The Microbiota-Gut-Brain Axis in Behaviour and Brain Disorders.

The gut, along with its microbiota (MB-gut), is the largest absorption organ and reservoir of bacteria in the human body [...].

Phenomic Microglia Diversity as a Druggable Target in the Hippocampus in Neurodegenerative Diseases.

Phenomics, the complexity of microglia phenotypes and their related functions compels the continuous study of microglia in disease animal models to find druggable targets for neurodegenerative disorders. Activation of microglia was long considered detrimental for neuron survival, but more recently it has become apparent that the real scenario of microglia morphofunctional diversity is far more complex. In this review, we discuss the recent literature on the alterations in microglia phenomics in the hippocampus of animal models of normal brain aging, acute neuroinflammation, ischemia, and neurodegenerative disorders, such as AD. Microglia undergo phenomic changes consisting of transcriptional, functional, and morphological changes that transform them into cells with different properties and functions. The classical subdivision of microglia into M1 and M2, two different, all-or-nothing states is too simplistic, and does not correspond to the variety of phenotypes recently discovered in the brain. We will discuss the phenomic modifications of microglia focusing not only on the differences in microglia reactivity in the diverse models of neurodegenerative disorders, but also among different areas of the brain. For instance, in contiguous and highly interconnected regions of the rat hippocampus, microglia show a differential, finely regulated, and region-specific reactivity, demonstrating that microglia responses are not uniform, but vary significantly from area to area in response to insults. It is of great interest to verify whether the differences in microglia reactivity may explain the differential susceptibility of different brain areas to insults, and particularly the higher sensitivity of CA1 pyramidal neurons to inflammatory stimuli. Understanding the spatiotemporal heterogeneity of microglia phenomics in health and disease is of paramount importance to find new druggable targets for the development of novel microglia-targeted therapies in different CNS disorders. This will allow interventions in three different ways: (i) by suppressing the pro-inflammatory properties of microglia to limit the deleterious effect of their activation; (ii) by modulating microglia phenotypic change to favor anti-inflammatory properties; (iii) by influencing microglia priming early in the disease process.

Cannabidiol inhibits microglia activation and mitigates neuronal damage induced by kainate in an in-vitro seizure model.

BACKGROUND: Epilepsy is one of the most common brain disorder and, despite the possible use of several therapeutic options, many patients continue to have seizures for their entire lifespan and they need new therapeutic approaches. In the last years the interest on the non-psychoactive compounds present in Cannabis sativa has massively increased, and cannabidiol (CBD) has been shown to be effective in the treatment of different types of neurological disorders and neurodegenerative diseases such as epilepsy, ischemia, multiple sclerosis and Alzheimer's Disease. METHODS: We investigated the effects of the selected cannabinoids, Δ9-tetrahydrocannabinol (THC), CBD and cannabigerol (CBG) in rat organotypic hippocampal slices exposed to kainate, an in vitro seizure model. Cell death in the cornu Ammonis 3 (CA3) hippocampal subregion was quantified by propidium iodide fluorescence. Morphological analysis and tissue organization were examined by immunohistochemistry and confocal microscopy and microglia activation and polarization was evaluated using flow cytometry and morphology analysis. RESULTS: When present in the incubation medium, cannabidiol reduced dose-dependent CA3 injury induced by kainate. Conversely, incubation with THC exacerbated hippocampal damage. The neuroprotective effects of cannabidiol were blocked by TRPV1, TRPV2, 5-HT1A, and PPARγ antagonists. Confocal microscopy confirmed that CBD but not THC had a significant protective effect against neuronal damage and tissue disorganization caused by kainate. Cannabidiol incubation significantly block the microglia activation from the M0 to M1 phenotype observed in the kainate in-vitro seizure model, pushing toward a transition from M0 to M2. CONCLUSIONS: Our results suggest that CBD mitigated neuronal damage induced by kainate and blocked the transition from the M0 to the M1 phenotype.

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.

The Protective Effect of CBD in a Model of In Vitro Ischemia May Be Mediated by Agonism on TRPV2 Channel and Microglia Activation.

