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Viviamo ormai in un mondo di plastica; moltissimi oggetti che usiamo tutti i giorni sono fatti di plastica. Il problema è che la plastica si degrada con molta difficoltà e, invece di essere correttamente riciclata e riutilizzata, viene spesso abbandonata nell’ambiente e finisce in mare. Le microplastiche, piccoli frammenti di plastica, possono essere ingerite dagli organismi acquatici e, attraverso la catena alimentare, possono arrivare direttamente nel nostro piatto. Spesso questi frammenti plastici accumulano e veicolano contaminanti chimici, tra cui gli interferenti endocrini, che possono alterare il sistema ormonale e avere effetti sulla salute degli animali, uomo incluso. Gli studi dei ricercatori si stanno focalizzando proprio per cercare di capire l’impatto delle microplastiche sulle singole cellule e sugli organismi interi.

Non-alcoholic fatty liver disease (NAFLD) is the most common cause of chronic liver disease; however, no specific pharmacological therapy has yet been approved for this condition. Plant-derived extracts can be an important source for the development of new drugs. The aim of this study was to investigate the effects of (E)-β-caryophyllene (BCP), a phytocannabinoid recently found to be beneficial against metabolic diseases, on HepG2 steatotic hepatocytes. Using a fluorescence-based lipid quantification assay and GC-MS analysis, we show that BCP is able to decrease lipid accumulation in steatotic conditions and to change the typical steatotic lipid profile by primarily reducing saturated fatty acids. By employing specific antagonists, we demonstrate that BCP action is mediated by multiple receptors: CB2 cannabinoid receptor, peroxisome proliferator-activated receptor α (PPARα) and γ (PPARγ). Interestingly, BCP was able to counteract the increase in CB2 and the reduction in PPARα receptor expression observed in steatotic conditions. Moreover, through immunofluorescence and confocal microscopy, we demonstrate that CB2 receptors are mainly intracellularly localized and that BCP is internalized in HepG2 cells with a maximum peak at 2 h, suggesting a direct interaction with intracellular receptors. The results obtained with BCP in normal and steatotic hepatocytes encourage future applications in the treatment of NAFLD.

The 3T3-L1 murine pre-adipocyte line is an established cell culture model for screening Metabolism Disrupting Chemicals (MDCs). Despite a need to accurately identify MDCs for further evaluation, relatively little research has been performed to comprehensively evaluate reproducibility across laboratories, assess factors that might contribute to varying degrees of differentiation between laboratories (media additives, plastics, cell source, etc.), or to standardize protocols. As such, the goals of this study were to assess interlaboratory variability of efficacy and potency outcomes for triglyceride accumulation and pre-adipocyte proliferation using the mouse 3T3-L1 pre-adipocyte cell assay to test chemicals. Ten laboratories from five different countries participated. Each laboratory evaluated one reference chemical (rosiglitazone) and three blinded test chemicals (tributyltin chloride, pyraclostrobin, and bisphenol A) using: 1) their Laboratory-specific 3T3-L1 Cells (LC) and their Laboratory-specific differentiation Protocol (LP), 2) Shared 3T3-L1 Cells (SC) with LP, 3) LC with a Shared differentiation Protocol (SP), and 4) SC with SP. Blinded test chemical responses were analyzed by the coordinating laboratory. The magnitude and range of bioactivities reported varied considerably across laboratories and test conditions, though the presence or absence of activity for each tested chemical was more consistent. Triglyceride accumulation activity determinations for rosiglitazone ranged from 90 to 100% across test conditions, but 30–70 % for pre-adipocyte proliferation; this was 40–80 % for triglyceride accumulation induced by pyraclostrobin, 80–100 % for tributyltin, and 80–100 % for bisphenol A. Consistency was much lower for pre-adipocyte proliferation, with 30–70 % active determinations for pyraclostrobin, 30–50 % for tributyltin, and 20–40 % for bisphenol A. Greater consistency was observed for the SC/SP assessment. As such, working to develop a standardized adipogenic differentiation protocol represents the best strategy for improving consistency of adipogenic responses using the 3T3-L1 model to reproducibly identify MDCs and increase confidence in reported outcomes. Over-arching project supported by grants [R01 ES016099 to HMS; R00 ES030405 to CDK] from the National Institute of Environmental Health Sciences (NIEHS); University of Turin; European Union's Horizon 2020 research and innovation program under grant agreement GOLIATH No. 825489; Brunel University London; NIEHS (1K22ES026208 and R01ES027863); NIEHS (Z0ES102785); Spanish Institute of Health Carlos III (grant FIS-PI16/01812).

The endocannabinoid system (ECS) is involved in the modulation of several basic biological processes, having widespread roles in neurodevelopment, neuromodulation, immune response, energy homeostasis and reproduction. In the adult central nervous system (CNS) the ECS mainly modulates neurotransmitter release, however, a substantial body of evidence has revealed a central role in regulating neurogenesis in developing and adult CNS, also under pathological conditions. Due to the complexity of investigating ECS functions in neural progenitors in vivo, we tested the suitability of the ST14A striatal neural progenitor cell line as a simplified in vitro model to dissect the role and the mechanisms of ECS-regulated neurogenesis, as well as to perform ECS-targeted pharmacological approaches. We report that ST14A cells express various ECS components, supporting the presence of an active ECS. While CB1 and CB2 receptor blockade did not affect ST14A cell number, exogenous administration of the endocannabinoid 2-AG and the synthetic CB2 agonist JWH133 increased ST14A cell proliferation. Phospholipase C (PLC), but not PI3K pharmacological blockade negatively modulated CB2-induced ST14A cell proliferation, suggesting that a PLC pathway is involved in the steps downstream to CB2 activation. On the basis of our results, we propose ST14A neural progenitor cells as a useful in vitro model for studying ECS modulation of neurogenesis, also in prospective in vivo pharmacological studies.

