Mixing energy drinks and alcohol during adolescence impairs brain function: A study of rat hippocampal plasticity.

In the last decades, the consumption of energy drinks has risen dramatically, especially among young people, adolescents and athletes, driven by the constant search for ergogenic effects, such as the increase in physical and cognitive performance. In parallel, mixed consumption of energy drinks and ethanol, under a binge drinking modality, under a binge drinking modality, has similarly grown among adolescents. However, little is known whether the combined consumption of these drinks, during adolescence, may have long-term effects on central function, raising the question of the risks of this habit on brain maturation. Our study was designed to evaluate, by behavioral, electrophysiological and molecular approaches, the long-term effects on hippocampal plasticity of ethanol (EtOH), energy drinks (EDs), or alcohol mixed with energy drinks (AMED) in a rat model of binge-like drinking adolescent administration. The results show that AMED binge-like administration produces adaptive hippocampal changes at the molecular level, associated with electrophysiological and behavioral alterations, which develop during the adolescence and are still detectable in adult animals. Overall, the study indicates that binge-like drinking AMED adolescent exposure represents a habit that may affect permanently hippocampal plasticity.

New perspective for an old drug: Can naloxone be considered an antioxidant agent?

Background: Experimental evidence indicates that Naloxone (NLX) holds antioxidant properties. The present study aims at verifying the hypothesis that NLX could prevent oxidative stress induced by hydrogen peroxide (H2O2) in PC12 cells. Methods: To investigate the antioxidant effect of NLX, initially, we performed electrochemical experiments by means of platinum-based sensors in a cell-free system. Subsequently, NLX was tested in PC12 cells on H2O2-induced overproduction of intracellular levels of reactive-oxygen-species (ROS), apoptosis, modification of cells' cycle distribution and damage of cells' plasma membrane. Results: This study reveals that NLX counteracts intracellular ROS production, reduces H2O2-induced apoptosis levels, and prevents the oxidative damage-dependent increases of the percentage of cells in G2/M phase. Likewise, NLX protects PC12 cells from H2O2- induced oxidative damage, by preventing the lactate dehydrogenase (LDH) release. Moreover, electrochemical experiments confirmed the antioxidant properties of NLX. Conclusion: Overall, these findings provide a starting point for studying further the protective effects of NLX on oxidative stress.

Withania somnifera influences MDMA-induced hyperthermic, cognitive, neurotoxic and neuroinflammatory effects in mice.

Withania somnifera (WS) is utilized in Ayurvedic medicine owing to its central and peripheral beneficial properties. Several studies have accrued indicating that the recreational amphetamine-related drug (+/-)- 3,4-methylenedioxymethamphetamine (MDMA; Ecstasy) targets the nigrostriatal dopaminergic system in mice, inducing neurodegeneration and gliosis, causing acute hyperthermia and cognitive impairment. This study aimed to investigate the effect of a standardized extract of W. somnifera (WSE) on MDMA-induced neurotoxicity, neuroinflammation, memory impairment and hyperthermia. Mice received a 3-day pretreatment with vehicle or WSE. Thereafter, vehicle- and WSE-pretreated mice were randomly divided into four groups: saline, WSE, MDMA alone, WSE plus MDMA. Body temperature was recorded throughout treatment, and memory performance was assessed by a novel object recognition (NOR) task at the end of treatment. Thereafter, immunohistochemistry was performed to evaluate in the substantia nigra pars compacta (SNc) and striatum the levels of tyrosine hydroxylase (TH), as marker of dopaminergic degeneration, and of glial fibrillary acidic protein (GFAP) and TMEM119, as markers of astrogliosis or microgliosis, respectively. MDMA-treated mice showed a decrease in TH-positive neurons and fibers in the SNc and striatum respectively, an increase in gliosis and body temperature, and a decrease in NOR performance, irrespective of vehicle or WSE pretreatment. Acute WSE plus MDMA counteracted the modifications in TH-positive cells in SNc, GFAP-positive cells in striatum, TMEM in both areas and NOR performance, as compared to MDMA alone, while no differences were observed as compared to saline. Results indicate that WSE acutely administered in combination with MDMA, but not as pretreatment, protects mice against the noxious central effects of MDMA.

Effects of docosanyl ferulate, a constituent of Withania somnifera, on ethanol- and morphine-elicited conditioned place preference and ERK phosphorylation in the accumbens shell of CD1 mice.

