the rats' vSTR and the influence of minocycline on MMP-9 expression in mephedrone exposed rats. Unpaired Student's t-test was applied to evaluate the effect of mephedrone treatment on the expression of NMDAR subunits in the rats vSTR.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author.

Conflicts of Interest:
The authors declare no conflict of interest.