Building momentum through networks: Bioimaging across the Americas.

In September 2023, the two largest bioimaging networks in the Americas, Latin America Bioimaging (LABI) and BioImaging North America (BINA), came together during a 1-week meeting in Mexico. This meeting provided opportunities for participants to interact closely with decision-makers from imaging core facilities across the Americas. The meeting was held in a hybrid format and attended in-person by imaging scientists from across the Americas, including Canada, the United States, Mexico, Colombia, Peru, Argentina, Chile, Brazil and Uruguay. The aims of the meeting were to discuss progress achieved over the past year, to foster networking and collaborative efforts among members of both communities, to bring together key members of the international imaging community to promote the exchange of experience and expertise, to engage with industry partners, and to establish future directions within each individual network, as well as common goals. This meeting report summarises the discussions exchanged, the achievements shared, and the goals set during the LABIxBINA2023: Bioimaging across the Americas meeting.

Adrenoreceptor phylogeny and novel functions of nitric oxide in ascidian immune cells.

Nitric oxide (NO) is a simple molecule involved in many biological processes and functions in the cardiovascular, neural, and immune systems. In recent years, NO has also been recognized as a crucial messenger in communication between the nervous and immune systems. Together with NO, catecholamines are the main group of neurotransmitters involved in cross-talk between the nervous and immune systems. Catecholamines such as noradrenaline, can act on immune cells through adrenoreceptors (ARs) present on the cell surface, and NO can cross the cell membrane and interact with secondary messengers, modulating catecholamine production. Here, we analyzed the mutual modulation by noradrenaline and NO in Phallusia nigra immune cells for specific subtypes of ARs. We also investigated the involvement of protein kinases A and C as secondary messengers to these specific subtypes of ARs in the adrenergic signaling pathway that culminates in NO modulation, and the phylogenetic distribution of ARs in deuterostome genomes. This analysis provided evidence for single-copy orthologs of α1, α2 and ß-AR in ascidian genomes, suggesting that NO and NA act on a less diverse set of ARs in urochordates. Pharmacological assays showed that high levels of NO can induce ascidian immune cells to produce catecholamines. We also observed that protein kinases A and C are the secondary messengers involved in downstream modulation of NO production through an ancestral ß-AR. Taken together, these results provide new information on NO as a modulator of immune cells, and reveal the molecules involved in the signaling pathway of ARs. The results also indicate that ARs may participate in NO modulation. Finally, our results suggest that the common ancestor of urochordates possessed a less complex system of ARs required for immune action and diverse pharmacological responses, since the α-ARs are phylogenetically more related to D1-receptors than are the ß-ARs.

Use of invertebrates to model chemically induced parkinsonism-symptoms.

The prevalence of neurological diseases is currently growing due to the combination of several factor, including poor lifestyle and environmental imbalance which enhance the contribution of genetic factors. Parkinson's disease (PD), a chronic and progressive neurological condition, is one of the most prevalent neurodegenerative human diseases. Development of models may help to understand its pathophysiology. This review focuses on studies using invertebrate models to investigate certain chemicals that generate parkinsonian-like symptoms models. Additionally, we report some preliminary results of our own research on a crustacean (the crab Ucides cordatus) and a solitary ascidian (Styela plicata), used after induction of parkinsonism with 6-hydroxydopamine and the pesticide rotenone, respectively. We also discuss the advantages, limits, and drawbacks of using invertebrate models to study PD. We suggest prospects and directions for future investigations of PD, based on invertebrate models.

Central nervous system regeneration in ascidians: cell migration and differentiation.