BACKGROUND: Docosanyl ferulate (DF) is a behaviourally active GABAA receptor complex (GABAAR) agonist, recently isolated from the standardized methanolic extract of Withania somnifera Dunal (WSE) root. Previous studies have shown that WSE prevents both ethanol- and morphine-dependent acquisition and expression of conditioned place preference (CPP) and stimulation of dopamine release in the nucleus accumbens shell (AcbSh). AIMS: The study aimed at determining (a) whether DF contributes to WSE's ability to affect the acquisition and expression of ethanol- and morphine-elicited CPP and, given that phosphorylation of extracellular signal-regulated kinase (pERK) in the AcbSh is involved in associative learning and motivated behaviours, (b) whether WSE and DF may affect ethanol- and morphine-induced ERKs phosphorylation in the AcbSh. METHODS: In adult male CD1 mice, DF's effects on the acquisition and expression of ethanol- and morphine-elicited CPP were evaluated by a classical place conditioning paradigm, whereas the effects of WSE and DF on ethanol- and morphine-elicited pERK in the AcbSh were evaluated by immunohistochemistry. RESULTS AND CONCLUSIONS: The study shows that DF, differently from WSE, affects only the acquisition but not the expression of ethanol- and morphine-induced CPP. Moreover, the study shows that both WSE and DF can prevent ethanol- and morphine-elicited pERK expression in the AcbSh. Overall, these results highlight subtle but critical differences for the role of GABAARs in the mechanism by which WSE affects these ethanol- and morphine-dependent behavioural and molecular/cellular responses and support the suggestion of WSE and DF for the control of different components of drug addiction.

Ethanol-Dependent Synthesis of Salsolinol in the Posterior Ventral Tegmental Area as Key Mechanism of Ethanol's Action on Mesolimbic Dopamine.

Abnormal consumption of ethanol, the ingredient responsible for alcoholic drinks' addictive liability, causes millions of deaths yearly. Ethanol's addictive potential is triggered through activation, by a still unknown mechanism, of the mesolimbic dopamine (DA) system, part of a key motivation circuit, DA neurons in the posterior ventral tegmental area (pVTA) projecting to the ipsilateral nucleus accumbens shell (AcbSh). The present in vivo brain microdialysis study, in dually-implanted rats with one probe in the pVTA and another in the ipsilateral or contralateral AcbSh, demonstrates this mechanism. As a consequence of the oral administration of a pharmacologically relevant dose of ethanol, we simultaneously detect a) in the pVTA, a substance, 1-methyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline (salsolinol), untraceable under control conditions, product of condensation between DA and ethanol's first by-product, acetaldehyde; and b) in the AcbSh, a significant increase of DA release. Moreover, such newly generated salsolinol in the pVTA is responsible for increasing AcbSh DA release via µ opioid receptor (µOR) stimulation. In fact, inhibition of salsolinol's generation in the pVTA or blockade of pVTA µORs prevents ethanol-increased ipsilateral, but not contralateral, AcbSh DA release. This evidence discloses the long-sought key mechanism of ethanol's addictive potential and suggests the grounds for developing preventive and therapeutic strategies against abnormal consumption.

The biologically active compound of Withania somnifera (L.) Dunal, docosanyl ferulate, is endowed with potent anxiolytic properties but devoid of typical benzodiazepine-like side effects.

BACKGROUND: Clinical and experimental studies support the therapeutic potential of Withania somnifera (WS) (L.) Dunal on anxiety disorders. This potential is attributable to components present in different plant extracts; however, the individual compound(s) endowed with specific anxiolytic effects and potential modulatory activity of the GABAA receptor complex (GABAAR) have remained unidentified until the recent isolation from a WS methanolic root extract of some GABAAR-active compounds, including the long alkyl-chain ferulic acid ester, docosanyl ferulate (DF). AIMS: This study was designed to assess whether DF (0.05, 0.25 and 2 mg/kg), similarly to diazepam (2 mg/kg), may exert anxiolytic effects, whether these effects may be significantly blocked by the benzodiazepine antagonist flumazenil (10 mg/kg) and whether DF may lack some of the benzodiazepines' typical motor, cognitive and motivational side effects. METHODS: The behavioural paradigms Elevated Plus Maze, Static Rods, Novel Object Recognition, Place Conditioning and potentiation of ethanol-induced Loss of Righting Reflex were applied on male CD-1 mice. RESULTS: Similarly to diazepam, DF exerts anxiolytic effects that are blocked by flumazenil. Moreover, at the full anxiolytic dose of 2 mg/kg, DF lacks typical benzodiazepine-like side effects on motor and cognitive performances and on place conditioning. Moreover, DF fails to potentiate ethanol's (3 g/kg) depressant activity at the ethanol-induced Loss of Righting Reflex paradigm. CONCLUSIONS: These data point to DF as an effective benzodiazepine-like anxiolytic compound that, in light of its lack of motor, mnemonic and motivational side effects, could be a suitable candidate for the treatment of anxiety disorders.

