Differential pattern of neurotoxicity induced by the gliadin peptides p31-43 and p57-68 in in vitro model of epilepsy.

Epilepsy is a central nervous system (CNS) disorder causing repeated seizures due to a transient excessive or synchronous alteration in the electrical activity of the brain. Several neurological disorders have been associated to gluten-related diseases (GRD), including epilepsy. However, the molecular mechanisms that associate GRD and epileptogenesis are still unknown. Our previous data have shown that the gliadin peptide 31-43 (p31-43) enhanced number and duration of seizures induced by kainate in mice and exacerbated CA3-kainate-induced neurotoxicity in organotypic hippocampal slices. Here, we investigated whether another important gliadin peptide p57-68 may exerts effects similar to p31-43 on kainate-induced neurotoxicity. We find that both peptides exacerbate kainate-induced damage in the CA3 region once simultaneously challenged. However, after pre-incubation, p31-43 additionally exacerbates neurotoxicity in the CA1 region, while p57-68 does not. These data suggested differential intracellular mechanisms activated by the peptides. Indeed, analysing intracellular signalling pathways we discover that p31-43 induces significant intracellular changes, including increased phosphorylation of Akt, Erk1/2, and p65, decreased p38 phosphorylation, and deacetylation of nuclear histone-3. Based on these observations, we demonstrate that p31-43 likely activates specific intracellular signaling pathways involved in neuronal excitability, inflammation, and epigenetic regulation, which may contribute to its exacerbation of kainate-induced neurotoxicity. In contrast, p57-68 appears to exert its effects through different mechanisms. Further research is necessary to elucidate the precise mechanisms by which these peptides influence neurotoxicity and understand their implications for neurological disorders.

Comparative Study of Mycobacterium bovis and Mycobacterium avium subsp. paratuberculosis In Vitro Infection in Bovine Bone Marrow Derived Macrophages: Preliminary Results.

Bovine tuberculosis and paratuberculosis are endemic in many areas worldwide. This work aims to study cytokines production and gene expression profiles of bovine macrophages infected with Mycobacterium bovis and Mycobacterium paratuberculosis subsp. avium (MAP) strains to identify potential diagnostic biomarkers. Bovine bone marrow stem cells were differentiated into macrophages and subsequently infected in vitro with different spoligotypes of M. bovis and MAP field strains (as single infections and coinfections), using different multiplicity of infection. Supernatant and cell pellets were collected 24 h, 48 h, and one week post-infection. Preliminarily, gene expression on cell pellets of IL-1ß, IL-2, INFγ, IL-6, IL-10, IL-12, and TNFα was assessed by qRT-PCR one week p.i. Subsequently, IL-1ß and IL-6 were measured by ELISA and qRT-PCR to investigated their production retrospectively 24 h and 48 h p.i. A variability in macrophages response related to the concentration of mycobacteria, the coinfection with MAP, and M. bovis spoligotypes was identified. An early and constant IL-6 increase was observed in the M. bovis infection. A lower increase in IL-1ß was also detected at the highest concentration of the two M. bovis spoligotypes one week post-infection. IL-6 and IL-1 ß production was reduced and differently expressed in the MAP infection. IL-6 appeared to be the earliest cytokines produced by bovine macrophages infected with M. bovis.

Ethanol-induced AMPA alterations are mediated by mGLU5 receptors through miRNA upregulation in hippocampal slices.

