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Official websites use. Share sensitive information only on official, secure websites. Although MDPV poses public health risks, its neuropharmacological profile remains poorly explored. This study aimed to provide evidence on that direction. Locomotor, exploratory, and emotional behavior, in addition to striatal neurotoxicity and glial signature, were assessed within 18—24 h, a known time-window encompassing classical amphetamine dopaminergic neurotoxicity. MDPV resulted in unchanged locomotor activity open field test and emotional behavior elevated plus maze, splash test, tail suspension test. Expectedly, and in sheer contrast with MDPV, METH resulted in decrease general locomotor activity paralleled by a significant striatal TH depletion, astrogliosis, and microglia arborization alterations Sholl analysis. This comparative study newly highlights that binge MDPV-exposure comes without evident behavioral, neurochemical, and glial changes at a time-point where METH-induced striatal neurotoxicity is clearly evident. Nevertheless, neuropharmacological MDPV signature needs further profiling at different time-points, regimens, and brain regions. Over the last decade, the illicit drug market has become increasingly complex and dynamic with the speed of appearance of new psychoactive substances NPSs \[ 1 \]. These are synthetic derivations of the naturally occurring stimulant cathinone found in the khat plant amphetamine structural analogue \[ 2 , 8 \]. They may be either N-alkylated or the nitrogen atom is part of a pyrrolidine ring, as is the case of 3,4-Methylenedioxypyrovalerone MDPV. Synthetic cathinones have also a phenyl ring, which is an important site for chemical modifications. MDPV is considered to have increased potency and abuse potential \[ 3 , 7 \]. MDPV high lipophilicity may be held responsible for its enhanced blood—brain barrier BBB permeability \[ 2 , 3 \] and drug brain levels \[ 5 \], hence contributing to its potency at DAT and, therefore, to the dose-dependent increase of extracellular dopamine DA in the nucleus accumbens NAc , often associated with locomotor and rewarding effects \[ 7 , 12 , 13 \]. MDPV has been mostly studied for its psychostimulant features with abuse potential \[ 6 \], namely the locomotor activity \[ 12 , 13 , 14 , 15 , 16 , 17 , 18 , 19 , 20 \], self-administration behavior \[ 14 , 19 , 21 , 22 \], conditioned place preference \[ 20 , 23 \], and drug discrimination \[ 14 , 17 , 24 \] parameters. Undoubtedly, this synthetic cathinone acts as a potent CNS stimulant, which raises obvious concerns about its potential neurotoxic effects, and possibly amphetamine-like effects, due to their structural similarity. However, data on the underlying mechanisms of MDPV induced neurotoxicity is not abundant \[ 4 , 22 , 26 , 27 \] and mostly circumscribed to in vitro studies \[ 28 , 29 , 30 , 31 , 32 , 33 , 34 \], hampering proper risk characterization and adverse effect management \[ 2 , 5 , 25 \]. Indeed, the scarce data available suggests that synthetic cathinones may exert differential neurotoxic properties on monoaminergic neurons and elicit more complex responses than non-keto amphetamines e. Contrastingly, a great deal of attention has been devoted to the aspects underlying METH-induced neurotoxicity. Additionally, METH induced neuroinflammation has been widely reported either in vitro \[ 36 , 37 , 38 \] or in vivo \[ 35 , 36 , 37 , 39 \], seemingly mediated by glial activation \[ 35 \]. Although neuroimmune modulation has been claimed responsible \[ 25 , 35 , 40 \], some authors have recently stood in contrast to those claims of excessive and detrimental neuroinflammation believed to contribute and exacerbate METH neurotoxicity \[ 41 \]. Accordingly, new avenues are being explored. There is a growing body of evidence showing that a group of pattern recognition receptors PRRs that detect and respond to exogenous pathogen associated molecular patterns PAMPs and endogenous danger associated molecular patterns DAMPs , the toll-like receptors TLRs , may be implicated in psychostimulant induced innate neuroimmune responses \[ 36 , 40 , 42 , 43 \]. TLR4 signaling seems to be involved in drug reward-associated behaviors and proinflammatory responses, contributing to the development of drug addiction \[ 36 , 43 \]. Additionally, the receptor for advanced glycation endproducts RAGE , described to recognize glycosylated proteins and lipids in the forms of advanced glycation endproducts AGEs , also detecting DAMPs \[ 42 \], seems to play a pivotal role in synaptic function, glial response, and inflammation homeostasis when upregulated \[ 45 \]. In fact, our group previously showed that striatum dopaminergic neurotoxicity was associated with increased RAGE transcription and total protein levels \[ 46 \]. Therefore, it is also envisaged as a potential player on striatal