Adult ascidians have the capacity to regenerate the central nervous system (CNS) and are therefore excellent models for studies on neuroregeneration. The possibility that undifferentiated blood cells are involved in adult neuroregeneration merits investigation. We analyzed the migration, circulation, and role of hemocytes of the ascidian Styela plicata in neuroregeneration. Hemocytes were removed and incubated with superparamagnetic iron oxide nanoparticles (SPION), and these SPION-labeled hemocytes were injected back into the animals (autologous transplant), followed by neurodegeneration with the neurotoxin 3-acetylpyridine (3AP). Magnetic resonance imaging showed that 1, 5, and 10 days after injury, hemocytes migrated to the intestinal region, siphons, and CNS. Immunohistochemistry revealed that the hemocytes that migrated to the CNS were putative stem cells (P-element-induced wimpy testis + or PIWI + cells). In the cortex of the neural ganglion, migrated hemocytes started to lose their PIWI labeling 5 days after injury, and 10 days later started to show ß-III tubulin labeling. In the neural gland, however, the hemocytes remained undifferentiated during the entire experimental period. Transmission electron microscopy revealed regions in the neural gland with characteristics of neurogenic niches, not previously reported in ascidians. These results showed that migration of hemocytes to the hematopoietic tissue and to the 3AP-neurodegenerated region is central to the complex mechanism of neuroregeneration.

Cell proliferation in the central nervous system of an adult semiterrestrial crab.

Neurogenesis occurs in adults of most organisms, both vertebrates and invertebrates. In semiterrestrial crabs of the infraorder Brachyura, the deutocerebrum, where neurogenesis occurs, processes the olfactory sensory information from the antennae. The deutocerebrum is composed of a pair of olfactory lobes associated with cell clusters 9 and 10 (Cl 9 and Cl 10), containing proliferating cells. Because the location of the neurogenic niche in brachyuran semiterrestrial crabs has not been defined, here we describe a neurogenic niche in the central olfactory system of the crab Ucides cordatus and report two types of glial cells in the deutocerebrum, based on different markers. Serotonin (5-hydroxytryptamine) labeling was used to reveal neuroanatomical aspects of the central olfactory system and the neurogenic niche. The results showed a zone of proliferating neural cells within Cl 10, which also contains III beta-tubulin (Tuj1)+ immature neurons, associated with a structure that has characteristics of the neurogenic niche. For the first time, using two glial markers, glial fibrillary acidic protein (GFAP) and glutamine synthetase (GS), we identified two types of astrocyte-like cells in different regions of the deutocerebrum. This study adds to the understanding of neurogenesis in a brachyuran semiterrestrial crustacean and encourages comparative studies between crustaceans and vertebrates, including mammals, based on shared aspects of both mechanisms of neurogenesis and regenerative potentials.

Lipid Rafts from Olfactory Ensheathing Cells: Molecular Composition and Possible Roles.

Olfactory ensheathing cells (OECs) are specialized glial cells of the olfactory system, believed to play a role in the continuous production of olfactory neurons and ensheathment of their axons. Although OECs are used in therapeutic applications, little is known about the cellular mechanisms underlying their migratory behavior. Recently, we showed that OEC migration is sensitive to ganglioside blockage through A2B5 and Jones antibody in OEC culture. Gangliosides are common components of lipid rafts, where they participate in several cellular mechanisms, including cell migration. Here, we characterized OEC lipid rafts, analyzing the presence of specific proteins and gangliosides that are commonly expressed in motile neural cells, such as young neurons, oligodendrocyte progenitors, and glioma cells. Our results showed that lipid rafts isolated from OECs were enriched in cholesterol, sphingolipids, phosphatidylcholine, caveolin-1, flotillin-1, gangliosides GM1 and 9-O-acetyl GD3, A2B5-recognized gangliosides, CNPase, α-actinin, and ß1-integrin. Analysis of the actin cytoskeleton of OECs revealed stress fibers, membrane spikes, ruffled membranes and lamellipodia during cell migration, as well as the distribution of α-actinin in membrane projections. This is the first description of α-actinin and flotillin-1 in lipid rafts isolated from OECs and suggests that, together with ß1-integrin and gangliosides, membrane lipid rafts play a role during OEC migration. This study provides new information on the molecular composition of OEC membrane microdomains that can impact on our understanding of the role of OEC lipid rafts under physiological and pathological conditions of the nervous system, including inflammation, hypoxia, aging, neurodegenerative diseases, head trauma, brain tumor, and infection.