Prenatal alcohol exposure (PAE) affects neuronal networks and brain development causing a range of physical, cognitive and behavioural disorders in newborns that persist into adulthood. The array of consequences associated with PAE can be grouped under the umbrella-term 'fetal alcohol spectrum disorders' (FASD). Unfortunately, there is no cure for FASD as the molecular mechanisms underlying this pathology are still unknown. We have recently demonstrated that chronic EtOH exposure, followed by withdrawal, induces a significant decrease in AMPA receptor (AMPAR) expression and function in developing hippocampus in vitro. Here, we explored the EtOH-dependent pathways leading to hippocampal AMPAR suppression. Organotypic hippocampal slices (2 days in cultures) were exposed to EtOH (150 mM) for 7 days followed by 24 h EtOH withdrawal. Then, the slices were analysed by means of RT-PCR for miRNA content, western blotting for AMPA and NMDA related-synaptic proteins expression in postsynaptic compartment and electrophysiology to record electrical properties from CA1 pyramidal neurons. We observed that EtOH induces a significant downregulation of postsynaptic AMPA and NMDA subunits and relative scaffolding protein expression and, accordingly, a decrease of AMPA-mediated neurotransmission. Simultaneously, we found that chronic EtOH induced-upregulation of miRNA 137 and 501-3p and decreased AMPA-mediated neurotransmission are prevented by application of the selective mGlu5 antagonist MPEP during EtOH withdrawal. Our data indicate mGlu5 via miRNA137 and 501-3p expression as key factors in the regulation of AMPAergic neurotransmission that may contribute, at least in part, to the pathogenesis of FASD.

Oral and fecal microbiota perturbance in cocaine users: Can rTMS-induced cocaine abstinence support eubiosis restoration?

The effects of cocaine on microbiota have been scarcely explored. Here, we investigated the gut (GM) and oral (OM) microbiota composition of cocaine use disorder (CUD) patients and the effects of repetitive transcranial magnetic stimulation (rTMS). 16S rRNA sequencing was used to characterize GM and OM, whereas PICRUST2 assessed functional changes in microbial communities, and gas-chromatography was used to evaluate fecal short and medium chain fatty acids. CUD patients reported a significant decrease in alpha diversity and modification of the abundances of several taxa in both GM and OM. Furthermore, many predicted metabolic pathways were differentially expressed in CUD patients' stool and saliva samples, as well as reduced levels of butyric acid that appear restored to normal amounts after rTMS treatment. In conclusion, CUD patients showed a profound dysbiotic fecal and oral microbiota composition and function and rTMS-induced cocaine abstinence determined the restoration of eubiotic microbiota.

Urinary Tract Infections: The Current Scenario and Future Prospects.

Urinary tract infections (UTIs) are among the most common bacterial infections worldwide, occurring in both community and healthcare settings. Although the clinical symptoms of UTIs are heterogeneous and range from uncomplicated (uUTIs) to complicated (cUTIs), most UTIs are usually treated empirically. Bacteria are the main causative agents of these infections, although more rarely, other microorganisms, such as fungi and some viruses, have been reported to be responsible for UTIs. Uropathogenic Escherichia coli (UPEC) is the most common causative agent for both uUTIs and cUTIs, followed by other pathogenic microorganisms, such as Klebsiella pneumoniae, Proteus mirabilis, Enterococcus faecalis, and Staphylococcus spp. In addition, the incidence of UTIs caused by multidrug resistance (MDR) is increasing, resulting in a significant increase in the spread of antibiotic resistance and the economic burden of these infections. Here, we discuss the various factors associated with UTIs, including the mechanisms of pathogenicity related to the bacteria that cause UTIs and the emergence of increasing resistance in UTI pathogens.

Role of the Intestinal Microbiota in the Genesis of Major Depression and the Response to Antidepressant Drug Therapy: A Narrative Review.

A major depressive disorder is a serious mental illness characterized by a pervasive low mood that negatively concerns personal life, work life, or education, affecting millions of people worldwide. To date, due to the complexity of the disease, the most common and effective treatments consist of a multi-therapy approach, including psychological, social, and pharmacological support with antidepressant drugs. In general, antidepressants are effective in correcting chemical imbalances of neurotransmitters in the brain, but recent evidence has underlined the pivotal role of gut microbiota (GM) also in the regulation of their pharmacokinetics/pharmacodynamics, through indirect or direct mechanisms. The study of these complex interactions between GM and drugs is currently under the spotlight, and it has been recently named "pharmacomicrobiomics". Hence, the purpose of this review is to summarize the contribution of GM and its metabolites in depression, as well as their role in the metabolism and activity of antidepressant drugs, in order to pave the way for the personalized administration of antidepressant therapies.

Antidepressant-induced increase in GluA2 expression does not translate in changes of AMPA receptor-mediated synaptic transmission at CA3/CA1 synapses in rats.