Impact of Caffeine on Ethanol-Induced Stimulation and Sensitization: Changes in ERK and DARPP-32 Phosphorylation in Nucleus Accumbens.

BACKGROUND: Caffeine is frequently consumed with ethanol to reduce the impairing effects induced by ethanol, including psychomotor slowing or incoordination. Both drugs modulate dopamine (DA)-related markers in accumbens (Acb), and Acb DA is involved in voluntary locomotion and locomotor sensitization. The present study determined whether caffeine can affect locomotion induced by acute and repeated ethanol administration in adult male CD-1 mice. METHODS: Acute administration of caffeine (7.5 to 30.0 mg/kg) was evaluated for its effects on acute ethanol-induced (1.5 to 3.5 g/kg) changes in open-field horizontal locomotion, supported rearing, and rearing not supported by the wall. DA receptor-dependent phosphorylation markers were assessed: extracellular signal-regulated kinase (pERK), and dopamine-and cAMP-regulated phosphoprotein Mr32kDa phosphorylated at threonine 75 site (pDARPP-32-Thr75) in Acb core and shell. Acutely administered caffeine was also evaluated in ethanol-sensitized (1.5 g/kg) mice. RESULTS: Acute ethanol decreased both types of rearing. Caffeine increased supported rearing but did not block ethanol -induced decreases in rearing. Both substances increased horizontal locomotion in a biphasic manner, and caffeine potentiated ethanol-induced locomotion. Although ethanol administered repeatedly induced sensitization of locomotion and unsupported rearing, acute administration of caffeine to ethanol-sensitized mice in an ethanol-free state resulted in blunted stimulant effects compared with those seen in ethanol-naïve mice. Ethanol increased pERK immunoreactivity in both subregions of the Acb, but coadministration with caffeine blunted this increase. There were no effects on pDARPP-32(Thr75) immunoreactivity. CONCLUSIONS: The present results demonstrated that, after the first administration, caffeine potentiated the stimulating actions of ethanol, but did not counteract its suppressant or ataxic effects. Moreover, our results show that caffeine has less activating effects in ethanol-sensitized animals.

Effects of caffeine on ethanol-elicited place preference, place aversion and ERK phosphorylation in CD-1 mice.

BACKGROUND: Epidemiological studies indicate a rise in the combined consumption of caffeinated and alcoholic beverages, which can lead to increased risk of alcoholic-beverage overconsumption. However, the effects of the combination of caffeine and ethanol in animal models related to aspects of drug addiction are still underexplored. AIMS: To characterize the pharmacological interaction between caffeine and ethanol and establish if caffeine can affect the ability of ethanol (2 g/kg) to elicit conditioned place preference and conditioned place aversion, we administered caffeine (3 or 15 mg/kg) to male CD-1 mice before saline or ethanol. Moreover, we determined if these doses of caffeine could affect ethanol (2 g/kg) elicited extracellular signal-regulated kinase phosphorylation in brain areas, nucleus accumbens, bed nucleus of stria terminalis, central nucleus of the amygdala, and basolateral amygdala, previously associated with this type of associative learning. RESULTS: In the place-conditioning paradigm, caffeine did not have an effect on its own, whereas ethanol elicited significant conditioned-place preference and conditioned-place aversion. Caffeine (15 mg/kg) significantly prevented the acquisition of ethanol-elicited conditioned-place preference and, at both doses, also prevented the acquisition of ethanol-elicited conditioned-place aversion. Moreover, both doses of caffeine also prevented ethanol-elicited extracellular signal-regulated kinase phosphorylation expression in all brain areas examined. CONCLUSIONS: The present data indicate a functional antagonistic action of caffeine and ethanol on associative learning and extracellular signal-regulated kinase phosphorylation after an acute interaction. These results could provide exciting grounds for further studies, also in a translational perspective, of their pharmacological interaction modulating other processes involved in drug consumption and addiction.