(E)-β-caryophyllene (BCP) is a bicyclic sesquiterpene widely distributed in the plant kingdom, where it contributes a unique aroma to essential oils and has a pivotal role in the survival and evolution of higher plants. Recent studies provided evidence for protective roles of BCP in animal cells, highlighting its possible use as a novel therapeutic tool. Experimental results show the ability of BCP to reduce pro-inflammatory mediators such as tumor necrosis factor-alfa (TNF-α), interleukin-1β (IL-1β), interleukin-6 (IL-6), nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), thus ameliorating chronic pathologies characterized by inflammation and oxidative stress, in particular metabolic and neurological diseases. Through the binding to CB2 cannabinoid receptors and the interaction with members of the family of peroxisome proliferator-activated receptors (PPARs), BCP shows beneficial effects on obesity, non-alcoholic fatty liver disease/nonalcoholic steatohepatitis (NAFLD/NASH) liver diseases, diabetes, cardiovascular diseases, pain and other nervous system disorders. This review describes the current knowledge on the biosynthesis and natural sources of BCP, and reviews its role and mechanisms of action in different inflammation-related metabolic and neurologic disorders.

(E)-β-caryophyllene (BCP) is a bicyclic sesquiterpene widely distributed in the plant kingdom, where it contributes a unique aroma to essential oils and has a pivotal role in the survival and evolution of higher plants. Recent studies provided evidence for protective roles of BCP in animal cells, highlighting its possible use as a novel therapeutic tool. Experimental results show the ability of BCP to reduce pro-inflammatory mediators such as tumor necrosis factor-alfa (TNF-α), interleukin-1β (IL-1β), interleukin-6 (IL-6), nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), thus ameliorating chronic pathologies characterized by inflammation and oxidative stress, in particular metabolic and neurological diseases. Through the binding to CB2 cannabinoid receptors and the interaction with members of the family of peroxisome proliferator-activated receptors (PPARs), BCP shows beneficial effects on obesity, non-alcoholic fatty liver disease/nonalcoholic steatohepatitis (NAFLD/NASH) liver diseases, diabetes, cardiovascular diseases, pain and other nervous system disorders. This review describes the current knowledge on the biosynthesis and natural sources of BCP, and reviews its role and mechanisms of action in different inflammation-related metabolic and neurologic disorders.

The G protein-coupled cannabinoid receptors type 1 (CB1R) and type 2 (CB2R), and their endocannabinoid (eCBs) ligands, have been implicated in several aspects of brain wiring during development. Here we aim to assess whether interfering with CB1R affects development, neuritogenesis and pathfinding of GnRH and AgRP neurons, forebrain neurons that control respectively reproduction and appetite. We pharmacologically and genetically interfered with CB1R in zebrafish strains with fluorescently labeled GnRH3 and the AgRP1 neurons. By applying CB1R antagonists we observed a reduced number of GnRH3 neurons, fiber misrouting and altered fasciculation. Similar phenotypes were observed by CB1R knockdown. Interfering with CB1R also resulted in a reduced number, misrouting and poor fasciculation of the AgRP1 neuron&rsquo, s axonal projections. Using a bioinformatic approach followed by qPCR validation, we have attempted to link CB1R functions with known guidance and fasciculation proteins. The search identified stathmin-2, a protein controlling microtubule dynamics, previously demonstrated to be coexpressed with CB1R and now shown to be downregulated upon interference with CB1R in zebrafish. Together, these results raise the likely possibility that embryonic exposure to low doses of CB1R-interfering compounds could impact on the development of the neuroendocrine systems controlling sexual maturation, reproduction and food intake.

Bisphenol A (BPA) is one of the most widely used chemicals worldwide, e.g., as a component of plastic containers for food and water. It is considered to exert an estrogenic effect, by mimicking estradiol (E2) action. Because of this widespread presence, it has attracted the interest and concern of researchers and regulators. Despite the vast amount of related literature, the potential adverse effects of environmentally significant doses of BPA are still object of controversy, and the mechanisms by which it can perturb endocrine functions, and particularly the neuroendocrine axis, are not adequately understood. One of the ways by which endocrine disruptors (EDCs) can exert their effects is the perturbation of calcium signaling mechanisms. In this study, we addressed the issue of the impact of BPA on the neuroendocrine system with an in vitro approach, using a consolidated model of immortalized Gonadotropin-Releasing Hormone (GnRH) expressing neurons, the GT1&ndash, 7 cell line, focusing on the calcium signals activated by the endocrine disruptor. The investigation was limited to biologically relevant doses (nM&ndash, &micro, M range). We found that BPA induced moderate increases in intracellular calcium concentration, comparable with those induced by nanomolar doses of E2, without affecting cell survival and with only a minor effect on proliferation.

Even though microplastic (MP) pollution in aquatic environment is nowadays widely studied, a huge gap of knowledge exists on their actual biological effects. In this study we first reported environmental baseline data on the occurrence and characterization of floating MPs in Italian coastal waters of the Central Adriatic Sea by using a standardized monitoring protocol. Further, we analyzed the concentrations of MP-associated chemicals and evaluated their potential adipogenic effects using 3T3-L1 preadipocytes. MPs were found in each sampling stations showing the highest abundance (1.88 ± 1.78 items/m3) in the sites more distant from the coast with fragments as the most common shape category. All targeted organic pollutants (i.e. polychlorinated biphenyls - PCBs, polycyclic aromatic hydrocarbons -PAHs, organophosphorus - OP, and organochlorine - OC pesticides) have been detected on the surface of the collected MPs. The highest concentrations of PAHs were found on MPs from inshore (i.e. 6 NM) waters (10.37 ng/g plastic). Among pesticides, all measured OPs were detected in each sample analyzed with pirimiphos-methyl as the most representative compound. For OCs, the sum of all concentrations of congeners was higher in coastal with respect to offshore waters. Moreover, in vitro 3T3-L1 screening of MP extracts indicated potential metabolic effects resulting in both adipogenesis and lipid uptake/storage.