Chronic treatment with serotonin selective reuptake inhibitors or tryciclic antidepressant drugs in rodents has been shown to increase the expression of GluA1 and/or GluA2 AMPA receptor (AMPAR) subunits in several brain areas, including the hippocampus. These changes in AMPAR composition have been suggested to result in increased glutamatergic neurotransmission and possibly underlie enhanced hippocampal synaptic plasticity through the increased availability of calcium-permeable AMPARs, specifically at CA3/CA1 synapses. However, the possibility that chronic treatment with antidepressants actually results in strengthened glutamatergic neurotransmission in CA1 has poorly been investigated. Here, we studied whether chronic treatment with the multimodal antidepressant drug trazodone mimicked the effect of paroxetine on the expression of AMPAR subunits in male wistar rat hippocampus and whether these drugs produced a parallel facilitation of field excitatory postsynaptic potentials (fEPSP) responses evoked by activation of CA3/CA1 synapses in dorsal hippocampal slices. In addition, we investigated whether the quality of glutamatergic AMPARs involved in basal neurotransmission was changed by altered subunit expression, e.g. leading to appearance of calcium-permeable AMPARs. We found a significant increase in GluA2 subunit expression following treatment with trazodone or paroxetine for twenty-one days, but not after seven-days treatment. In contrast, we did not find any significant changes in fEPSP responses supporting either a facilitation of glutamatergic neurotransmission in basal conditions or the appearance of functional calcium-permeable AMPARs at CA3/CA1 pyramidal neuron synapses. Thus, neurochemically-detected increases in the expression of AMPAR subunits cannot directly be extrapolated in increased number of functioning receptors and/or facilitated basal neurotransmission.

SARS-CoV-2 Infection in Patients with Cystic Fibrosis: What We Know So Far.

Respiratory infections are the most common and most frequent diseases, especially in children and the elderly, characterized by a clear seasonality and with an incidence that usually tends to decrease with increasing age. These infections often resolve spontaneously, usually without the need for antibiotic treatment and/or with the possible use of symptomatic treatments aimed at reducing overproduction of mucus and decreasing coughing. However, when these infections occur in patients with weakened immune systems and/or underlying health conditions, their impact can become dramatic and in some cases life threatening. The rapid worldwide spread of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection has caused concern for everyone, becoming especially important for individuals with underlying lung diseases, such as CF patients, who have always paid close attention to implementing protective strategies to avoid infection. However, adult and pediatric CF patients contract coronavirus infection like everyone else. In addition, although numerous studies were published during the first wave of the pandemic on the risk for patients with cystic fibrosis (CF) to develop severe manifestations when infected with SARS-CoV-2, to date, a high risk has been found only for patients with poorer lung function and post-transplant status. In terms of preventive measures, vaccination remains key. The best protection for these patients is to strengthen preventive measures, such as social distancing and the use of masks. In this review, we aim to summarize and discuss recent advances in understanding the susceptibility of CF individuals to SARS-CoV-2 infection.

The Neuroprotective Effects of mGlu1 Receptor Antagonists Are Mediated by an Enhancement of GABAergic Synaptic Transmission via a Presynaptic CB1 Receptor Mechanism.

In this study, we investigated the cross-talk between mGlu1 and CB1 receptors in modulating GABA hippocampal output in whole-cell voltage clamp recordings in rat hippocampal acute slices, in organotypic hippocampal slices exposed to oxygen and glucose deprivation (OGD) and in gerbils subjected to global ischemia. CB1 receptor expression was studied using immunohistochemistry and the CA1 contents of anandamide (AEA) and 2-arachidonoylglycerol (2-AG) were measured by LC-MS/MS. Our results show that mGlu1 receptor antagonists enhance sIPSCs in CA1 pyramidal cells and the basal and ischemic hippocampal release of GABA in vivo in a manner that is mediated by CB1 receptor activation. In hippocampal slices exposed to OGD and in ischemic gerbils, mGlu1 receptor antagonists protected CA1 pyramidal cells against post-ischemic injury and this effect was reduced by CB1 receptor activation. OGD induced a transient increase in the hippocampal content of AEA and this effect is prevented by mGlu1 receptor antagonist. Finally, OGD induced a late disruption of CB1 receptors in the CA1 region and the effect was prevented when CA1 pyramidal cells were protected by mGlu1 antagonists. Altogether, these results suggest a cooperative interaction between mGlu1 receptors and the endocannabinoid system in the mechanisms that lead to post-ischemic neuronal death.