Inhibition of Morphine- and Ethanol-Mediated Stimulation of Mesolimbic Dopamine Neurons by Withania somnifera.

Morphine- and ethanol-induced stimulation of neuronal firing of ventral tegmental area (VTA) dopaminergic neurons and of dopamine (DA) transmission in the shell of the nucleus accumbens (AcbSh) represents a crucial electrophysiological and neurochemical response underlying the ability of these compounds to elicit motivated behaviors and trigger a cascade of plasticity-related biochemical events. Previous studies indicate that the standardized methanolic extract of Withania somnifera roots (WSE) prevents morphine- and ethanol-elicited conditioned place preference and oral ethanol self-administration. Aim of the present research was to investigate whether WSE may also interfere with the ability of morphine and ethanol to stimulate VTA dopaminergic neurons and thus AcbSh DA transmission as assessed in male Sprague-Dawley rats by means of patch-clamp recordings in mesencephalic slices and in vivo brain microdialysis, respectively. Morphine and ethanol significantly stimulated spontaneous firing rate of VTA neurons and DA transmission in the AcbSh. WSE, at concentrations (200-400 µg/ml) that significantly reduce spontaneous neuronal firing of VTA DA neurons via a GABAA- but not GABAB-mediated mechanism, suppressed the stimulatory actions of both morphine and ethanol. Moreover, in vivo administration of WSE at a dose (75 mg/kg) that fails to affect basal DA transmission, significantly prevented both morphine- and ethanol-elicited increases of DA in the AcbSh. Overall, these results highlight the ability of WSE to interfere with morphine- and ethanol-mediated central effects and suggest a mechanistic interpretation of the efficacy of this extract to prevent the motivational properties of these compounds.

Not Just from Ethanol. Tetrahydroisoquinolinic (TIQ) Derivatives: from Neurotoxicity to Neuroprotection.

The 1,2,3,4-tetrahydroisoquinolines (TIQs) are compounds frequently described as alkaloids that can be found in the human body fluids and/or tissues including the brain. In most circumstances, TIQs may be originated as a consequence of reactions, known as Pictet-Spengler condensations, between biogenic amines and electrophilic carbonyl compounds, including ethanol's main metabolite, acetaldehyde. Several TIQs may also be synthesized enzymatically whilst others may be formed in the body as by-products of other compounds including TIQs themselves. The biological actions of TIQs appear critically dependent on their metabolism, and nowadays, among TIQs, 1-methyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline (salsolinol), N-methyl-1-methyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline (N-methyl-(R)-salsolinol), 1-[(3,4-dihydroxyphenyl)methyl]-1,2,3,4-tetrahydroisoquinoline-6,7-diol (norlaudanosoline or tetrahydropapaveroline or THP) and 1-benzyl-1,2,3,4-tetrahydroisoquinoline (1BnTIQ) are considered as those endowed with the most potent neurotoxic actions. However, it remains to be established whether a continuous exposure to TIQs or to their metabolites might carry toxicological consequences in the short- or long-term period. Remarkably, recent findings suggest that some TIQs such as (1-[(4-hydroxyphenyl)methyl]-1,2,3,4-tetrahydroisoquinoline-6,7-diol) (higenamine) and 1-methyl-1,2,3,4-tetrahydroisoquinoline (1-MeTIQ) as well as N-methyl-tetrahydroisoquinoline (N-methyl-TIQ) exert unique neuroprotective and neurorestorative actions. The present review article provides an overview on these aspects of TIQs and summarizes those that presently appear the most significant highlights on this puzzling topic.

Ferulic Acid Esters and Withanolides: In Search of Withania somnifera GABAA Receptor Modulators.