METH neurotoxicity \[ 25 , 40 \], an intriguingly overlooked setting. Our group was pioneer in reporting preserved striatal RAGE levels, three days after a single neurotoxic METH dose \[ 47 \], and further studies are needed to deepen knowledge on different METH regimens, doses, and time-points. This poor pharmacological characterization of MDPV poses a high risk for public health, evident from the reported NPS use intoxications and fatalities \[ 2 , 3 , 4 , 25 \]. Therefore, the present work aimed to provide a novel and integrative characterization of the fundamental impact of MDPV on neurotoxicity, exploring classical neuronal and glial markers in addition to putative innate immune modulators and emotional behavior. METH was meant to be used both as a comparison measure same monoaminergic selectivity and also to deepen current knowledge on METH neurotoxicity. Our results show, for the first time, that a binge MDPV-exposure comes without evident behavioral and glial changes, when METH-induced striatal neurotoxicity striatal TH depletion, astrogliosis, and microglia arborization alterations is already present. Nevertheless, the characterization of neuropharmacological MDPV signature is critical for understanding its neurotoxic potential at different time-points, doses, and regimens. Experimental design. The EPM test was performed to assess stress-induced mice anxiety-like behavior Figure 3. The locomotor alterations seen in this apparatus hinder any assumption regarding these behavior alterations in the open arms, which probably reflect diminished total arm entries compared to SAL Figure 3 c. TH is the rate-limiting enzyme in DA synthesis and is the golden marker for dopaminergic terminals in striatum. To unravel the effect and possible differences between MDPV and METH on glial reactivity, a possible contributor to psychostimulant induced neurotoxicity, we evaluated astrocytic GFAP and microglial Iba-1 markers in striatal coronal sections Figure 5. Nevertheless, we were intrigued by the apparent misshape found in microglia in METH-treated mice Iba-1 seems to exhibit a higher concentration of signal in microglia somas than in surrounding ramifications, Figure 5 a. Accordingly, microglia activation is translated in dynamic changes over time, starting from retraction of the processes and further enlargement of the soma \[ 51 \]. Therefore, a Sholl analysis was performed to explore if at the present time-point 24 h , there were subtle changes in microglia morphology between groups. In fact, the obtained results confirmed our hypothesis, showing an early state of microglial reactivity in METH-treated mice. This seemed to happen without changing the number of primary branches Np Figure 5 d. Once again, these results are consistent with METH induced neurotoxicity, astrogliosis, and unique microglial alterations not observed for MDPV at the present time-point. The suggestion of astrogliosis and early microglia activation induced by METH-exposure, prompted us to further analyze other immune parameters that might be associated with this glial activation. PPRs have been discussed as involved in psychostimulant neurotoxicity, and our group already linked striatum dopaminergic neurotoxicity with increased RAGE \[ 46 \]. Therefore, as a complementary approach, we evaluated the impact of these dopaminergic toxins on the overall isoform-level of RAGE Figure 6. As previously described \[ 46 \], protein quantification through western blot allowed us to distinguish RAGE-isoforms, using antibodies specific to each terminal domain. Data not statistically compared. The main study novelty was the assessment of both emotional behavior and neurotoxicity related glial reactivity and neuroimmune modulation, poorly elucidated after both MDPV- and METH-exposure. Additionally, it falls well within the dose range used in similar MDPV published studies \[ 4 , 12 , 17 , 19 , 26 \]. Overall, the obtained results indicated minor MDPV alterations at this time-point. On the other side, results were clearer on the METH induced neurotoxicity, triggering evident hypolocomotion accompanied by striatal TH depletion, astrogliosis, and unique microglia alterations. We must clarify that no significant differences were observed in terms of food and water intake, or in body weight along the 24 h after exposure to both MDPV and METH. Regarding the behavioral analysis, the significant hypolocomotion found in METH mice and the absent alterations in the locomotor profile of MDPV mice, 24 h after the last dose, are in agreement with the results reported by others for these psychostimulants. Consistently, MDPV displays rapid pharmacokinetics, with peak plasma concentrations achieved at 10—20 min and declining quickly thereafter \[ 11 \]. In addition, MDPV reached the brain 5 min after a subcutaneous administration and peaked 20—25 min later, and the striatum elimination half-life occurred 