Cannabinoids, used for centuries for recreational and medical purposes, have potential therapeutic value in stroke treatment. Cannabidiol (CBD), a non-psychoactive compound and partial agonist of TRPV2 channels, is efficacious in many neurological disorders. We investigated the effects of CBD or Δ9-tetrahydrocannabinol (THC) in rat organotypic hippocampal slices exposed to oxygen-glucose deprivation (OGD), an in vitro model of ischemia. Neuronal TRPV2 expression decreased after OGD, but it increased in activated, phagocytic microglia. CBD increased TRPV2 expression, decreased microglia phagocytosis, and increased rod microglia after OGD. THC had effects contrary to those of CBD. Our results show that cannabinoids have different effects in ischemia. CBD showed neuroprotective effects, mediated, at least in part, by TRPV2 channels, since the TRPV2 antagonist tranilast blocked them, while THC worsened the neurodegeneration caused by ischemia. In conclusion, our results suggest that different cannabinoid molecules play different roles in the mechanisms of post-ischemic neuronal death. These different effects of cannabinoid observed in our experiments caution against the indiscriminate use of cannabis or cannabinoid preparations for recreational or therapeutic use. It was observed that the positive effects of CBD may be counteracted by the negative effects caused by high levels of THC.

Neuroprotective Effects of Cannabidiol but Not Δ9-Tetrahydrocannabinol in Rat Hippocampal Slices Exposed to Oxygen-Glucose Deprivation: Studies with Cannabis Extracts and Selected Cannabinoids.

(1) Background: Over the past 10 years, a number of scientific studies have demonstrated the therapeutic potential of cannabinoid compounds present in the Cannabis Sativa and Indica plants. However, their role in mechanisms leading to neurodegeneration following cerebral ischemia is yet unclear. (2) Methods: We investigated the effects of Cannabis extracts (Bedrocan, FM2) or selected cannabinoids (Δ9-tetrahydrocannabinol (THC), cannabidiol (CBD), and cannabigerol) in rat organotypic hippocampal slices exposed to oxygen-glucose deprivation (OGD), an in vitro model of forebrain global ischemia. Cell death in the CA1 subregion of slices was quantified by propidium iodide fluorescence, and morphological analysis and tissue organization were examined by immunohistochemistry and confocal microscopy. (3) Results: Incubation with the Bedrocan extract or THC exacerbated, whereas incubation with the FM2 extract or cannabidiol attenuated CA1 injury induced by OGD. Δ9-THC toxicity was prevented by CB1 receptor antagonists, the neuroprotective effect of cannabidiol was blocked by TRPV2, 5-HT1A, and PPARγ antagonists. Confocal microscopy confirmed that CBD, but not THC, had a significant protective effect toward neuronal damage and tissue disorganization caused by OGD in organotypic hippocampal slices. (4) Conclusions: Our results suggest that cannabinoids play different roles in the mechanisms of post-ischemic neuronal death. In particular, appropriate concentrations of CBD or CBD/THC ratios may represent a valid therapeutic intervention in the treatment of post-ischemic neuronal death.

Microglial distribution, branching, and clearance activity in aged rat hippocampus are affected by astrocyte meshwork integrity: evidence of a novel cell-cell interglial interaction.

Aging and neurodegenerative diseases share a condition of neuroinflammation entailing the production of endogenous cell debris in the CNS that must be removed by microglia ( i.e., resident macrophages), to restore tissue homeostasis. In this context, extension of microglial cell branches toward cell debris underlies the mechanisms of microglial migration and phagocytosis. Amoeboid morphology and the consequent loss of microglial branch functionality characterizes dysregulated microglia. Microglial migration is assisted by another glial population, the astroglia, which forms a dense meshwork of cytoplasmic projections. Amoeboid microglia and disrupted astrocyte meshwork are consistent traits in aged CNS. In this study, we assessed a possible correlation between microglia and astroglia morphology in rat models of chronic neuroinflammation and aging, by 3-dimensional confocal analysis implemented with particle analysis. Our findings suggest that a microglia-astroglia interaction occurs in rat hippocampus via cell-cell contacts, mediating microglial cell branching in the presence of inflammation. In aged rats, the impairment of such an interaction correlates with altered distribution, morphology, and inefficient clearance by microglia. These data support the idea that generally accepted functional boundaries between microglia and astrocytes should be re-evaluated to better understand how their functions overlap and interact.-Lana, D., Ugolini, F., Wenk, G. L., Giovannini, M. G., Zecchi-Orlandini, S., Nosi, D. Microglial distribution, branching, and clearance activity in aged rat hippocampus are affected by astrocyte meshwork integrity: evidence of a novel cell-cell interglial interaction.