Recent studies suggest that exposure to some plasticizers, such as Bisphenol A (BPA), play a role in endocrine/metabolic dispruption and can affect lipid accumulation in adipocytes. Here, we investigated the adipogenic activity and nuclear receptor interactions of four plasticizers approved for the manufacturing of food-contact materials (FCMs) and currently considered safer alternatives. Differentiating 3T3-L1 mouse preadipocytes were exposed to scalar concentrations (0.01–25 μM) of DiNP (Di-iso-nonyl-phthalate), DiDP (Di-iso-decyl-phthalate), DEGDB (Diethylene glycol dibenzoate), or TMCP (Tri-m-cresyl phosphate). Rosiglitazone, a well-known pro-adipogenic peroxisome proliferator activated receptor gamma (PPARγ) agonist, and the plasticizer BPA were included as reference compounds. All concentrations of plasticizers were able to enhance lipid accumulation, with TMCP being the most effective one. Accordingly, when comparing in silico the ligand binding efficiencies to the nuclear receptors PPARγ and retinoid-X-receptor-alpha (RXRα), TMPC displayed the highest affinity to both receptors. Differently from BPA, the four plasticizers were most effective in enhancing lipid accumulation when added in the mid-late phase of differentiation, thus suggesting the involvement of different intracellular signalling pathways. In line with this, TMCP, DiDP, DiNP and DEGDB were able to activate PPARγ in transient transfection assays, while previous studies demonstrated that BPA acts mainly through other nuclear receptors. qRT-PCR studies showed that all plasticizers were able to increase the expression of CCAAT/enhancer binding protein β (Cebpβ) in the early steps of adipogenesis, and the adipogenesis master gene Pparγ2 in the middle phase, with very similar efficacy to that of Rosiglitazone. In addition, TMCP was able to modulate the expression of both Fatty Acid Binding Protein 4/Adipocyte Protein 2 (Fabp4/Ap2) and Lipoprotein Lipase (Lpl) transcripts in the late phase of adipogenesis. DEGDB increased the expression of Lpl only, while the phthalate DiDP did not change the expression of either late-phase marker genes Fabp4 and Lpl. Taken together, our results suggest that exposure to low, environmentally relevant doses of the plasticizers DiNP, DiDP, DEGDB and TMCP increase lipid accumulation in 3T3-L1 adipocytes, an effect likely mediated through activation of PPARγ and interference at different levels with the transcriptional cascade driving adipogenesis.

Trans-&beta, caryophyllene (BCP) is a natural sesquiterpene hydrocarbon with several important pharmacological activities, including antioxidant, anti-inflammatory, anticancer, and cardioprotective functions. These properties are mainly due to its selective interaction with the peripherally expressed cannabinoid receptor 2. In addition, BCP activates peroxisome proliferated activator receptors &alpha, and &gamma, and inhibits the Toll-like receptor signaling pathway. Given the growing scientific interest in BCP, the aim of our study was to investigate the metabolic effects of a black pepper extract (PipeNig&reg, FL), containing a high standardized content of BCP. In particular our interest was focused on its potential activity on lipid accumulation and glucose uptake. The extract PipeNig&reg, FL was chemically characterized by gas chromatography&ndash, mass spectrometry (GC&ndash, MS) and gas chromatography with flame-ionization detection (GC&ndash, FID), confirming a high content (814 mg/g) of BCP. Experiments were performed on 3T3-L1 preadipocytes and on C2C12 myotubes. Lipid content following 3T3-L1 adipogenic differentiation was quantified with AdipoRed fluorescence staining. Glucose uptake and GLUT4 membrane translocation were studied in C2C12 myotubes with the fluorescent glucose analog 2-NBDG and by immunofluorescence analysis. Here we show that PipeNig&reg, FL reduces 3T3-L1 adipocyte differentiation and lipid accumulation. Moreover, acute exposure of C2C12 myotubes to PipeNig&reg, FL improves glucose uptake activity and GLUT4 migration. Taken together, these results reveal interesting and novel properties of BCP, suggesting potential applications in the prevention of lipid accumulation and in the improvement of glucose uptake.

New cells are continuously added to the rodent olfactory bulb (OB), throughout development and in adults. These cells migrate tangentially from the subventricular zone along the rostral migratory stream to the OB, where they migrate radically from the center to periphery of the OB. Although different modalities of radial migration have been described in other brain regions, the mechanisms governing radial migration in the OB are still mostly unknown. Here, we identify a new modality of migration in which neuronal precursors migrate along blood vessels toward their destination. Our results show that half of the radially migrating cells associate with the vasculature in the granule cell layer of the OB, andin vivotime-lapse imaging demonstrates that they use blood vessels as a scaffold for their migration through an interaction with the extracellular matrix and perivascular astrocyte end feet. The present data provide evidence that a new modality of migration, vasophilic migration, is occurring in the adult brain and reveals a novel role of brain vasculature.

© 2015 Heindel et al. A multidisciplinary group of experts gathered in Parma Italy for a workshop hosted by the University of Parma, May 16-18, 2014 to address concerns about the potential relationship between environmental metabolic disrupting chemicals, obesity and related metabolic disorders. The objectives of the workshop were to: 1. Review findings related to the role of environmental chemicals, referred to as "metabolic disruptors", in obesity and metabolic syndrome with special attention to recent discoveries from animal model and epidemiology studies; 2. Identify conclusions that could be drawn with confidence from existing animal and human data; 3. Develop predictions based on current data; and 4. Identify critical knowledge gaps and areas of uncertainty. The consensus statements are intended to aid in expanding understanding of the role of metabolic disruptors in the obesity and metabolic disease epidemics, to move the field forward by assessing the current state of the science and to identify research needs on the role of environmental chemical exposures in these diseases. We propose broadening the definition of obesogens to that of metabolic disruptors, to encompass chemicals that play a role in altered susceptibility to obesity, diabetes and related metabolic disorders including metabolic syndrome.

