The effects of heavy metal exposure on brain and gut microbiota: A systematic review of animal studies.

The gut-brain axis is a crucial interface between the central nervous system and the gut microbiota. Recent evidence shows that exposure to environmental contaminants, such as heavy metals, can cause dysbiosis in gut microbiota, which may affect the gut-brain communication, impacting aspects of brain function and behavior. This systematic review of the literature aims to evaluate whether deleterious effects on brain function due to heavy metal exposure could be mediated by changes in the gut microbiota profile. Animal studies involving exposure to heavy metals and a comparison with a control group that evaluated neuropsychological outcomes and/or molecular outcomes along with the analysis of microbiota composition were reviewed. The authors independently assessed studies for inclusion, extracted data and assessed risk of bias using the protocol of Systematic Review Center for Laboratory Animal Experimentation (SYRCLE) for preclinical studies. A search in 3 databases yielded 16 eligible studies focused on lead (n = 10), cadmium (n = 1), mercury (n = 3), manganese (n = 1), and combined exposure of lead and manganese (n = 1). The animal species were rats (n = 7), mice (n = 4), zebrafish (n = 3), carp (n = 1) and fruit fly (n = 1). Heavy metals were found to adversely affect cognitive function, behavior, and neuronal morphology. Moreover, heavy metal exposure was associated with changes in the abundance of specific bacterial phyla, such as Firmicutes and Proteobacteria, which play crucial roles in gut health. In some studies, these alterations were correlated with learning and memory impairments and mood disorders. The interplay of heavy metals, gut microbiota, and brain suggests that heavy metals can induce direct brain alterations and indirect effects through the microbiota, contributing to neurotoxicity and the development of neuropsychological disorders. However, the small number of papers under review makes it difficult to draw definitive conclusions. Further research is warranted to unravel the underlying mechanisms and evaluate the translational implications for human health.

Genetic Load of SARS-CoV-2 in Aerosols Collected in Operating Theaters.

After the outbreak of COVID-19, additional protocols have been established to prevent the transmission of the SARS-CoV-2 from the patient to the health personnel and vice versa in health care settings. However, in the case of emergency surgeries, it is not always possible to ensure that the patient is not infected with SARS-CoV-2, assuming a potential source of transmission of the virus to health personnel. This work aimed to evaluate the presence of the SARS-CoV-2 and quantify the viral load in indoor air samples collected inside operating rooms, where emergency and scheduled operations take place. Samples were collected for 3 weeks inside two operating rooms for 24 h at 38 L/min in quartz filters. RNA was extracted from the filters and analyzed using RT-qPCR targeting SARS-CoV-2 genes E, N1 and N2 regions. SARS-CoV-2 RNA was detected in 11.3% of aerosol samples collected in operating rooms, despite with low concentrations (not detected at 13.5 cg/m3 and 10.5 cg/m3 in the scheduled and emergency operating rooms, respectively). Potential sources of airborne SARS-CoV-2 could be aerosolization of the virus during aerosol-generating procedures and in open surgery from patients that might have been recently infected with the virus, despite presenting a negative COVID-19 test. Another source could be related to health care workers unknowingly infected with the virus and exhaling SARS-CoV-2 virions into the air. These results highlight the importance of reinforcing preventive measures against COVID-19 in operating rooms, such as the correct use of protective equipment, screening programs for health care workers, and information campaigns. IMPORTANCE Operating rooms are critical environments in which asepsis must be ensured. The COVID-19 pandemic entailed the implementation of additional preventative measures in health care settings, including operating theaters. Although one of the measures is to operate only COVID-19 free patients, this measure cannot be always implemented, especially in emergency interventions. Therefore, a surveillance campaign was conducted during 3 weeks in two operating rooms to assess the level of SARS-CoV-2 genetic material detected in operating theaters with the aim to assess the risk of COVID-19 transmission during operating procedures. SARS-CoV-2 genetic material was detected in 11% of aerosol samples collected in operating rooms, despite with low concentrations. Plausible SARS-CoV-2 sources have been discussed, including patients and health care personnel infected with the virus. These results highlight the importance of reinforcing preventive measures against COVID-19 in operating rooms, such as the correct use of protective equipment, screening programs for health care workers and information campaigns.

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.

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.

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.