Changes of Oxidative Stress-Related Gene Expression in an in vitro Model of Neonatal Hypoxic-Ischemic Encephalopathy.

BACKGROUND: We recently demonstrated that oxygen-glucose deprivation (OGD) and unconjugated bilirubin (UCB) can damage mature and immature organotypic hippocampal slices and induce an oxidative stress similar to what occurs in jaundiced term and preterm infants with hypoxic-ischemic encephalopathy (HIE). OBJECTIVES: To assess the effects of OGD and UCB on the expression of heme-oxygenase 1 (HO-1) and oxidative stress-related enzymes in an in vitro model of HIE. METHODS: Mature and immature organotypic hippocampal slices were exposed to 30-min OGD and to 24 h UCB or UCB plus human serum albumin (HSA). The expression of HO-1, superoxide dismutase 1 (SOD1), catalase (CAT), glutathione peroxidase (GPX), and nuclear factor erythroid-related factor 2 were analyzed by real-time PCR. RESULTS: In mature slices, OGD did not affect the expression of HO-1 and oxidative stress-induced enzymes. The addition of UCB was associated with the upregulation of HO-1 and Nrf2 that is abolished by the presence of equimolar amount of HSA. In immature slices, OGD induced the downregulation of CAT, GPX, and Nrf2 expression and the addition of UCB further decreased GPX. The addition of UCB and HSA reverted the effects of OGD and UCB on gene expression. CONCLUSIONS: In an in vitro model of HIE in term infants, we did not observe neuroprotective changes of the expression of HO-1 and genes involved in antioxidant defenses. Conversely, in an in vitro model of HIE in preterm infants, we observed a harmful decrease of the expression of genes encoding for antioxidant enzymes.

Recent Advances in the Use of Molecular Methods for the Diagnosis of Bacterial Infections.

Infections caused by bacteria have a major impact on public health-related morbidity and mortality. Despite major advances in the prevention and treatment of bacterial infections, the latter continue to represent a significant economic and social burden worldwide. The WHO compiled a list of six highly virulent multidrug-resistant bacteria named ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) responsible for life-threatening diseases. Taken together with Clostridioides difficile, Escherichia coli, Campylobacter spp., (C. jejuni and C. coli), Legionella spp., Salmonella spp., and Neisseria gonorrhoeae, all of these microorganisms are the leading causes of nosocomial infections. The rapid and accurate detection of these pathogens is not only important for the early initiation of appropriate antibiotic therapy, but also for resolving outbreaks and minimizing subsequent antimicrobial resistance. The need for ever-improving molecular diagnostic techniques is also of fundamental importance for improving epidemiological surveillance of bacterial infections. In this review, we aim to discuss the recent advances on the use of molecular techniques based on genomic and proteomic approaches for the diagnosis of bacterial infections. The advantages and limitations of each of the techniques considered are also discussed.

Hypoxia/Ischemia-Induced Rod Microglia Phenotype in CA1 Hippocampal Slices.

The complexity of microglia phenotypes and their related functions compels the continuous study of microglia in diseases animal models. We demonstrated that oxygen-glucose deprivation (OGD) induced rapid, time- and space-dependent phenotypic microglia modifications in CA1 stratum pyramidalis (SP) and stratum radiatum (SR) of rat organotypic hippocampal slices as well as the degeneration of pyramidal neurons, especially in the outer layer of SP. Twenty-four h following OGD, many rod microglia formed trains of elongated cells spanning from the SR throughout the CA1, reaching the SP outer layer where they acquired a round-shaped amoeboid phagocytic head and phagocytosed most of the pyknotic, damaged neurons. NIR-laser treatment, known to preserve neuronal viability after OGD, prevented rod microglia formation. In CA3 SP, pyramidal neurons were less damaged, no rod microglia were found. Thirty-six h after OGD, neuronal damage was more pronounced in SP outer and inner layers of CA1, rod microglia cells were no longer detectable, and most microglia were amoeboid/phagocytic. Damaged neurons, more numerous 36 h after OGD, were phagocytosed by amoeboid microglia in both inner and outer layers of CA1. In response to OGD, microglia can acquire different morphofunctional phenotypes which depend on the time after the insult and on the subregion where microglia are located.