ErbB-4 is expressed by the periglomerular and the mitral/tufted cells of the adult mouse olfactory bulb (OB) and in the present work we tested whether this expression is regulated by the olfactory nerve input to the OB. Reversible zinc sulphate lesions of the olfactory mucosa were made in adult mice and the deafferented OB analysed by immunohistochemistry, Western blotting and semiquantitative RT-PCR. Following deafferentation, the expression of erbB-4, erbB-2 and neuregulin-1 (NRG-1) mRNAs in the OB was altered. At early stages (7-14 days) after lesion the levels of expression of olfactory marker protein (OMP), tyrosine hydroxylase (TH), erbB-4 and NRG-1 mRNAs were decreased, whilst expression of erbB-2 increased and that of NRG-2 was not significantly altered. We observed at least two distinct time courses for these expression changes. The lowest amounts of mRNA for erbB-4 and NRG-1 were observed at day 7 after lesion, whilst mRNAs for TH and OMP were lowest at day 14. At day 28 after the lesion, when olfactory receptor neuron axons had reinnervated the olfactory bulb, the expression levels of OMP, TH, erbB-2, erbB-4 and NRG-1 were identical to control values. These results indicate that the expression of erbB-4 mRNA and protein in periglomerular and mitral cells is controlled by peripheral olfactory innervation. The tight correlation in NRG-1 and erbB-4 expression levels also suggests a possible functional link that deserves further exploration.

The interplay between neuregulins and the ErbB receptor family has a pivotal role in the development of several tissues, including the nervous system, and is maintained in the adult olfactory system where extensive plasticity and neurogenesis are retained. In the present work we show the immunohistochemical localization of ErbB-3 and ErbB-4 in the olfactory system of adult normal and lesioned mice. The expression of ErbB-3 is demonstrated to be restricted to the ensheathing cells of the olfactory nerve and to a few substentacular cells of the olfactory epithelium (OE). ErbB-3 staining circumvents the glomeruli but is never observed inside them or elsewhere in the adult olfactory bulb. Conversely, ErbB-4 immunoreactivity is found in all the periglomerular and mitral/tufted cells of the olfactory bulb (OB) and to a minor extent in the olfactory neurons and basal cells of the OE. Interestingly enough, cells coming out from the rostral migratory stream of the subependymal layer (SEL), as well as isolated cells in the granule cell layer, possibly migrating cells, strongly stained for ErbB-4 expression. Lesions of the olfactory epithelium have been performed by unilateral intranasal irrigation with ZnSO4 and 3 weeks after the irrigation, the olfactory bulbs were analyzed for olfactory marker protein (OMP), tyrosine hydroxylase (TH), ErbB-3 and ErbB-4 expression. In the deafferented OB, the drastic loss of immunoreactivity for OMP was accompanied by a strong reduction of ErbB-3 expression. Most of the deafferented dopaminergic interneurons switched off TH expression. In the deafferented periglomerular and mitral/tufted cells ErbB-4 expression was turned off and down, respectively. No differences were noted at the granular cell layer level in the deafferented OB with respect to control. Taken together our results suggest that, in normal conditions, neuregulins are involved in the survival of the ensheathing cells of the olfactory nerve through ErbB-3 activation and in the functional activity of postsynaptic neurons through ErbB-4 activation.

Chemical lesion of olfactory neuroepitheium induced an up-regulation of the mGlu1a metabotropic glutamate receptor protein in the olfactory bulb, as shown by Western blot analysis. At 2 days after the lesion, the increase in the receptor protein was associated with an increase in mGlu1a mRNA levels; in contrast, at longer times after the lesion (16 days), mRNA levels were reduced in spite of the high expression of the receptor protein, perhaps as a result of product-inhibition of mGlu1 gene expression. Immunohistochemical analysis showed that the increase in mGlu1a induced by olfactory denervation was confined to the glomeruli, which occupy the external portion of the olfactory bulb. Within these structures, mGlu1a receptors are mainly localized on the distal dendrites of mitral cells, which are innervated by the glutamatergic axons of the olfactory nerve. These results demonstrate that the expression of mGlu1a receptors is up-regulated in response to glutamatergic deafferentation, supporting a role for this particular receptor subtype in the physiology of synaptic transmission.

Endocannabinoids are natural lipids regulating a large array of physiological functions and behaviors in vertebrates. The endocannabinoid system is highly conserved in evolution and comprises several specific receptors (type-1 and type-2 cannabinoid receptors), their endogenous ligands (e.g. anandamide and 2-arachidonoylglycerol), and a number of biosynthetic and degradative enzymes. In the last few years, endocannabinoids have been described as critical signals in the control of male and female reproduction at multiple levels: centrally, by targeting hypothalamic Gonadotropin-Releasing-Hormone secreting neurons and pituitary, and locally, with direct effects on the gonads. These functions are supported by the extensive localization of cannabinoid receptors and endocannabinoid metabolic enzymes at different levels of the hypothalamic-pituitary-gonadal axis in mammals, as well as bonyfish and amphibians. In vivo and in vitro studies indicate that endocannabinoids centrally regulate gonadal functions by modulating the Gonadotropin Releasing Hormone-gonadotropin-steroid network through direct and indirect mechanisms. Several proofs of local endocannabinoid regulation have been found in the testis and male genital tracts, since endocannabinoids control Sertoli and Leydig cells activity, germ cell progression, as well as the acquisition of sperm functions. A comparative approach usually is a key step in the study of physiological events leading to the building of a general model. Thus, in this review we summarize the action of endocannabinoids at different levels of the male reproductive axis, with special emphasis, where appropriate, on data from non-mammalian vertebrates.