Space-Dependent Glia-Neuron Interplay in the Hippocampus of Transgenic Models of ß-Amyloid Deposition.

This review is focused on the description and discussion of the alterations of astrocytes and microglia interplay in models of Alzheimer's disease (AD). AD is an age-related neurodegenerative pathology with a slowly progressive and irreversible decline of cognitive functions. One of AD's histopathological hallmarks is the deposition of amyloid beta (Aß) plaques in the brain. Long regarded as a non-specific, mere consequence of AD pathology, activation of microglia and astrocytes is now considered a key factor in both initiation and progression of the disease, and suppression of astrogliosis exacerbates neuropathology. Reactive astrocytes and microglia overexpress many cytokines, chemokines, and signaling molecules that activate or damage neighboring cells and their mutual interplay can result in virtuous/vicious cycles which differ in different brain regions. Heterogeneity of glia, either between or within a particular brain region, is likely to be relevant in healthy conditions and disease processes. Differential crosstalk between astrocytes and microglia in CA1 and CA3 areas of the hippocampus can be responsible for the differential sensitivity of the two areas to insults. Understanding the spatial differences and roles of glia will allow us to assess how these interactions can influence the state and progression of the disease, and will be critical for identifying therapeutic strategies.

A2B Adenosine Receptors: When Outsiders May Become an Attractive Target to Treat Brain Ischemia or Demyelination.

Adenosine is a signaling molecule, which, by activating its receptors, acts as an important player after cerebral ischemia. Here, we review data in the literature describing A2BR-mediated effects in models of cerebral ischemia obtained in vivo by the occlusion of the middle cerebral artery (MCAo) or in vitro by oxygen-glucose deprivation (OGD) in hippocampal slices. Adenosine plays an apparently contradictory role in this receptor subtype depending on whether it is activated on neuro-glial cells or peripheral blood vessels and/or inflammatory cells after ischemia. Indeed, A2BRs participate in the early glutamate-mediated excitotoxicity responsible for neuronal and synaptic loss in the CA1 hippocampus. On the contrary, later after ischemia, the same receptors have a protective role in tissue damage and functional impairments, reducing inflammatory cell infiltration and neuroinflammation by central and/or peripheral mechanisms. Of note, demyelination following brain ischemia, or autoimmune neuroinflammatory reactions, are also profoundly affected by A2BRs since they are expressed by oligodendroglia where their activation inhibits cell maturation and expression of myelin-related proteins. In conclusion, data in the literature indicate the A2BRs as putative therapeutic targets for the still unmet treatment of stroke or demyelinating diseases.

Clasmatodendrosis and ß-amyloidosis in aging hippocampus.

Alterations of the tightly interwoven neuron/astrocyte interactions are frequent traits of aging, but also favor neurodegenerative diseases, such as Alzheimer disease (AD). These alterations reflect impairments of the innate responses to inflammation-related processes, such as ß-amyloid (Aß) burdening. Multidisciplinary studies, spanning from the tissue to the molecular level, are needed to assess how neuron/astrocyte interactions are influenced by aging. Our study addressed this requirement by joining fluorescence-lifetime imaging microscopy/phasor multiphoton analysis with confocal microscopy, implemented with a novel method to separate spectrally overlapped immunofluorescence and Aß autofluorescence. By comparing data from young control rats, chronically inflamed rats, and old rats, we identified age-specific alterations of neuron/astrocyte interactions in the hippocampus. We found a correlation between Aß aggregation (+300 and +800% of aggregated Aß peptide in chronically inflamed and oldvs.control rats, respectively) and fragmentation (clasmatodendrosis) of astrocyte projections (APJs) (+250 and +1300% of APJ fragments in chronically inflamed and oldvs.control rats, respectively). Clasmatodendrosis, in aged rats, associates with impairment of astrocyte-mediated Aß clearance (-45% of Aß deposits on APJs, and +33% of Aß deposits on neurons in oldvs.chronically inflamed rats). Furthermore, APJ fragments colocalize with Aß deposits and are involved in novel Aß-mediated adhesions between neurons. These data define the effects of Aß deposition on astrocyte/neuron interactions as a key topic in AD biology.-Mercatelli, R., Lana, D., Bucciantini, M., Giovannini, M. G., Cerbai, F., Quercioli, F., Zecchi-Orlandini, S., Delfino, G., Wenk, G. L., Nos, D. Clasmatodendrosis and ß-amyloidosis in aging hippocampus.