Extended-spectrum ß-lactamase & carbapenemase-producing fermentative Gram-negative bacilli in clinical isolates from a University Hospital in Southern Italy.

The aim of this study was to determine the prevalence of extended-spectrum ß-lactamases (ESBLs)- and carbapenemase-producing fermentative Gram-negative bacteria (FGNB) in a University Hospital in Southern Italy. These bacteria have the potential to disseminate bacterial resistance in healthcare settings and cause untreatable and prolonged infections associated with high rates of mortality. A retrospective observational study was carried out in a University Hospital in Sicily from January to December 2019. A total of 1046 FGNB were recovered from different clinical samples among which 40%, 15% and 37% were, respectively, MDR, carbapenemase and ESBL producers. Antibiotic resistance profile of FGNB against the first-line drugs was remarkably high. K. pneumoniae (57%) followed by E. coli (27%) were found here as the major sources of ESBL producers. The highest proportion of ESBL producers was from ICU ward (72%), and were isolated from urine samples (63.6%) followed by blood samples (54%). Carbapenemase production among the FGNB in our study was about 0.9%, which is more than twice than the prevalence rate reported by the European Antimicrobial Resistance Surveillance Network (ECDC) (0.4%). To our knowledge, this is the first report on the prevalence of ESBL and carbapenemase-producing FGNB in this region. Our data clearly indicate the importance of implementing antibiotic stewardship strategies in our region to reduce the unnecessary use of antibiotics.

Bacterial Antibiotic Resistance: The Most Critical Pathogens.

Antibiotics have made it possible to treat bacterial infections such as meningitis and bacteraemia that, prior to their introduction, were untreatable and consequently fatal. Unfortunately, in recent decades overuse and misuse of antibiotics as well as social and economic factors have accelerated the spread of antibiotic-resistant bacteria, making drug treatment ineffective. Currently, at least 700,000 people worldwide die each year due to antimicrobial resistance (AMR). Without new and better treatments, the World Health Organization (WHO) predicts that this number could rise to 10 million by 2050, highlighting a health concern not of secondary importance. In February 2017, in light of increasing antibiotic resistance, the WHO published a list of pathogens that includes the pathogens designated by the acronym ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) to which were given the highest "priority status" since they represent the great threat to humans. Understanding the resistance mechanisms of these bacteria is a key step in the development of new antimicrobial drugs to tackle drug-resistant bacteria. In this review, both the mode of action and the mechanisms of resistance of commonly used antimicrobials will be examined. It also discusses the current state of AMR in the most critical resistant bacteria as determined by the WHO's global priority pathogens list.

NIR Laser Photobiomodulation Induces Neuroprotection in an In Vitro Model of Cerebral Hypoxia/Ischemia.

Brain photobiomodulation (PBM) is an innovative treatment for a variety of neurological conditions, including cerebral ischemia. However, the capability of PBM for ischemic stroke needs to be further explored and its mechanisms of action remain currently unclear. The aim of the present research was to identify a treatment protocol capable of inducing neuroprotection and to investigate the molecular mechanisms activated by a dual-wavelength near infrared (NIR) laser source in an organotypic hippocampal slice model of hypoxia/ischemia. Hippocampal slices were exposed to oxygen and glucose deprivation (OGD) for 30 min followed by NIR laser light (fluence 3.71, 7.42, or 14.84 J/cm2; wavelengths 808 nm and 905 nm) delivered immediately or 30 min or 60 min after OGD, in order to establish a therapeutic window. Neuronal injury was assessed by propidium iodide fluorescence 24 h later. Our results show that NIR laser irradiation attenuates OGD neurotoxicity once applied immediately or 30 min after OGD. Western blot analysis of proteins involved in neuroinflammation (iNOS, COX-2, NFkB subunit p65, and Bcl-2) and in glutamatergic-mediated synaptic activity (vGluT1, EAAT2, GluN1, and PSD95) showed that the protein modifications induced by OGD were reverted by NIR laser application. Moreover, CA1 confocal microscopy revealed that the profound morphological changes induced by OGD were reverted by NIR laser radiation. In conclusion, NIR laser radiation attenuates OGD neurotoxicity in organotypic hippocampal slices through attenuation of inflammatory mechanisms. These findings shed light on molecular definition of NIR neuroprotective mechanisms, thus underlining the potential benefit of this technique for the treatment of cerebral ischemia.