The endocannabinoid system (ECS) has a well-documented pivotal role in the control of mammalian reproductive functions, by acting at multiple levels, that is, central (CNS) and local (gonads) levels. Since studies performed in animal models other than mammals might provide further insight into the biology of these signalling molecules, in the present paper we review the comparative data pointing toward the endocannabinoid involvement in the reproductive control of non-mammalian vertebrates, focussing in particular on the central regulation of teleost and amphibian reproduction. The morphofunctional distribution of brain cannabinoid receptors will be discussed in relation to other crucial signalling molecules involved in the control of reproductive functions, such as GnRH, dopamine, aromatase, and pituitary gonadotropins.

In this study, we analysed the molecular heterogeneity and synaptic localization of the N-methyl-D-aspartate receptor subunit 1 and the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptor subunit 1 in the olfactory bulb glomerular synaptic circuitry. Semiquantitative reverse transcriptase polymerase chain reaction showed that approximately 40% of the N-methyl-D-aspartate receptor subunit 1 messenger RNA splice variants contain the N1 exon, which conveys specific functional properties on the channel. In other forebrain and hindbrain regions that we examined, the ratio of the N1-containing (receptor subunit 1(1XX)) to N1-lacking (receptor subunit 1(0XX)) N-methyl-D-aspartate receptor subunit 1 messenger RNAs varied considerably. The cellular and subcellular distribution of N-methyl-D-aspartate receptor subunit 1 and AMPA receptor subunit 1 was investigated with antibodies generated against the C-terminal domain of the individual subunits [Petralia R. S. et al. (1994) J. Neurosci. 14, 667 696; Wenthold R. J. et al. (1992) J. biol Chem. 267, 501 507]. Both N-methyl-D-aspartate receptor subunit 1 and AMPA receptor subunit 1 were localized to the postsynaptic density of asymmetric synapses established by olfactory receptor neuron terminals with the dendrites of mitral and tufted cells. Not all of these synapses, however, were labelled. These results are consistent with the notion that glutamate is the neurotransmitter at the olfactory nerve to mitral and tufted cell synapses, and suggest a high heterogeneity in the expression of the postsynaptic glutamate receptors.

Cannabinoids, the bioactive constituents of Cannabis sativa, and endocannabinoids, among which the most important are anandamide and 2-arachidonoylglycerol, control various biological processes by binding to specific G protein-coupled receptors, namely CB1 and CB2 cannabinoid receptors. While a vast amount of information on the mammalian endocannabinoid system does exist, few data have been reported on bony fish. In the goldfish, Carassius auratus, the CB1 receptor has been cloned and its distribution has been analyzed in the retina, brain and gonads, while CB2 had not yet been isolated. In the present paper we cloned the goldfish CB2 receptor and show that it presents a quite high degree of amino acid identity with zebrafish Danio rerio CB2A and CB2B receptors, while the percentage of identity is lower with the pufferfish Fugu rubripes CB2, as also confirmed by the phylogenetic analysis. The sequence identity becomes much lower when comparing the goldfish and the mammalian CB2 sequences; as for other species, goldfish CB2 and CB1 amino acid sequences share moderate levels of identity. Western-blotting analysis shows the CB2 receptor as two major bands of about 53 kDa and 40 kDa, and other faint bands with apparent molecular masses around 70 kDa, 57 kDa and 55 kDa. Since the distribution of a receptor could give information on its physiological role, we evaluated and compared CB1 and CB2 mRNA expression in different goldfish organs by means of quantitative Real-Time PCR. Our results show that both CB1 and CB2 receptors are widely expressed in the goldfish, displaying some tissue specificities, thus opening the way for further functional studies on bony fish and other non-mammalian vertebrates.

Using a competitive polymerase chain reaction (PCR) and appropriate internal standards, we have analyzed absolute amounts of the alpha 6 GABAA receptor subunit mRNA in the postnatally developing cerebellum and neocortex. The PCR data have shown that absolute amounts of the alpha 6 receptor subunit mRNA in the cerebellum increase dramatically (nearly 100-fold) during the second postnatal week, reaching maximal levels by postnatal day 21 (1 fmol/microgram total RNA). The absolute amount of the alpha 6 GABAA receptor subunit mRNA in the cortex at postnatal day 1 was 2 amol/microgram total RNA and increased to 7 amol/micrograms total RNA by postnatal day 14. No further increase in alpha 6 mRNA expression in the adult cortex was observed. Microscopic analysis of emulsion coated and counterstained sections indicated that alpha 6 GABAA receptor subunit mRNA labeling was only detected in the internal granule cell layer and not in either the external granule cell layer or in migrating granule cells. The alpha 1 GABAA receptor subunit mRNA increased in the cerebellar cortex with a similar temporal profile, although its distribution extended to additional cell types (Purkinje cells, stellate/basket cells and possibly cerebellar astrocytes). The temporal expression of these two GABAA receptor subunit mRNAs is coincident with the formation of synaptic contacts in the granule cell dendrites suggesting that afferent pathways innervating these neurons following cell migration may play a critical role in increasing the expression of mRNAs encoding the alpha 1 and alpha 6 GABAA receptor subunits.

Alternative splicing of the metabotropic glutamate receptor 1 (mGluR1) receptor gene generates two major receptor isoforms, mGluR1a and mGluR1b, differing in intracellular function and distribution. However, little is known on the expression profiles of these variants during development. We examined the mRNA expression profile of mGluR1a/b in microdissected layers and acutely isolated mitral cells in the developing mouse olfactory bulb. This analysis showed that the two mGluR1 variants are differentially regulated within each bulb layer. During the first postnatal week, the mGluR1a isoform replaces GluR1b in the microdissected mitral cell layer (MCL) and in isolated identified mitral cells, coinciding with a developmental epoch of mitral cell dendritic reorganization. Although mGluR1a mRNA is expressed at high levels in both the adult external plexiform layer (EPL) and MCL, Western blotting analysis reveals a marked reduction of the mGluR1a protein in the MCL, where mitral cell bodies are located, and strong labeling in the EPL, which contains mitral cell dendrites. This suggests that there is increased dendritic trafficking efficiency of the receptor in adult. The temporal and spatial shift in mGluR1b/a expression suggests distinct roles of the mGluR1 isoforms, with mGluR1b potentially involved in the early mitral cell maturation and mGluR1a in dendritic and synapse function.