Effects of morphine on place conditioning and ERK1/2 phosphorylation in the nucleus accumbens of psychogenetically selected Roman low- and high-avoidance rats.

RATIONALE: Extracellular signal-regulated kinase (ERK1/2) phosphorylation is critical for neuronal and behavioural functions; in particular, phosphorylated ERK1/2 (pERK1/2) expression in the nucleus accumbens (Acb) of the rat is stimulated by addictive drugs with the exception of morphine, which decreases accumbal ERK1/2 phosphorylation in the Sprague-Dawley and Wistar rats. The psychogenetically selected Roman low- (RLA) and high-avoidance (RHA) rats differ behaviourally and neurochemically in many responses to addictive drugs. In particular, morphine elicits a greater increment in locomotor activity and in dopamine transmission in the Acb of RHA vs RLA rats. However, the effects of morphine on place conditioning (conditioned place preference (CPP)) and ERK1/2 phosphorylation in the Roman lines remain unknown. OBJECTIVES AND METHODS: To characterize in the Roman lines the reinforcing properties of morphine (i.e. morphine-elicited CPP acquisition) and the relationship between these properties and its effects on ERK1/2 phosphorylation in the Acb, the behavioural effects of morphine were evaluated in a place-conditioning apparatus and ERK1/2 phosphorylation was assessed by immunohistochemistry in the shell and core subregions of the Acb of rats both acutely administered with morphine or undergoing conditioning. RESULTS: Morphine elicited CPP in both Roman lines and decreased pERK1/2 expression in the Acb of RLA but not RHA rats. Such decrease was prevented by conditioning. CONCLUSIONS: These findings indicate that the selective breeding of the Roman lines has generated a divergence, in terms of morphine-elicited pERK1/2 expression but not of morphine-elicited CPP, between RLA and RHA rats and sustain the observation that changes in pERK1/2 expression in the Acb are not a requisite for the reinforcing effects of morphine.

Is catalase involved in the effects of systemic and pVTA administration of 4-methylpyrazole on ethanol self-administration?

The oxidative metabolism of ethanol into acetaldehyde involves several enzymes, including alcohol dehydrogenase (ADH) and catalase-hydrogen peroxide (H2O2). In this regard, while it is well known that 4-methylpyrazole (4-MP) acts by inhibiting ADH in the liver, little attention has been placed on its ability to interfere with fatty acid oxidation-mediated generation of H2O2, a mechanism that may indirectly affect catalase whose enzymatic activity requires H2O2. The aim of our investigation was twofold: 1) to evaluate the effect of systemic (i.p. [intraperitoneal]) and local (into the posterior ventral tegmental area, pVTA) administration of 4-MP on oral ethanol self-administration, and 2) to assess ex vivo whether or not systemic 4-MP affects liver and brain H2O2 availability. The results show that systemic 4-MP reduced ethanol but not acetaldehyde or saccharin self-administration, and decreased the ethanol deprivation effect. Moreover, local intra-pVTA administration of 4-MP reduced ethanol but not saccharin self-administration. In addition, although unable to affect basal catalase activity, systemic administration of 4-MP decreased H2O2 availability both in liver and in brain. Overall, these results indicate that 4-MP interferes with ethanol self-administration and suggest that its behavioral effects could be due to a decline in catalase-H2O2 system activity as a result of a reduction of H2O2 availability, thus highlighting the role of central catalase-mediated metabolism of ethanol and further supporting the key role of acetaldehyde in the reinforcing properties of ethanol.

Mystic Acetaldehyde: The Never-Ending Story on Alcoholism.

After decades of uncertainties and drawbacks, the study on the role and significance of acetaldehyde in the effects of ethanol seemed to have found its main paths. Accordingly, the effects of acetaldehyde, after its systemic or central administration and as obtained following ethanol metabolism, looked as they were extensively characterized. However, almost 5 years after this research appeared at its highest momentum, the investigations on this topic have been revitalized on at least three main directions: (1) the role and the behavioral significance of acetaldehyde in different phases of ethanol self-administration and in voluntary ethanol consumption; (2) the distinction, in the central effects of ethanol, between those arising from its non-metabolized fraction and those attributable to ethanol-derived acetaldehyde; and (3) the role of the acetaldehyde-dopamine condensation product, salsolinol. The present review article aims at presenting and discussing prospectively the most recent data accumulated following these three research pathways on this never-ending story in order to offer the most up-to-date synoptic critical view on such still unresolved and exciting topic.