A Balancing Act: The Immune System Supports Neurodegeneration and Neurogenesis.

Decapod crustaceans, like mammals, retain the ability to make new neurons throughout life. In mammals, immune cells are closely associated with stem cells that generate adult-born neurons. In crayfish, evidence suggests that immune cells (hemocytes) originating in the immune system travel to neurogenic regions and transform into neural progenitor cells. This nontraditional immune activity takes place continuously under normal physiological conditions, but little is known under pathological conditions (neurodegeneration). In this study, the immune system and its relationship with neurogenesis were investigated during neurodegeneration (unilateral antennular ablation) in adult crayfish. Our experiments show that after ablation (1) Proliferating cells decrease in neurogenic areas of the adult crayfish brain; (2) The immune response, but not neurogenesis, is ablation-side dependent; (3) Inducible nitric oxide synthase (iNOS) plays a crucial role in the neurogenic niche containing neural progenitors during the immune response; (4) Brain areas targeted by antennular projections respond acutely (15 min) to the lesion, increasing the number of local immune cells; (5) Immune cells are recruited to the area surrounding the ipsilateral neurogenic niche; and (6) The vasculature in the niche responds acutely by dilation and possibly also neovascularization. We conclude that immune cells are important in both neurodegeneration and neurogenesis by contributing in physiological conditions to the maintenance of the number of neural precursor cells in the neurogenic niche (neurogenesis), and in pathological conditions (neurodegeneration) by coordinating NO release and vascular responses associated with the neurogenic niche. Our data suggest that neural damage and recovery participate in a balance between these competing immune cell roles.

The Neurotropic Parasite Toxoplasma gondii Induces Sustained Neuroinflammation with Microvascular Dysfunction in Infected Mice.

Toxoplasmosis is one of the leading parasitic diseases worldwide. Some data suggest that chronic acquired toxoplasmosis could be linked to behavioral alterations in humans. The parasite infects neurons, forming immunologically silent cysts. Cerebral microcirculation homeostasis is determinant to brain functions, and pathologic states can alter capillarity or blood perfusion, leading to neurodegeneration and cognitive deficits. Albino mice were infected with Toxoplasma gondii (ME49 strain) and analyzed after 10, 40, and 180 days. Infected mice presented decreased cerebral blood flow at 10 and 40 days post infection (dpi), which were restored at 180 dpi, as shown by laser speckle contrast imaging. Intravital microscopy demonstrated that infection led to significant capillary rarefaction, accompanied by neuroinflammation, with microglial activation and increased numbers of rolling and adherent leukocytes to the wall of cerebral capillaries. Acetylcholine-induced vasodilation was altered at all time points, and blood brain barrier permeability was evident in infected animals at 40 dpi. Infection reduced angiogenesis, with a decreased number of isolectin B4-stained blood vessels and a decrease in length and branching of laminin-stained capillaries. Sulfadiazine reduced parasite load and partially repaired microvascular damages. We conclude that T. gondii latent infection causes a harmful insult in the brain, promoting neuroinflammation and microcirculatory dysfunction in the brain, with decreased angiogenesis and can contribute to a neurodegenerative process.

A role for gangliosides and ß1-integrin in the motility of olfactory ensheathing glia.