Methamphetamine produces locomotor activation and typical stereotyped motor patterns, which are commonly related with increased catecholamine activity within the basal ganglia, including the dorsal and ventral striatum. Since the cerebellum is critical for movement control, and for learning of motor patterns, we hypothesized that cerebellar catecholamines might be a target of methamphetamine. To test this experimental hypothesis we injected methamphetamine into C57 Black mice at the doses of 5 mg/kg two or three times, 2 h apart. This dosing regimen is known to be toxic for striatal dopamine terminals. However, we found that in the cerebellum, methamphetamine increased the expression of the primary transcript of the tyrosine hydroxylase (TH) gene, followed by an increased expression of the TH protein. Increased TH was localized within Purkinje cells, where methamphetamine increased the number of TH-immunogold particles, and produced a change in the distribution of the enzyme by increasing the cytoplasmic percentage. Increased TH expression was accompanied by a slight increase in noradrenaline content. This effect was highly site-specific for the cortex of posterior vermal lobules, while only slight effects were detectable in the hemispheres. The present data indicate that the cerebellum does represent a target of methamphetamine, which produces specific and fine alterations of the catecholamine system involving synthesis, amount, and compartmentalization of TH as well as increased noradrenaline levels. This may be relevant for motor alterations induced by methamphetamine. In line with this, inherited cerebellar movement disorders in various animal species including humans are associated with increased TH immunoreactivity within intrinsic neurons of the same lobules of the cerebellar cortex.

In the rodent brain neural progenitor cells are born in the subventricular zone and migrate along a pathway called the rostral migratory stream (RMS) into the olfactory bulb where they differentiate into several classes of interneurones. In the adult, tangential migration in the RMS takes place in 'chains' of cells contained within glial tubes. In contrast, neonatal neuroblasts along the RMS lack these defined glial tubes and chains, migrating instead as individual cells. Time-lapse confocal microscopy of neuroblasts at each of these ages shows that individual cells migrate in a saltatory manner with bursts of high speed followed by periods of slower speed. Tangential migration within a glial tube is 20% faster than migration as individual cells. Neuroblasts may also interact and modify the extracellular matrix during migration through expression of a family of proteins, the matrix metalloproteinases (MMPs). MMPs are present and active along the subventricular zone-olfactory bulb pathway. In the presence of inhibitors of MMPs, neuroblast migration rates were reduced only when cells migrate individually. Chain migration in the adult was unaffected by MMP inhibitors. Taken together, these data suggest that MMPs only influence migration as individual cells and not as chains.

Neuregulins (NRGs), and their cognate receptors (ErbBs), play essential roles in numerous aspects of neural development and adult synaptic plasticity. The goal of this study was to investigate the developmental expression profiles of these molecules during the olfactory bulb (OB) maturation. The OB is a highly organized structure with cell types and synaptic connections segregated into discrete anatomical layers. We employed a novel approach by combining single-layer microdissection at different development ages, with isoform-specific semi-quantitative RT-PCR and Western blotting to monitor layer-specific developmental profiles of these molecules and alternate splice variants. Layer and age specific regulation was observed for the ErbB4 splice variants JMa/JMb and NRG-1-beta1/beta2 forms. With the exception of the outermost (nerve) layer, ErbB4-JMb and NRG-1-beta1 are expressed throughout the OB and their expressions decrease in the adult age in most layers. In contrast both ErbB4-JMa and NRG-1-beta2 are highly expressed in the granule cell layer in the early postnatal OB. This early postnatal expression correlates with the dramatic change from radial glia to astrocytes and appearance of the bulk of granule cells occurring at this developmental stage.

Olfactory bulb interneurons are continuously generated throughout development and in adulthood. These neurons are born in the subventricular zone (SVZ) and migrate along the rostral migratory stream into the olfactory bulb where they differentiate into local interneurons. To investigate the differentiation of GABAergic interneurons of the olfactory bulb we used a transgenic mouse which expresses green fluorescent protein (GFP) under the control of the glutamic acid decarboxylase 65 kDa (GAD65) promoter. During development and in adulthood GFP was expressed by cells in the SVZ and along the entire length of its rostral extension including the distal portion within the olfactory bulb. The occurrence of GAD65 mRNA in these zones was confirmed by PCR analysis on microdissected regions along the pathway. Polysialic acid neural cell adhesion molecule, a marker of migrating neuroblasts in adults, was coexpressed by the majority of the GFP-positive SVZ-derived progenitor cells. Cell tracer injections into the SVZ indicated that approximately 26% of migrating progenitor cells expressed GFP. These data show the early differentiation of migrating SVZ-derived progenitors into a GAD65-GFP-positive phenotype. These cells could represent a restricted lineage giving rise to GAD65-positive GABAergic olfactory bulb interneurons.

The present study has analyzed the effect of progesterone and its derivatives (dihydroprogesterone and tetrahydroprogesterone) on the gene expression of the peripheral myelin protein 22 utilizing in vivo and in vitro models, The data obtained indicate that tetrahydroprogesterone is able to stimulate the gene expression of peripheral myelin protein 22 both in vivo (in adult but not in old animals) and in Schwann cell cultures. An effect of this steroid, which is known to interact with the GABA(A) receptor, would not be surprising, since in the present study we show the presence in Schwann cells and in the sciatic nerve of the messengers for several subunits (alpha 2, alpha 3, beta 1, beta 2, and beta 3) of the GABA(A) receptor. An effect of tetrahydroprogesterone is also evident on the gene expression of another myelin protein, the peripheral myelin protein zero. However, in this case also dihydroprogesterone, which is able to bind the progesterone receptor, is involved, both in old and adult animals, in the stimulation of messengers levels of this myelin protein, In conclusion, the present data show that the gene expression of two important peripheral myelin proteins can be influenced by progesterone derivatives. The hypothesis has been put forward that part of their effects might occur not through the classical progesterone receptor, but rather via an interaction with the GABA(A) receptor. J. Neurosci, Res. 56:349-357, 1999, (C) 1999 Wiley-Liss, Inc.