Nine compounds, including two undescribed withanolides, withasomniferolides A and B (1 and 2), three known withanolides (3-5), a ferulic acid dimeric ester (6), and an inseparable mixture of three long alkyl chain ferulic acid esters (7-9), were isolated from a GABAA receptor positive activator methanol extract of the roots of Withania somnifera. The structures of the isolated compounds were elucidated based on NMR, MS, and ECD data analysis. In order to bioassay the single ferulic acid derivatives, compounds 6-9 were also synthesized. The most active compound, docosanyl ferulate (9), was able to enhance the GABAA receptor inhibitory postsynaptic currents with an IC50 value of 7.9 µM. These results, by showing an ability to modulate the GABAA receptor function, cast fresh light on the biological activities of the secondary metabolites of W. somnifera roots.

Active avoidance learning differentially activates ERK phosphorylation in the primary auditory and visual cortices of Roman high- and low-avoidance rats.

Understanding the mechanisms underlying conditioned avoidance is a critical step toward the development of novel treatments of anxiety. In this context, the two-way active avoidance (2WAA) task is a validated paradigm to investigate uncontrolled avoidance, a hallmark of anxiety disorders. The outbred Roman high- (RHA) and low-avoidance (RLA) rat lines are selected for respectively rapid vs. poor acquisition of active avoidant behavior, and emotional reactivity appears to be the most prominent behavioral difference between the two lines, with RLA rats being more fearful/anxious than their RHA counterparts. This study was aimed at assessing the relationship between the different performance of RHA and RLA rats in the 2WAA task and the number of phosphorylated ERK positive (pERK+) neurons in the primary auditory and visual cortices, in three sub-nuclei of the amygdala, as well as in the nucleus accumbens, and the prefrontal cortex. The results indicate that: (1) RHA rats, but not their RLA counterparts, learn very rapidly to avoid mild electric foot-shocks by crossing to the opposite compartment of the shuttle-box during the presentation of the conditioned stimulus and (2) the different behavior of the Roman lines during active avoidance training is associated with differential changes in the number of pERK+ neurons in the primary auditory and visual cortices (where the proactive coping of RHA rats is associated with increased ERK phosphorylation), but not in the other brain areas examined. These results are consistent with the hypothesis that the activation of the ERK signaling cascade in the auditory and visual cortices may be involved in the acquisition of aversive learning in RHA rats.

Evidence of a PPARγ-mediated mechanism in the ability of Withania somnifera to attenuate tolerance to the antinociceptive effects of morphine.

Notwithstanding the experimental evidence indicating Withania somnifera Dunal roots extract (WSE) ability to prolong morphine-elicited analgesia, the mechanisms underlying this effect are largely unknown. With the aim of evaluating a PPARγ-mediated mechanism in such WSE effects, we verified the ability of the PPARγ antagonist GW-9662 to modulate WSE actions. Further, we evaluated the influence of GW-9662 upon WSE / morphine interaction in SH-SY5Y cells since we previously reported that WSE hampers the morphine-induced µ-opioid receptor (MOP) receptor down-regulation. Nociceptive thresholds / tolerance development were assessed in different groups of rats receiving vehicles (control), morphine (10 mg/kg; i.p.), WSE (100 mg/kg, i.p.) and PPARγ antagonist GW-9662 (1 mg/kg; s.c.) in acute and chronic schedules of administration. Moreover, the effects of GW-9662 (5 and 10 µM) applied alone and in combination with morphine (10 µM) and/or WSE (0.25 and 1.00 mg/mL) on the MOP gene expression were investigated in cell cultures. Data analysis revealed a functional effect of the PPARγ antagonist in attenuating the ability of WSE to prolong morphine analgesic effect and to reduce tolerance development after repeated administration. In addition, molecular experiments demonstrated that the blockade of PPARγ by GW-9662 promotes MOP mRNA down-regulation and counteracts the ability of 1.00 mg/mL of WSE to keep an adequate MOP receptor availability. In conclusion, our results support the involvement of a PPARγ-mediated mechanism in the WSE effects on morphine-mediated nociception and the likely usefulness of WSE in lengthening the analgesic efficacy of opioids in chronic therapy.

Standardized phytotherapic extracts rescue anomalous locomotion and electrophysiological responses of TDP-43 Drosophila melanogaster model of ALS.