Olfactory ensheathing glia (OEG) are found in the olfactory mucosa, nerve and bulb, and provide in vivo ensheathment for the unmyelinated olfactory axons within the central and peripheral nervous system domains. OEG cells are able to migrate long distances within the neuropil of the central nervous system. Because gangliosides such as 9-O-acetyl GD3 have crucial regulatory roles in neuronal migration during development, we analyzed whether OEG in organotypical cultures are revealed by anti-9-O-acetyl GD3 and/or gangliosides are recognized by the A2B5 antibody (G-A2B5), and whether these gangliosides are involved in OEG migration. Our results showed that all OEG migrating out of a section of olfactory bulb onto a laminin substrate bound to the 9-O-acetyl GD3 and A2B5 antibodies, and that 2',3'-cyclic nucleotide phosphodiesterase (CNPase) colocalized with 9-O-acetyl GD3 and with G-A2B5. Additionally, we showed that the immune blockade of 9-O-acetyl GD3 or G-A2B5 reduced the migration of OEG on laminin, and that 9-O-acetyl GD3 and G-A2B5 colocalized with the ß1-integrin subunit. We also confirmed the phenotype of in-vitro-grown OEG cells derived from adult rats, showing that they express CNPase, and also α-smooth muscle actin, which is not expressed by Schwann cells. Our data showed that the gangliosides 9-O-acetyl GD3 and G-A2B5 participate in the migratory activity of OEG cells, and that the ß1-integrin subunit colocalizes with these gangliosides. These results suggest a new role for ß1-integrin and gangliosides in the polarized migration of OEG cells, and provide new information on the molecules controlling OEG motility and behavior.

Catecholamines are produced by ascidian immune cells: The involvement of PKA and PKC in the adrenergic signaling pathway.

The stress response is a complex mechanism, which includes changes in the immune system to enable organisms to maintain homeostasis. The neurohormones dopamine, noradrenaline (NA) and adrenalin are responsible for the physiological modulations that occur during acute stress. In the present study, we analyzed the effects of NA on the immune system specific to nitric-oxide (NO) production by subpopulations of immune cells (hemocytes) of the ascidian Phallusia nigra. We also investigated the capability of immune cells to produce catecholamine (CA). Finally, we tested the involvement of protein kinase A (PKA) and C (PKC) in the NA downstream signaling pathway. The results revealed that NA can reduce NO production by P. nigra hemocytes threefold, and that signet-ring cells, univacuolar refractile granulocytes and morula cells are the cell types most involved in this event. A challenge effected with Zymosan A induced CA production, and co-incubation with both inhibitors of the second messengers PKA and PKC revealed the involvement of these molecules in the adrenergic pathway of P. nigra hemocytes. Taken together, these results suggest that NO production can be down-regulated by NA through α- and ß-adrenoceptors via the second messengers PKA and PKC.

Developmental effects of exposure to ultraviolet B radiation on the freshwater prawn Macrobrachium olfersi: Mitochondria as a target of environmental UVB radiation.

In South America, increased UVB radiation has become an important environmental issue that is potentially threatening aquatic ecosystems. Considering that species exhibit different degrees of sensitivity to UVB radiation and that embryos are more sensitive than organisms at later life stages, the aim of this study was to characterize the effects of UVB radiation on subcellular compartments of embryos of the freshwater prawn Macrobrachium olfersi. This species lives and reproduces in clear and shallow waters, where UV radiation can fully penetrates. Embryos were irradiated with a UVB 6W lamp for 30min and examined after 1h, 12h, 24h and 48h of exposure. The irradiance of the UVB used simulates the UV radiation that embryos receive in the natural environment. The subcellular compartment most affected by the UVB radiation was the mitochondria, which exhibited a circular shape, a decrease in mitochondrial cristae, rupture of membranes and a morphology compatible with fission. These impairments were observed simultaneously with increased ROS production, just after 1h of UVB exposure. Thus, we investigated proteins related to mitochondrial fission (Drp-1) and fusion (Mfn-1), which are essential to cell maintenance. We found a significant increase in Drp-1 expression at all analyzed time-points and a significant decrease in Mfn-1 expression only after 24h of UVB exposure. Additionally, a decrease in embryonic cell viability was verified via the mitochondrial integrity assay. To conclude, we observed important mitochondrial dysfunctions against the environmental stress caused by UVB radiation. Moreover, the cellular responses found are critical and should not be disregarded, because they impact embryos that can potentially compromise the aquatic ecosystems.