Differential effects of the MEK inhibitor SL327 on the acquisition and expression of ethanol-elicited conditioned place preference and aversion in mice.

The involvement of mitogen-activating extracellular kinase (MEK) in place conditioning may vary depending on the motivational sign (positive or negative) and nature (pharmacological or nociceptive) of the unconditioned stimulus (US) and on the phase (acquisition or expression) of the learning process. This study investigated the role of MEK on the acquisition and expression of ethanol-elicited (given 2 g/kg) backward (preference, CPP) and forward (aversion, CPA) place conditioning. The MEK inhibitor SL327 (50 mg/kg for CPP, and 50 and 100 mg/kg for CPA) was administered to CD-1 mice 60 minutes before an ethanol dose (acquisition) or 60 minutes before the post-conditioning tests (expression). Ethanol significantly elicited CPP and CPA; SL327 (50 mg/kg) significantly blocked the acquisition of ethanol-elicited CPP, but not that of CPA. Moreover, SL327 (50 and 100 mg/kg) significantly reduced the expression of ethanol-elicited CPP, but not that of CPA. Finally, SL327 also prevented ethanol-elicited (given 2 g/kg) increases of phosphorylated extracellular signal regulated kinase (pERK)-positive neurons in the nucleus accumbens and other nuclei of the extended amygdala. Overall, these results confirmed the differential involvement of MEK in the acquisition and expression of drug-elicited place conditioning and suggested its differential involvement in distinct behavioral outcomes, depending on the motivational sign of the (same) US and on the significance of the experimental phase of the learning process.

From Ethanol to Salsolinol: Role of Ethanol Metabolites in the Effects of Ethanol.

In spite of the global reputation of ethanol as the psychopharmacologically active ingredient of alcoholic drinks, the neurobiological basis of the central effects of ethanol still presents some dark sides due to a number of unanswered questions related to both its precise mechanism of action and its metabolism. Accordingly, ethanol represents the interesting example of a compound whose actions cannot be explained as simply due to the involvement of a single receptor/neurotransmitter, a scenario further complicated by the robust evidence that two main metabolites, acetaldehyde and salsolinol, exert many effects similar to those of their parent compound. The present review recapitulates, in a perspective manner, the major and most recent advances that in the last decades boosted a significant growth in the understanding on the role of ethanol metabolism, in particular, in the neurobiological basis of its central effects.

Role of nucleus accumbens µ opioid receptors in the effects of morphine on ERK1/2 phosphorylation.

RATIONALE: Despite the critical role attributed to phosphorylated extracellular signal regulated kinase (pERK1/2) in the nucleus accumbens (Acb) in the actions of addictive drugs, the effects of morphine on ERK1/2 phosphorylation in this area are still controversial. OBJECTIVES: In order to investigate further this issue, we studied (1) the ability of morphine to affect ERK1/2 phosphorylation in the shell (AcbSh) and core (AcbC) of Sprague-Dawley and Wistar rats and of CD-1 and C57BL/6J mice and (2) the role of dopamine D1 and µ-opioid receptors in Sprague-Dawley rats and CD-1 mice. METHODS: The pERK1/2 expression was assessed by immunohistochemistry. RESULTS: In rats, morphine decreased AcbSh and AcbC pERK1/2 expression, whereas in mice, increased it preferentially in the AcbSh compared with the AcbC. Systemic SCH 39166 decreased pERK1/2 expression on its own in the AcbSh and AcbC of Sprague-Dawley rats and CD-1 mice; furthermore, in rats, SCH 39166 disclosed the ability of morphine to stimulate pERK1/2 expression. Systemic (rats and mice) and intra-Acb (rats) naltrexone prevented both decreases, in rats, and increases, in mice. CONCLUSIONS: These findings confirm the differential effects of morphine in rats and mice Acb and that D1 receptors exert a facilitatory role on ERK1/2 phosphorylation; furthermore, they indicate that, in rats, removal of the D1-dependent pERK1/2 expression discloses the stimulatory influence of morphine on ERK1/2 phosphorylation and that the morphine's ability to decrease pERK1/2 expression is mediated by Acb µ-opioid receptors. Future experiments may disentangle the psychopharmacological significance of the effects of morphine on pERK1/2 in the Acb.