The integrated role of ACh, ERK and mTOR in the mechanisms of hippocampal inhibitory avoidance memory.

The purpose of this review is to summarize the present knowledge on the interplay among the cholinergic system, Extracellular signal-Regulated Kinase (ERK) and Mammalian Target of Rapamycin (mTOR) pathways in the development of short and long term memories during the acquisition and recall of the step-down inhibitory avoidance in the hippocampus. The step-down inhibitory avoidance is a form of associative learning that is acquired in a relatively simple one-trial test through several sensorial inputs. Inhibitory avoidance depends on the integrated activity of hippocampal CA1 and other brain areas. Recall can be performed at different times after acquisition, thus allowing for the study of both short and long term memory. Among the many neurotransmitter systems involved, the cholinergic neurons that originate in the basal forebrain and project to the hippocampus are of crucial importance in inhibitory avoidance processes. Acetylcholine released from cholinergic fibers during acquisition and/or recall of behavioural tasks activates muscarinic and nicotinic acetylcholine receptors and brings about a long-lasting potentiation of the postsynaptic membrane followed by downstream activation of intracellular pathway (ERK, among others) that create conditions favourable for neuronal plasticity. ERK appears to be salient not only in long term memory, but also in the molecular mechanisms underlying short term memory formation in the hippocampus. Since ERK can function as a biochemical coincidence detector in response to extracellular signals in neurons, the activation of ERK-dependent downstream effectors is determined, in part, by the duration of ERK phosphorylation itself. Long term memories require protein synthesis, that in the synapto-dendritic compartment represents a direct mechanism that can produce rapid changes in protein content in response to synaptic activity. mTOR in the brain regulates protein translation in response to neuronal activity, thereby modulating synaptic plasticity and long term memory formation. Some studies demonstrate a complex interplay among the cholinergic system, ERK and mTOR. It has been shown that co-activation of muscarinic acetylcholine receptors and ß-adrenergic receptors facilitates the conversion of short term to long term synaptic plasticity through an ERK- and mTOR-dependent mechanism which requires translation initiation. It seems therefore that the complex interplay among the cholinergic system, ERK and mTOR is crucial in the development of new inhibitory avoidance memories in the hippocampus.

New orphan disease therapies from the proteome of industrial plasma processing waste- a treatment for aceruloplasminemia.

Plasma-derived therapeutic proteins are produced through an industrial fractionation process where proteins are purified from individual intermediates, some of which remain unused and are discarded. Relatively few plasma-derived proteins are exploited clinically, with most of available plasma being directed towards the manufacture of immunoglobulin and albumin. Although the plasma proteome provides opportunities to develop novel protein replacement therapies, particularly for rare diseases, the high cost of plasma together with small patient populations impact negatively on the development of plasma-derived orphan drugs. Enabling therapeutics development from unused plasma fractionation intermediates would therefore constitute a substantial innovation. To this objective, we characterized the proteome of unused plasma fractionation intermediates and prioritized proteins for their potential as new candidate therapies for human disease. We selected ceruloplasmin, a plasma ferroxidase, as a potential therapy for aceruloplasminemia, an adult-onset ultra-rare neurological disease caused by iron accumulation as a result of ceruloplasmin mutations. Intraperitoneally administered ceruloplasmin, purified from an unused plasma fractionation intermediate, was able to prevent neurological, hepatic and hematological phenotypes in ceruloplasmin-deficient mice. These data demonstrate the feasibility of transforming industrial waste plasma fraction into a raw material for manufacturing of new candidate proteins for replacement therapies, optimizing plasma use and reducing waste generation.

Hippocampal long term memory: effect of the cholinergic system on local protein synthesis.