61 min later, coincident with decreased psychostimulant effect \[ 18 \]. This immediate effect on locomotion seems to occur through a DA dependent mechanism, as pretreatment with both selective \[ 13 \] and non-selective \[ 18 \] DA receptor antagonists reversed those effects. Moreover, MDPV dose-dependent effects were also considered by several authors \[ 12 , 13 , 14 , 16 , 17 , 18 , 55 \]. While some saw a dose-dependent increase in increased horizontal and vertical activity, mostly observed within the first 1—2 h \[ 13 , 18 \], others have found no dose-dependent effect \[ 12 , 17 \] or even correlated increasing doses with decreased wheel activity \[ 16 \]. That hypolocomotion was also confirmed during the EPM test and, therefore, did not enable us to draw assertive conclusions regarding mice anxiety status. A depressive-like behavior could be suggested by the TST, and a stress-induced self-care disturbance was observed during the ST. Indeed, long-lasting depressive-like behavior was already reported by us \[ 50 \] and others \[ 58 , 59 \] following diverse METH regimens in rodents, and it is a commonly experienced withdrawal symptom in METH users. Nevertheless, we cannot exclude that this behavioral alteration may be, at least partially, attributed to the magnitude of the observed motor deficits. Regarding stress induced self-care dysfunction measures inferred during the ST, one might expect decreased grooming activity as a reflection form of motivational behavior, paralleled with some symptoms of depressive-like behavior such as apathy or an attenuation of sucrose preference \[ 20 \]. On the other hand, it may represent a typical displacement behavior, often increased in animals under conditions of stress and, thus, viewed as an indirect measure of an anxiogenic response to psychotropic drugs for instance \[ 60 \]. Indeed, anxiety is a negative emotional state critical to survival, but persistent and exaggerated apprehension causes substantial morbidity \[ 61 \]. Overall, our data further show that METH animals have locomotor and emotional impairments. The effects of MDPV administration on negative emotional states, namely on anxiety- EPM \[ 20 , 62 \], hedonic- sucrose preference test SPT \[ 20 \], and depressive-like behavior forced swim test FST \[ 63 \], have been poorly investigated to date. This cathinone did not cause an anxiety- and depressive-like behavior at this particular time-point. However, others using different dosing paradigms, time-points and species rat vs. These authors also reported alterations suggestive of a depressive-like behavior FST increased immobility , contrasting with our TST results. Our novel data outline a new behavioral profile for MDPV, 18 h after a high-dose binge regimen, where the stimulant effect is already absent, leaving behind apparent traces of emotional impairment as gauged by a reduction in time and distance in the center of the OFT apparatus, which is seemingly a stressful environment to animals \[ 64 \]. For METH, the present approach states the importance of having complete behavioral batteries to avoid hastening conclusions, since certain behaviors may be shaded by others. Therefore, the second aim of our study was to examine the effects on striatal molecular and cellular outcomes. The locomotor impairment observed has been often associated with METH induced early striatal neurotoxicity, characterized by depletion of dopaminergic—terminal markers TH and DAT and glial reactivity \[ 39 , 47 , 50 , 65 , 66 , 67 , 68 , 69 , 70 \]. Furthermore, our results also revealed that the dopaminergic insult was accompanied by increased GFAP-immunostaining of slightly enlarged astrocytes, suggesting an early stage of astrocytic activation. Consistently, similar drug-regimens showed a peak of astrogliosis over three days \[ 47 , 54 , 67 , 68 \]. Contrary to our expectations, no apparent activation of microglia Iba-1 immunostaining was found. Nevertheless, as Iba-1 staining was seemingly more evident around microglia somas when compared to SAL, we considered a Sholl analysis, as a highly sensitive approach to further analyze microglia morphology and complexity. Indeed, the results revealed morphological changes in microglia after METH-exposure, showing higher branching near the soma, together with a decrease in the number of processes along microglial cells. These data may represent early stages of microglial activation, before acquiring an amoeboid shape of the soma with retracted and shortened processes. In fact, microgliosis was already described 24 h after METH-exposure \[ 68 \]. On the other hand, little is known about the possible neurotoxicity produced by MDPV-exposure. As both toxins share selectivity for the dopaminergic system \[ 25 \], we hypothesized that the MDPV binge regimen could also induce striatal dopaminergic toxicity associated with TH depletion within the first 24 h. Our work