Selective stimulatory action of olfactory ensheathing glia-conditioned medium on oligodendroglial differentiation, with additional reference to signaling mechanisms.

We examined the effects of conditioned medium from olfactory ensheathing glia (OEGCM) on the differentiation of oligodendrocytes in mixed cultures of early postnatal hippocampi. Differentiation was judged from the numerical density (ND) of cells immunoreactive to 2'3' cyclic nucleotide 3'phosphodiesterase (CNPase) and O4 antibodies. NDs increased according to inverted-U dose-response curves, particularly for CNPase+ cells (9-fold at optimal dilution) and these changes were blocked by inhibitors of ERK1, p38-MAPK, and PI3K. Our results raise the possibility that OEG secreted factor(s) may counteract demyelination induced by trauma, neurodegenerative diseases, and advanced age, and should stimulate novel methods to deliver these factors and/or potentiating chemicals.

Vascular endothelial growth factor-like and its receptor in a crustacean optic ganglia: a role in neuronal differentiation?

The neural system appears before the vascular system in the phylogenetic tree. During evolution, vascular system generation takes advantage of the pre-existing vascular endothelial growth factor (VEGF) in order to form its networks. Nevertheless, the role of VEGF in neuronal and glial cells is not yet completely understood. In order to support the hypothesis of a neural role for VEGF, we searched for VEGF- and VEGF receptor (VEGFR)-like immunoreactivities (immunohisto/cytochemistry and Western blotting) in the eyestalk of the invertebrate Ucides cordatus (Crustacea, Brachyura, Ucididae). Our results showed that both neurons and glial cells expressed VEGF-immunoreactivity, and that VEGFR was evidenced in neural cells. This is the first report about the VEGF/VEGFR-like immunoreactivities in the nervous tissue of a crustacean, and enables U. cordatus to be included in the repertoire of animal models used for ascertaining the role of VEGF in the nervous system.

Evidence of involvement of the mannose receptor in the internalization of Streptococcus pneumoniae by Schwann cells.

BACKGROUND: The ability of S. pneumoniae to generate infections depends on the restrictions imposed by the host's immunity, in order to prevent the bacterium from spreading from the nasopharynx to other tissues, such as the brain. Some authors claim that strains of S. pneumoniae, which fail to survive in the bloodstream, can enter the brain directly from the nasal cavity by axonal transport through the olfactory and/or trigeminal nerves. However, from the immunological point of view, glial cells are far more responsive to bacterial infections than are neurons. This hypothesis is consistent with several recent reports showing that bacteria can infect glial cells from the olfactory bulb and trigeminal ganglia. Since our group previously demonstrated that Schwann cells (SCs) express a functional and appropriately regulated mannose receptor (MR), we decided to test whether SCs are involved in the internalization of S. pneumoniae via MR. RESULTS: Immediately after the interaction step, as well as 3 h later, the percentage of association was approximately 56.5%, decreasing to 47.2% and 40.8% after 12 and 24 h, respectively. Competition assays by adding a 100-fold excess of mannan prior to the S. pneumoniae infection reduced the number of infected cells at 3 and 24 h. A cytochemistry assay with Man/BSA-FITC binding was performed in order to verify a possible overlap between mannosylated ligands and internalized bacteria. Incubation of the SCs with Man/BSA-FITC resulted in a large number of intracellular S. pneumoniae, with nearly complete loss of the capsule. Moreover, the anti-pneumococcal antiserum staining colocalized with the internalized man/BSA-FITC, suggesting that both markers are present within the same endocytic compartment of the SC. CONCLUSIONS: Our data offer novel evidence that SCs could be essential for pneumococcal cells to escape phagocytosis and killing by innate immune cells. On the other hand, the results also support the idea that SCs are immunocompetent cells of the PNS that can mediate an efficient immune response against pathogens via MR.

Regulation of nitric-oxide production in hemocytes of the ascidian Phallusia nigra.