The present study was aimed at establishing a link between the cholinergic system and the pathway of mTOR and its downstream effector p70S6K, likely actors in long term memory encoding. We performed in vivo behavioral experiments using the step down inhibitory avoidance test (IA) in adult Wistar rats to evaluate memory formation under different conditions, and immunohistochemistry on hippocampal slices to evaluate the level and the time-course of mTOR and p70S6K activation. We also examined the effect of RAPA, inhibitor of mTORC1 formation, and of the acetylcholine (ACh) muscarinic receptor antagonist scopolamine (SCOP) or ACh nicotinic receptor antagonist mecamylamine (MECA) on short and long term memory formation and on the functionality of the mTOR pathway. Acquisition test was performed 30 min after i.c.v. injection of RAPA, a time sufficient for the drug to diffuse to CA1 pyramidal neurons, as demonstrated by MALDI-TOF-TOF imaging. Recall test was performed 1 h, 4 h or 24 h after acquisition. To confirm our results we performed in vitro experiments on live hippocampal slices: we evaluated whether stimulation of the cholinergic system with the cholinergic receptor agonist carbachol (CCh) activated the mTOR pathway and whether the administration of the above-mentioned antagonists together with CCh could revert this activation. We found that (1) mTOR and p70S6K activation in the hippocampus were involved in long term memory formation; (2) RAPA administration caused inhibition of mTOR activation at 1 h and 4 h and of p70S6K activation at 4 h, and long term memory impairment at 24 h after acquisition; (3) scopolamine treatment caused short but not long term memory impairment with an early increase of mTOR/p70S6K activation at 1 h followed by stabilization at longer times; (4) mecamylamine plus scopolamine treatment caused short term memory impairment at 1 h and 4 h and reduced the scopolamine-induced increase of mTOR/p70S6K activation at 1 h and 4 h; (5) mecamylamine plus scopolamine treatment did not impair long term memory formation; (6) in vitro treatment with carbachol activated mTOR and p70S6K and this effect was blocked by scopolamine and mecamylamine. Taken together our data reinforce the idea that distinct molecular mechanisms are at the basis of the two different forms of memory and are in accordance with data presented by other groups that there exist molecular mechanisms that underlie short term memory, others that underlie long term memories, but some mechanisms are involved in both.

Virtual Screening-Accelerated Discovery of a Phosphodiesterase 9 Inhibitor with Neuroprotective Effects in the Kainate Toxicity In Vitro Model.

In the central nervous system, some specific phosphodiesterase (PDE) isoforms modulate pathways involved in neuronal plasticity. Accumulating evidence suggests that PDE9 may be a promising therapeutic target for neurodegenerative diseases. In the current study, computational techniques were used to identify a nature-inspired PDE9 inhibitor bearing the scaffold of an isoflavone, starting from a database of synthetic small molecules using a ligand-based approach. Furthermore, docking studies supported by molecular dynamics investigations allowed us to evaluate the features of the ligand-target complex. In vitro assays confirmed the computational results, showing that the selected compound inhibits the enzyme in the nanomolar range. Additionally, we evaluated the expression of gene and protein levels of PDE9 in organotypic hippocampal slices, observing an increase following exposure to kainate (KA). Importantly, the PDE9 inhibitor reduced CA3 damage induced by KA in a dose-dependent manner in organotypic hippocampal slices. Taken together, these observations strongly support the potential of the identified nature-inspired PDE9 inhibitor and suggest that such a molecule could represent a promising lead compound to develop novel therapeutic tools against neurological diseases..

Morphofunctional Investigation in a Transgenic Mouse Model of Alzheimer's Disease: Non-Reactive Astrocytes Are Involved in Aß Load and Reactive Astrocytes in Plaque Build-Up.

The term neuroinflammation defines the reactions of astrocytes and microglia to alterations in homeostasis in the diseased central nervous system (CNS), the exacerbation of which contributes to the neurodegenerative effects of Alzheimer's disease (AD). Local environmental conditions, such as the presence of proinflammatory molecules, mechanical properties of the extracellular matrix (ECM), and local cell-cell interactions, are determinants of glial cell phenotypes. In AD, the load of the cytotoxic/proinflammatory amyloid ß (Aß) peptide is a microenvironmental component increasingly growing in the CNS, imposing time-evolving challenges on resident cells. This study aimed to investigate the temporal and spatial variations of the effects produced by this process on astrocytes and microglia, either directly or by interfering in their interactions. Ex vivo confocal analyses of hippocampal sections from the mouse model TgCRND8 at different ages have shown that overproduction of Aß peptide induced early and time-persistent disassembly of functional astroglial syncytium and promoted a senile phenotype of reactive microglia, hindering Aß clearance. In the late stages of the disease, these patterns were altered in the presence of Aß-plaques, surrounded by typically reactive astrocytes and microglia. Morphofunctional characterization of peri-plaque gliosis revealed a direct contribution of astrocytes in plaque buildup that might result in shielding Aß-peptide cytotoxicity and, as a side effect, in exacerbating neuroinflammation.