is pioneer in the assessment of striatal MDPV-induced toxicity with an administration protocol that replicates a METH binging paradigm that consistently causes striatal neurotoxicity. In agreement with the normal locomotor activity observed, no alterations were seen in striatal TH, GFAP, or Iba-1 protein semi-quantification or immunostaining. Therefore, no evidence of the classical markers of METH-neurotoxicity was found. Duart-Castells et al. This is in line with the observations of Lopez-Arnau et al. The authors argued that this effect takes place at the nerve terminal, and may be a response to the intense DAT blockade exerted by MDPV \[ 72 \]. In contrast to both Lopez-Arnau et al. The authors found altered 3-hydroxybutyric acid levels, which may reflect the activation of a neurotoxic pathway, but the increase in metabolites with neuroprotective properties seemed to counteract this change. Overall, previous results support the absence of striatal dopaminergic neurotoxic effects seen with MDPV, in contrast with METH-induced striatal profile. In fact, the observed METH effects on glial cell activity both astrocytes and microglia may be intrinsically linked with activation of inflammatory processes underlying neuronal damage \[ 13 \]. Thus, alterations in immune cell function can be held responsible for METH neuroinflammation and addictive effects \[ 40 \], which are strongly associated with neuronal impairment and generalized brain dysfunction usually present in drug abusers \[ 77 \]. MDPV is also expected to interfere with inflammatory pathways \[ 78 \]. Nevertheless, neuroinflammation markers have not been documented after MDPV in vivo administration. Accordingly, we aimed to characterize if PRR innate immune modulators, particularly the TLRs and RAGE may be altered, as both have been implied in microglia induced neurotoxicity and neuroinflammation in drug addiction \[ 25 , 36 , 40 , 42 , 43 , 45 , 46 , 79 \]. Unique attention has been given to TLR4 signaling: this is seemingly associated with METH induced activation of glial cells in hippocampus \[ 44 \], increased DA in the NAc shell \[ 36 \], enhanced cytokine expression in the cortex \[ 37 \], and reduced pro-inflammatory mediators in microglia-like cells upon LPS stimulation \[ 38 \]. These innate immune modulators were also unchanged following MDPV-exposure. This is in line with the absent neurotoxicity reported for this particular MDPV protocol, dose, time-point, and brain region. Overall, the link between neuroinflammatory pathways and psychostimulant neurotoxic effects needs to be further explored. The present study has some limitations that should be mentioned. First, only male mice were used and, therefore, results cannot be generalized for both sexes. Female mice were not used to avoid estrous cycle and hormonal impact on data variability, mostly on behavioral performance. Additionally, we cannot discard that MDPV impact in some neuronal signaling pathways, and glial activation might be sex-dependent. Second, we must highlight that using only a single time-point behavioral and neurochemical analysis is a major pitfall. Third, one should not discard that possible damages in other brain regions beyond striatum including hippocampus and prefrontal cortex, known targets for psychostimulants are in place at this time-point. Therefore, further research is warranted to highlight the complex CNS response to MDPV, namely including both sexes, different time-points, and brain regions. Although the present study contributes to a thorough characterization of an acute MDPV binge exposure in vivo, its translation to the clinics should be done with caution, for all the limitations mentioned, and also because of the complexity of drug abuse social and ethical issues. However, minor MDPV behavioral alterations seen with this acute paradigm suggest that the harm-reduction guidance at the clinical level should be done in accordance with that available for other stimulant and cathinone intoxication settings. All efforts were made to minimize animal suffering and to reduce the number of animals used. This binge-like treatment regimen was chosen because it results in extensive DA nerve ending damage and astrogliosis at 12—24 h, when used to inject substituted amphetamines \[ 67 \]. We decided to test MDPV in the same dose for direct comparison between both drugs tested and also because it falls well within the dose range used in similar MDPV in vivo studies \[ 12 , 17 , 19 , 26 \]. Finally, all animals survived this dosing regimen, and none showed convulsions or weight reductions. A battery of behavioral tests was conducted in three independent cohorts from 18 to 24 h after the last drug administration, in the following order: elevated plus maze test EPM , open field test OFT , splash test ST , and tail suspension test TST. Behavioral tests were carried out between 9 am and 1 pm in a sound-attenuated