Findings from studies using animal models expressing amyotrophic lateral sclerosis (ALS) mutations in RNA-binding proteins, such as Transactive Response DNA-binding protein-43 (TDP-43), indicate that this protein, which is involved in multiple functions, including transcriptional regulation and pre-mRNA splicing, represents a key candidate in ALS development. This study focuses on characterizing, in a Drosophila genetic model of ALS (TDP-43), the effects of Mucuna pruriens (Mpe) and Withania somnifera (Wse). Electrophysiological and behavioural data in TDP-43 mutant flies revealed anomalous locomotion (i.e. impaired climbing with unexpected hyperactivity) and sleep dysregulation. These features, in agreement with previous findings with a different ALS model, were at least partially, rescued by treatment with Mpe and Wse. In addition, electrophysiological recordings from dorsal longitudinal muscle fibers and behavioral observations of TDP-43 flies exposed to the volatile anaesthetics, diethyl ether or chloroform, showed paradoxical responses, which were normalized upon Mpe or Wse treatment. Hence, given the involvement of some potassium channels in the effects of anaesthetics, our results also hint toward a possible dysregulation of some potassium channels in the ALS-TDP-43 Drosophila model, that might shed new light on future therapeutic strategies pertaining to ALS.

Sex-specific differences in cannabinoid-induced extracellular-signal-regulated kinase phosphorylation in the cingulate cortex, prefrontal cortex, and nucleus accumbens of Lister Hooded rats.

Sex-dependent differences have been consistently described in cannabinoid addiction research. In particular, we recently reported that female Lister Hooded rats display greater self-administration of the cannabinoid CB1 receptor agonist WIN55,212-2 (WIN) and stronger reinstatement of cannabinoid-seeking behavior than males. Cannabinoids modulate the phosphorylation of the extracellular-signal-regulated kinase (ERK) pathway, leading to various forms of plasticity-related learning that likely affect operant behavior. However, whether or not the reported sex-dependent differences in cannabinoid-taking and cannabinoid-seeking behaviors may be related to a sexual dimorphic activation of the ERK pathway remains still to be determined. In the present study, we measured the level of phosphoERK-positive cells in the cingulate cortex (CG1), prefrontal cortex (PFCx), and nucleus accumbens of male and of intact (i.e. sham-operated) and ovariectomized female Lister Hooded rats 30 and 60 min after an acute, intravenous, injection of a dose of WIN (0.3 mg/kg) resembling the mean amount of drug daily self-administered by trained rats. We found that WIN significantly increased ERK activation in the CG1, PFCx, and nucleus accumbens in a sex time and, restricted to the cortical areas, layer-specific manner. Moreover, the comparison between intact and ovariectomized female rats revealed a significant role played by estrogens in WIN-elicited ERK activation. These results indicate, for the first time, the existence of a sexually dimorphic cannabinoid receptor-dependent ERK activation that, restricted to the CG1 and PFCx, is ovarian hormone-dependent.

The standardized Withania somnifera Dunal root extract alters basal and morphine-induced opioid receptor gene expression changes in neuroblastoma cells.

BACKGROUND: Behavioral studies demonstrated that the administration of Withania somnifera Dunal roots extract (WSE), prolongs morphine-elicited analgesia and reduces the development of tolerance to the morphine's analgesic effect; however, little is known about the underpinning molecular mechanism(s). In order to shed light on this issue in the present paper we explored whether WSE promotes alterations of µ (MOP) and nociceptin (NOP) opioid receptors gene expression in neuroblastoma SH-SY5Y cells. METHODS: A range of WSE concentrations was preliminarily tested to evaluate their effects on cell viability. Subsequently, the effects of 5 h exposure to WSE (0.25, 0.50 and 1.00 mg/ml), applied alone and in combination with morphine or naloxone, on MOP and NOP mRNA levels were investigated. RESULTS: Data analysis revealed that morphine decreased MOP and NOP receptor gene expression, whereas naloxone elicited their up-regulation. In addition, pre-treatment with naloxone prevented the morphine-elicited gene expression alterations. Interestingly, WSE was able to: a) alter MOP but not NOP gene expression; b) counteract, at its highest concentration, morphine-induced MOP down-regulation, and c) hamper naloxone-induced MOP and NOP up-regulation. CONCLUSION: Present in-vitro data disclose novel evidence about the ability of WSE to influence MOP and NOP opioid receptors gene expression in SH-SY5Y cells. Moreover, our findings suggest that the in-vivo modulation of morphine-mediated analgesia by WSE could be related to the hindering of morphine-elicited opioid receptors down-regulation here observed following WSE pre-treatment at its highest concentration.