Suppression of the cough reflex by inhibition of ERK1/2 activation in the caudal nucleus tractus solitarii of the rabbit.

The caudal nucleus tractus solitarii (cNTS), the predominant site of termination of cough-related afferents, has been shown to be a site of action of some centrally acting antitussive agents. A role of ERK1/2 has been suggested in acute central processing of nociceptive inputs. Because pain and cough share similar features, we investigated whether ERK1/2 activation could also be involved in the central transduction of tussive inputs. For this purpose, we undertook the present research on pentobarbital sodium-anesthetized, spontaneously breathing rabbits by using microinjections (30-50 nl) of an inhibitor of ERK1/2 activation (U0126) into the cNTS. Bilateral microinjections of 25 mM U0126 caused rapid and reversible reductions in the cough responses induced by both mechanical and chemical (citric acid) stimulation of the tracheobronchial tree. In particular, the cough number and peak abdominal activity decreased. Bilateral microinjections of 50 mM U0126 completely suppressed the cough reflex without affecting the Breuer-Hering inflation reflex, the pulmonary chemoreflex, and the sneeze reflex. These U0126-induced effects were, to a large extent, reversible. Bilateral microinjections of 50 mM U0124, the inactive analog of U0126, at the same cNTS sites had no effect. This is the first study that provides evidence that ERK1/2 activation within the cNTS is required for the mediation of cough reflex responses in the anesthetized rabbit. These results suggest a role for ERK1/2 in the observed effects via nontranscriptional mechanisms, given the short time involved. They also may provide hints for the development of novel antitussive strategies.

Neuronal and Astrocytic Morphological Alterations Driven by Prolonged Exposure with Δ9-Tetrahydrocannabinol but Not Cannabidiol.

Cannabis derivatives are largely used in the general population for recreational and medical purposes, with the highest prevalence among adolescents, but chronic use and abuse has raised medical concerns. We investigated the prolonged effects of Δ9-tetrahydrocannabinol (THC) and cannabidiol (CBD) in organotypic hippocampal slices from P7 rats cultured for 2 weeks. Cell death in the CA1 subregion of slices was quantified by propidium iodide (PI) fluorescence, pre-synaptic and post-synaptic marker proteins were analysed by Western blotting and neurodegeneration and astrocytic alterations by NeuN and GFAP by immunofluorescence and confocal laser microscopy. The statistical significance of differences was analysed using ANOVA with a post hoc Dunnett w-test (PI fluorescence intensities and Western blots) or Newman-Keuls (immunohistochemistry data) for multiple comparisons. A probability value (P) of < 0.05 was considered significant. Prolonged (72 h) THC or CBD incubation did not induce cell death but caused modifications in the expression of synaptic proteins and morphological alterations in neurons and astrocytes. In particular, the expression of PSD95 was reduced following incubation for 72 h with THC and was increased following incubation with CBD. THC for 72 h caused disorganisation of CA1 stratum pyramidalis (SP) and complex morphological modifications in a significant number of pyramidal neurons and in astrocytes. Our results suggest that THC or CBD prolonged exposure induce different effects in the hippocampus. In particular, 72 h of THC exposure induced neuronal and glia alterations that must draw our attention to the effects that relatively prolonged use might cause, especially in adolescents.

P2 receptor antagonists prevent synaptic failure and extracellular signal-regulated kinase 1/2 activation induced by oxygen and glucose deprivation in rat CA1 hippocampus in vitro.