GLUTAMATE (Glu) released by olfactory nerve axons acts on postsynaptic ionotropic and metabotropic glutamate receptors expressed by principal neurones and interneurones of the olfactory bulb (OB). Using ZnSO 4 lesioning of the rat olfactory mucosa and semiquantitative RT-PCR, we examined the effect of removal of the glutamatergic input to the OB on the expression of mGluRla, mGluR1b and GluR1 mRNAs. Two days after lesioning, mGluRla mRNA levels in OB increased by 45%. At this time, the expression of tyrosine hydroxylase (TH) mRNA, which is strictly dependent on olfactory nerve input, was still unchanged. In contrast, 16 days after lesioning, deafferented OB exhibited a decrease in both mGluRla (-30%) and TH (-40%) mRNAs. GluR1 and mGluR1b mRNA levels were not affected at either time point. These results suggest that alterations in glutamatergic input to OB selectively modulate the expression of the mGluR1 splicing form possessing a longer C-terminal domain.

Background A number of studies have separately shown that the neuregulin1 (NRG1)/ErbB4 system and NMDA-type glutamate receptors (NMDARs) are involved in several aspects of neuronal migration. In addition, intracellular calcium fluctuations play central roles in neuronal motility. Stable expression of the tyrosine kinase receptor ErbB4 promotes migratory activity in the neural progenitor cell line ST14A upon NRG1 stimulation. In this work we analyzed the potential interactions between the NRG1/ErbB4 system and NMDARs in the ST14A migratory process as well as its calcium dependence. Results RT-PCR studies have shown that both native ST14A cells (non-expressing ErbB4), as well as ErbB4-transfected cells express low levels of a restricted number of NMDAR subunits: NR1, NR2C, NR2D and NR3B. The resulting NMDAR would form Ca2+ channels characterized by low Mg2+-sensitivity and low Ca2+-permeability, generating small, long-lasting currents. Ca2+-imaging experiments showed slow [Ca2+]i increases in 45% of the cells following 8 μM NMDA stimulation. Basal migration of ErbB4-transfected ST14A cells was unaffected by 18 hrs NMDA incubation. However, over the same incubation time, NMDA was able to significantly enhance NRG1-induced migration. Pre-incubation with the intracellular calcium chelator BAPTA-AM reduced both NRG1- and NRG1/NMDA-stimulated migration, suggesting the involvement of Ca2+ in these processes. NRG1 stimulation of ErbB4-transfected ST14A cells induced a sustained, long-lasting increase in [Ca2+]i, in 99% of the cells. These intracellular Ca2+ signals could be ascribed to both release from intracellular stores and influx from the extracellular medium trough a mechanism of store-operated calcium entry (SOCE). Short-time co-incubation of NMDA and NRG1 did not substantially modify the NRG1-induced intracellular calcium signals. Conclusions In summary, NRG1 stimulation of the ErbB4 receptor exerts a sustained [Ca2+]i increase in ST14A neural progenitors; NRG1-induced migration is Ca2+-dependent and can be positively modulated by activation of the NMDA receptor.

The distribution of diazepam binding inhibitor (DBI), a multi-function peptide which has recently been discovered, was studied in the rat and human central nervous system and in peripheral organs of the rat by light and electron microscopical immunohistochemistry. In the central nervous system, DBI-LI was localized in many glial cells and glial tumors, and in some neurons. In the periphery, DBI-LI was found in many tissues but it was expressed selectively in specialized cell types. Intense DBI-LI was observed in some endocrine, steroid-producing cells such as glomerular cells of the adrenal gland and Leydig cells of the of the testis. Different types of epithelial cells, for instance distal convoluted tabular cells of the kidney and mucosal cells of the small intestine, displayed moderate DBI-LI. Some supporting cells, such as Schwann cells and Sertoli cells, were also immunopositive. The frequent localization of DBI in cells, also known to contain large amounts of mitochondrial benzodiazepine receptors, indicates that DBI may play an important role as an endogenous regulator of intracellular metabolic functions via the mitochondrial benzodiazepine receptor.

We studied the expression and distribution of the polypeptide diazepam binding inhibitor (DBI) in rat peripheral organs by immunocytochemistry, radioimmunoassay, Northern blot analysis and binding assay. Variable amounts of the DBI peptide and DBI mRNA were found in all the tissues examined (liver, duodenum, testis, kidney, adrenal gland, heart, ovary, lung, skeletal muscle and spleen), with the highest level of expression in liver (220 pmol of DBI/mg protein) and the lowest in spleen (11 pmol of DBI/mg protein). A good correlation between DBI-like immunoreactivity (DBI-LI) and mRNA content was found in all tissues except the heart. The immunohistochemical analysis revealed discrete localization of DBI-LI in cell types with specialized functions: for example, the highest DBI-LI content was found in steroid-producing cells (glomerulosa and fasciculata cells of adrenal cortex, Leydig cells of testis); lower DBI-LI immunostaining was found in epithelial cells specialized for water and electrolyte transport (intestinal mucosa, distal convoluted tubules of kidney). Hepatic cells contained moderate immunoreactivity however the total content of DBI in liver is relatively high and is due to the diffuse presence of DBI in every hepatocyte. Cells with high expression of DBI have been shown to contain a high density of mitochondrial benzodiazepine (BZ) binding sites. This observation led us to perform a competitive binding assay between DBI and [3H]PK11195 (a ligand for the mitochondrial BZ binding sites) on mitochondrial membranes of adrenal cortical cells. In this experiment, DBI yielded an apparent competitive inhibition of the binding of PK11195 to the BZ binding sites. Our data support a possible role for DBI as endogenous regulator of intracellular metabolic functions, such as steroidogenesis, via the mitochondrial BZ receptors.