Neurotoxicity of Unconjugated Bilirubin in Neonatal Hypoxic-Ischemic Brain Injury in vitro.

Background: The pathophysiology of bilirubin neurotoxicity in course of hypoxic-ischemic encephalopathy (HIE) in term and preterm infants is still poorly understood. We hypothesized that oxidative stress may be a common mechanism that link hyperbilirubinemia and HIE. Objectives: The objective of the present study was to evaluate whether unconjugated bilirubin (UCB) may enhance the HI brain injury by increasing oxidative stress and to test pioglitazone and allopurinol as new antioxidant therapeutic drugs in vitro. Methods: The effects of UCB were tested on organotypic hippocampal slices subjected to 30 min oxygen-glucose deprivation (OGD), used as in vitro model of HIE. The experiments were performed on mature (14 days in culture) and immature (7 days in culture) slices, to mimic the brains of term and preterm infants, respectively. Mature and immature slices were exposed to UCB, human serum albumin (HSA), pioglitazone, and/or allopurinol for 24 h, immediately after 30 min OGD. Neuronal injury was assessed using propidium iodide (PI) fluorescence. ROS formation was quantified by using the 2',7'-dichlorodihydrofluorescein diacetate (DCF-DA) method. Results: In mature slices, we found that the neurotoxicity, as well as oxidative stress, induced by OGD were enhanced by UCB. HSA significantly prevented UCB-increased neurotoxicity, but had a slight reduction on ROS production. Allopurinol, but not pioglitazone, significantly reduced UCB-increased neurotoxicity induced by OGD. In immature slices exposed to OGD, no increase of neuronal death was observed, whereas oxidative stress was detected after UCB exposure. HSA, pioglitazone and allopurinol have no protective effects on both OGD-induced neuronal death and on UCB-induced oxidative stress. For this reason, UCB, pioglitazone and allopurinol was also tested on ischemic preconditioning protocol. We found that UCB abolished the neuroprotection induced by preconditioning and increased oxidative stress. These effects were restored by allopurinol but not pioglitazone. Conclusions: UCB characterized a different path of neuronal damage and oxidative stress in mature and immature hippocampal slice model of HIE. Management of hyperbilirubinemia in a complex pathological condition, such as HIE and hyperbilirubinemia, should be very careful. Allopurinol could deserve attention as a novel pharmacological intervention for hyperbilirubinemia and HIE.

The Relevance of IL-1-Signaling in the Protection against Gram-Positive Bacteria.

Previous studies performed using a model of group B streptococcus (GBS)-induced peritoneal inflammation indicate that the interleukin-1 receptor (IL-1R) family plays an important role in the innate host defense against this encapsulated Gram-positive bacteria. Since the role of IL-1-dependent signaling in peritoneal infections induced by other Gram-positive bacteria is unknown, in the present study we sought to investigate the contribution of IL-1R signaling in host defenses against Streptococcus pyogenes (group A streptococcus or GAS) or Staphylococcus aureus, two frequent and global human Gram-positive extracellular pathogens. We analyzed here the outcome of GAS or S. aureus infection in IL-1R-deficient mice. After inoculated intraperitoneal (i.p.) inoculation with group A Streptococcus or S. aureus, all the wild-type (WT) control mice survived the challenge, while, respectively, 63% or 50% of IL-1-defective mice died. Lethality was due to the ability of both bacterial species to replicate and disseminate to the target organs of IL-1R-deficient mice. Moreover, the experimental results indicate that IL-1 signaling promotes the production of leukocyte attractant chemokines CXCL-1 and CXCL-2 and recruitment of neutrophils to bacterial infection sites. Accordingly, the reduced neutrophil recruitment in IL-1R-deficient mice was linked with decreased production of neutrophil chemokines. Collectively, our findings indicate that IL-1 signaling, as previously showed in host defense against GBS, plays a fundamental role also in controlling the progression and outcome of GAS or S. aureus disease.