To investigate the role of purinergic P2 receptors under ischemia, we studied the effect of P2 receptor antagonists on synaptic transmission and mitogen-activated protein kinase (MAPK) activation under oxygen and glucose deprivation (OGD) in rat hippocampal slices. The effect of the P2 antagonists pyridoxalphosphate-6-azophenyl-2',4'-disulfonate (PPADS, unselective, 30 µm), N( 6) -methyl-2'-deoxyadenosine-3',5'-bisphosphate (MRS2179, selective for P2Y(1) receptor, 10 µm), Brilliant Blue G (BBG, selective for P2X(7) receptor, 1 µm), and 5-[[[(3-phenoxyphenyl)methyl][(1S)-1,2,3,4-tetrahydro-1-naphthalenyl]amino]carbonyl]-1,2,4-benzenetricarboxylic acid (A-317491, selective for P2X(3) receptor, 10 µm), and of the newly synthesized P2X(3) receptor antagonists 2-amino-9-(5-iodo-2-isopropyl-4-methoxybenzyl)adenine (PX21, 1 µm) and 2-amino-9-(5-iodo-2-isopropyl-4-methoxybenzyl)-N( 6)-methyladenine (PX24, 1 µm), on the depression of field excitatory postsynaptic potentials (fEPSPs) and anoxic depolarization (AD) elicited by 7 min of OGD were evaluated. All antagonists significantly prevented these effects. The extent of CA1 cell injury was assessed 3 h after the end of 7 min of OGD by propidium iodide staining. Substantial CA1 pyramidal neuronal damage, detected in untreated slices exposed to OGD injury, was significantly prevented by PPADS (30 µm), MRS2179 (10 µm), and BBG (1 µm). Western blot analysis showed that, 10 min after the end of the 7 min of OGD, extracellular signal-regulated kinase (ERK)1/2 MAPK activation was significantly increased. MRS2179, BBG, PPADS and A-317491 significantly counteracted ERK1/2 activation. Hippocampal slices incubated with the ERK1/2 inhibitors 1,4-diamino-2,3-dicyano-1,4-bis(2-aminophenylthio)butadiene (U0126, 10 µm) and α-[amino[(4-aminophenyl)thio]methylene]-2-(trifluoromethyl) benzeneacetonitrile (SL327, 10 µm) showed significant fEPSP recovery after OGD and delayed AD, supporting the involvement of ERK1/2 in neuronal damage induced by OGD. These results indicate that subtypes of hippocampal P2 purinergic receptors have a harmful effect on neurotransmission in the CA1 hippocampus by participating in AD appearance and activation of ERK1/2.

The Microbiota-Gut-Brain Axis and Alzheimer Disease. From Dysbiosis to Neurodegeneration: Focus on the Central Nervous System Glial Cells.

The microbiota-gut system can be thought of as a single unit that interacts with the brain via the "two-way" microbiota-gut-brain axis. Through this axis, a constant interplay mediated by the several products originating from the microbiota guarantees the physiological development and shaping of the gut and the brain. In the present review will be described the modalities through which the microbiota and gut control each other, and the main microbiota products conditioning both local and brain homeostasis. Much evidence has accumulated over the past decade in favor of a significant association between dysbiosis, neuroinflammation and neurodegeneration. Presently, the pathogenetic mechanisms triggered by molecules produced by the altered microbiota, also responsible for the onset and evolution of Alzheimer disease, will be described. Our attention will be focused on the role of astrocytes and microglia. Numerous studies have progressively demonstrated how these glial cells are important to ensure an adequate environment for neuronal activity in healthy conditions. Furthermore, it is becoming evident how both cell types can mediate the onset of neuroinflammation and lead to neurodegeneration when subjected to pathological stimuli. Based on this information, the role of the major microbiota products in shifting the activation profiles of astrocytes and microglia from a healthy to a diseased state will be discussed, focusing on Alzheimer disease pathogenesis.

Neuroinflammation: Integrated Nervous Tissue Response through Intercellular Interactions at the "Whole System" Scale.

Different cell populations in the nervous tissue establish numerous, heterotypic interactions and perform specific, frequently intersecting activities devoted to the maintenance of homeostasis. Microglia and astrocytes, respectively the immune and the "housekeeper" cells of nervous tissue, play a key role in neurodegenerative diseases. Alterations of tissue homeostasis trigger neuroinflammation, a collective dynamic response of glial cells. Reactive astrocytes and microglia express various functional phenotypes, ranging from anti-inflammatory to pro-inflammatory. Chronic neuroinflammation is characterized by a gradual shift of astroglial and microglial phenotypes from anti-inflammatory to pro-inflammatory, switching their activities from cytoprotective to cytotoxic. In this scenario, the different cell populations reciprocally modulate their phenotypes through intense, reverberating signaling. Current evidence suggests that heterotypic interactions are links in an intricate network of mutual influences and interdependencies connecting all cell types in the nervous system. In this view, activation, modulation, as well as outcomes of neuroinflammation, should be ascribed to the nervous tissue as a whole. While the need remains of identifying further links in this network, a step back to rethink our view of neuroinflammation in the light of the "whole system" scale, could help us to understand some of its most controversial and puzzling features.