room under dim light 10 lx , following 1 h habituation. Mice behavior was scored by the same rater and monitored through a video camera. Afterwards, a blind analysis was performed by an external experienced researcher using the ANY Maze video tracking Stoelting Co. The EPM was used to evaluate the anxiety-related behavior in mice. The four arms measured 18 cm length and 6 cm wide, and two opposing arms were surrounded by opaque gray walls 15 cm height, closed arms , while the other two arms were devoid of walls open arms. Each animal was placed in the center of the apparatus, facing an open arm, and its exploratory behavior was measured during 5 min. The grooming behavior dorsal grooming of dirt coat was recorded for 5 min, as a validated tool to probe stress-induced self-care and motivational behavior \[ 60 \]. The TST is a predictive behavioral test of antidepressant activity and also other manipulations, including pharmacological e. The test is based on the inability of mice to exhibit escape-directed behavior, when subjected to a short-term stress, thus developing a still posture. Decreased immobility i. For that purpose, mice were suspended 50 cm above the floor using adhesive tape placed approximately 1 cm from the tip of the tail, for 6 min. The immobility time, when they hung passively and completely motionless, was further analyzed \[ 50 \]. RT-qPCR protocol was performed as previously described \[ 46 \]. Reactions were carried out in a thermocycler Eppendorf vapo. Non-template control reactions were performed for each gene, in order to assure that there was no unspecific amplification. The relative expression ratio of each of the target genes was computed based on its real-time PCR efficiencies E and the crossing point difference DCq for an unknown sample versus a control EDCq method. Results were obtained in normalized relative quantities and then converted to percentage using control group as reference. Total striatal protein extracts and western blot WB analysis were performed as previously described \[ 46 , 50 \]. Primary antibodies Table A1 and alkaline phosphatase-conjugated IgG secondary antibodies were used. All photograph areas were considered, and three different zones without staining black were used for background subtraction. Using the Image J Sholl plugin \[ 82 \], we analyzed 40 cells of each experimental condition. For each cell, we removed surrounding processes manually using Fiji Software. Through the line segment tool, we draw a line from the center of each soma to the tip of its longest process, thus providing the maximum process length. We determined the number of intersections made by microglia branching processes with each successive increasing circle to create a Sholl plot. From these data, the maximum number of intersections Nm, the highest number of intersections regardless the radius value , the critical value at which Nm occurred Cr , and the number of primary branches Np, the number of branches that originated from the microglia soma were determined. METH was able to reproduce previous findings of locomotor and emotional impairment, striatal dopaminergic—terminal damage, and astrogliosis. We additionally provided for the first time a distinct microglial morphological profile using Sholl analysis, which is suggestive of microgliosis. Therefore, we further consolidate METH neurotoxic profile. This study also provides a new and integrative insight on the effect of an acute MDPV binge paradigm on behavior, neurochemical, and glial parameters. Regardless of the non-neurotoxic effects produced by a MDPV dosing paradigm similar to the METH toxic protocol used herein , our research highlights the need of further research exploring other time-points and alternative mechanistic pathways in different brain regions where MDPV may have detrimental effects. Conceptualization: S. All authors have read and agreed to the published version of the manuscript. This section collects any data citations, data availability statements, or supplementary materials included in this article. As a library, NLM provides access to scientific literature. Pharmaceuticals Basel. Find articles by Luciana Fernandes. Find articles by Cristina Lemos. Ali fda. Find articles by Syed F Ali. Find articles by Paulo Rodrigues-Santos. Find articles by Carlos A Fontes-Ribeiro. Find articles by Edna Soares. Find articles by Sofia D Viana. Find articles by Frederico C Pereira. Antoni Camins Espuny : Academic Editor. Open in a new tab. Primary and secondary antibodies used for western blot and immunohistochemistry. Similar articles. Add to Collections. Create a new collection. Add to an existing collection. Choose a collection Unable to load your collection due to an error Please try again. Add Cancel. Primary Antibodies. Santa Cruz. Secondary Antibodies. Alexa , anti-goat. Life Technologies.

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