Ethanol neurotoxicity is mediated by changes in expression, surface localization and functional properties of glutamate AMPA receptors.

Modifications in the subunit composition of AMPA receptors (AMPARs) have been linked to the transition from physiological to pathological conditions in a number of contexts, including EtOH-induced neurotoxicity. Previous work from our laboratory showed that EtOH withdrawal causes CA1 pyramidal cell death in organotypic hippocampal slices and changes in the expression of AMPARs. Here, we investigated whether changes in expression and function of AMPARs may be causal for EtOH-induced neurotoxicity. To this aim, we examined the subunit composition, localization and function of AMPARs in hippocampal slices exposed to EtOH by using western blotting, surface expression assay, confocal microscopy and electrophysiology. We found that EtOH withdrawal specifically increases GluA1 protein signal in total homogenates, but not in the post-synaptic density-enriched fraction. This is suggestive of overall increase and redistribution of AMPARs to the extrasynaptic compartment. At functional level, AMPA-induced calcium influx was unexpectedly reduced, whereas AMPA-induced current was enhanced in CA1 pyramidal neurons following EtOH withdrawal, suggesting that increased AMPAR expression may lead to cell death because of elevated excitability, and not for a direct contribution on calcium influx. Finally, the neurotoxicity caused by EtOH withdrawal was attenuated by the non-selective AMPAR antagonist 2,3-dioxo-6-nitro-1,2,3,4-tetrahydrobenzo[f]quinoxaline-7-sulfonamide disodium salt as well as by the selective antagonist of GluA2-lacking AMPARs 1-naphthyl acetyl spermine. We conclude that EtOH neurotoxicity involves changes in expression, surface localization and functional properties of AMPARs, and propose GluA2-lacking AMPARs as amenable specific targets for the development of neuroprotective drugs in EtOH-withdrawal syndrome.

Differential mechanisms of tolerance induced by NMDA and 3,5-dihydroxyphenylglycine (DHPG) preconditioning.

We investigated the molecular events triggered by NMDA and 3,5-dihydroxyphenylglycine (DHPG) preconditioning, that lead to neuroprotection against excitotoxic insults (AMPA or oxygen and glucose deprivation) in rat organotypic hippocampal slices, with particular attention on glutamate receptors and on cannabinoid system. We firstly evaluated the protein expression of NMDA and AMPA receptor subunits after preconditioning using western blot analysis performed in post-synaptic densities. We observed that following NMDA, but not DHPG preconditioning, the expression of GluA1 was significantly reduced and this reduction appeared to be associated with the internalization of AMPA receptors. Whole-cell voltage clamp recordings on CA1 pyramidal neurons of organotypic slices show that 24 hr after exposure to NMDA and DHPG preconditioning, AMPA-induced currents were significantly reduced. To clarify the mechanisms induced by DHPG preconditioning, we then investigated the involvement of the endocannabinoid system. Exposure of slices to the CB1 antagonist AM251 prevented the development of tolerance to AMPA toxicity induced by DHPG but not NMDA. Accordingly, the MAG-lipase inhibitor URB602, that increases arachidonoylglycerol (2-AG) content, but not the FAAH inhibitor URB597, that limits the degradation of anandamide, was also able to induce tolerance versus AMPA and OGD toxicity, suggesting that 2-AG is responsible for the DHPG-induced tolerance. In conclusion, preconditioning with NMDA or DHPG promotes differential neuroprotective mechanisms: NMDA by internalization of GluA1-AMPA receptors, DHPG by producing the endocannabinoid 2-AG.