In the present work we characterized both the presynaptic and postsynaptic components of cholinergic transmission in a primary culture of corticostriatal neurons prepared from newborn rat brain. This culture preparation contains a small population of choline acetyltransferase (ChAT) immunoreactive neurons, corresponding to approximately 3% of the total cell number, and synthesizes increasing amounts of acetylcholine (ACh) from the third day in vitro (DIV), which reaches a plateau around the 10 day of culture. Muscarinic cholinergic receptors (mAChR), measured by the binding of the muscarinic antagonist [3H]quinuclidinyl benzilate ([3H]QNB), are detectable from the fifth DIV and increase linearly during the time of culture. At the twelfth DIV, the density of mAChRs (approximately 600 fmol/mg protein) is comparable to the density of mAChR in adult rat cortex. These receptors are coupled to second messenger systems, since muscarinic agonists inhibit adenylate cyclase activity and stimulate phosphoinositide breakdown with efficacies and potencies similar to those found in adult rat cortex. Moreover, by using the reverse transcriptase-polymerase chain reaction (RT-PCR) technique, we were able to demonstrate the presence of the m1, m3, and m4 mAChR subtype mRNAs in this neuronal culture at 12 DIV. Our data suggest that corticostriatal neuronal cultures develop in vitro ACh-synthesizing neurons and functionally active cholinergic receptors. This therefore makes them ideally suited to study the development and properties of brain mAChR subtypes.

Diazepam Binding Inhibitor (DBI) is an endogenous 11-kDa peptide originally isolated from rat brain. In rat brain DBI coexists with at least three different processing products and the members of this peptide family have been shown to displace benzodiazepines and beta carbolines from recognition sites located on the allosteric modulatory centers of GABAA receptors. Immunocytochemical methods were used to study the location of DBI and two of the processing products, octadecaneuropeptide (ODN) DBI 33-50 and triakontatetraneuropeptide (TTN) DBI 17-50, in rat brain. DBI-LI was found in selected neuronal perikarya and in many glia and glial-like cells. All circumventricular organs displayed a strong DBI like immunoreactivity (LI). The distribution and cellular location of the ODN-LI and TTN-LI differed from that of DBI because they were preferentially associated with DBI in neurons, but not in glia or glial-like cells. The presence of DBI, but not of its processing products, in glial cells, circumventricular organs, and cells of peripheral tissues suggests that the function of this peptide may extend to other yet unknown function in addition to an action on the allosteric modulatory center of GABAA receptors located in neurons.

Diazepam binding inhibitor (DBI), an endogenous 10-kDa polypeptide was isolated from rat and human brain by monitoring displacement of radioactive diazepam bound to specific recognition sites in brain synaptic and mitochondrial membranes. The cellular location of DBI mRNA was studied in rat brain and selected peripheral tissues by in situ hybridization histochemistry with a 35S-labeled single-stranded complementary RNA probe. DBI mRNA was heterogeneously distributed in rat brain, with particularly high levels in the area postrema, the cerebellar cortex, and ependyma of the third ventricle. Intermediate levels were found in the olfactory bulb, pontine nuclei, inferior colliculi, arcuate nucleus, and pineal gland. Relatively low but significant levels of silver grains were observed overlying many mesencephalic and telencephalic areas that have previously been shown to contain numerous DBI-immunoreactive neurons and a high density of central benzodiazepine receptors. In situ hybridizations also revealed high levels of DBI mRNA in the posterior lobe of the pituitary gland, liver, and germinal center of the white pulp of spleen, all tissues that are rich in peripheral benzodiazepine binding sites. The tissue-specific pattern of DBI gene expression described here could be exploited to further understand the physiological function of DBI in the brain and periphery.

Olfactory neuroepithelial (OE) cells were dissociated from late stage embryonic mice and analysed for carnosine expression. The yield of carnosine neurones was twice as high when the OE cells were seeded along with the olfactory bulb cells. Carnosine neurones resulted from both in vitro survival and neurogenesis, and were associated with clusters of underlying flat cells immunopositive for keratin. Our results demonstrate that olfactory neurones expressing their neurotransmitter carnosine can be studied in culture, and the close association with keratin-immunopositive basal cells suggests that they are dependent on these cells for survival and/or differentiation.

Previously, we have shown that erbB-3 expression is restricted to the ensheathing cells of the olfactory nerve layer, while erbB-4 is found in the periglomerular and mitral/tufted cells of the olfactory bulb and in cells coming out from the rostral migratory stream of the subependymal layer. In the present work, we have treated adult mice with zinc sulfate intranasal irrigation and analyzed erbB-3 and erbB-4 expression in the deafferented olfactory bulb. Following treatment, olfactory axons undergo degeneration, as indicated by the loss of OMP expression in the deafferented olfactory bulb. The thickness of the olfactory nerve layer is reduced, but the specific intensity of erbB-3 labeling in the remaining olfactory nerve layer is increased with respect to control. Interestingly, following deafferentation, erbB-4 immunoreactivity decreases specifically in cell types that normally make synaptic contacts with primary olfactory neurons in the glomeruli, i.e. periglomerular and mitral/tufted cells. Partial lesion of the olfactory epithelium allows regenerative axon growth of olfactory neurons to the olfactory bulb. Following olfactory axon regeneration, erbB-3 and erbB-4 immunoreactivity in the olfactory bulb is similar to control. Thus, like tyrosine hydroxylase, the down regulation of erbB-4 expression in the periglomerular cells is reversible.