Nitric oxide (NO) production in ascidians is related to immune responses of blood cells, and also to events such as egg fertilization and notochord regression. However, the signaling pathway for NO generation has been little investigated in this animal model. The present contribution identifies the cells involved in NO production and provides new information about a pathway for NO signaling. We were able to identify eight types of blood cells in the hemolymph of the ascidian Phallusia nigra, of which signet ring cells, univacuolar refractile granulocytes, and morula cells were involved in NO production. Zymosan A and lipopolysaccharide (LPS) enhanced NO production by blood cells, and the compound N-nitro-L-arginine-methyl ester (L-NAME) reduced NO production. Finally, the application of protein kinase A (PKA) and protein kinase C (PKC) inhibitors revealed that these molecules participate, together with NFκB, in the regulation of NO production by blood cells of P. nigra. This is the first report to show that PKA and PKC are involved in a signaling pathway that leads to NO production in ascidian blood cells.

Melatonin as a signaling molecule for metabolism regulation in response to hypoxia in the crab Neohelice granulata.

Melatonin has been identified in a variety of crustacean species, but its function is not as well understood as in vertebrates. The present study investigates whether melatonin has an effect on crustacean hyperglycemic hormone (CHH) gene expression, oxygen consumption (VO2) and circulating glucose and lactate levels, in response to different dissolved-oxygen concentrations, in the crab Neohelice granulata, as well as whether these possible effects are eyestalk- or receptor-dependent. Melatonin decreased CHH expression in crabs exposed for 45 min to 6 (2, 200 or 20,000 pmol·crab-1) or 2 mgO2·L-1 (200 pmol·crab-1). Since luzindole (200 nmol·crab-1) did not significantly (p > 0.05) alter the melatonin effect, its action does not seem to be mediated by vertebrate-typical MT1 and MT2 receptors. Melatonin (200 pmol·crab-1) increased the levels of glucose and lactate in crabs exposed to 6 mgO2·L-1, and luzindole (200 nmol·crab-1) decreased this effect, indicating that melatonin receptors are involved in hyperglycemia and lactemia. Melatonin showed no effect on VO2. Interestingly, in vitro incubation of eyestalk ganglia for 45 min at 0.7 mgO2·L-1 significantly (p < 0.05) increased melatonin production in this organ. In addition, injections of melatonin significantly increased the levels of circulating melatonin in crabs exposed for 45 min to 6 (200 or 20,000 pmol·crab-1), 2 (200 and 20,000 pmol·crab-1) and 0.7 (200 or 20,000 pmol·crab-1) mgO2·L-1. Therefore, melatonin seems to have an effect on the metabolism of N. granulata. This molecule inhibited the gene expression of CHH and caused an eyestalk- and receptor-dependent hyperglycemia, which suggests that melatonin may have a signaling role in metabolic regulation in this crab.

Neuroprotective Effects of Metformin Through AMPK Activation in a Neurotoxin-Based Model of Cerebellar Ataxia.

Cerebellar ataxia is a heterogeneous group of neural disorders clinically characterized by cerebellar dysfunction. The diagnosis of patients with progressive cerebellar ataxia is complex due to the direct correlation with other neuron diseases. Although there is still no cure for this pathological condition, some metabolic, hereditary, inflammatory, and immunological factors affecting cerebellar ataxia are being studied and may become therapeutic targets. Advances in studying the neuroanatomy, pathophysiology, and molecular biology of the cerebellum (CE) contribute to a better understanding of the mechanisms behind the development of this disorder. In this study, Wistar rats aged 30 to 35 days were injected intraperitoneally with 3-acetylpyridine (3-AP) and/or metformin (for AMP-activated protein kinase (AMPK) enzyme activation) and euthanized in 24 hours and 4 days after injection. We analyzed the neuromodulatory role of the AMPK on cerebellar ataxia induced by the neurotoxin 3-AP in the brain stem (BS) and CE, after pre-treatment for 7 and 15 days with metformin, a pharmacological indirect activator of AMPK. The results shown here suggest that AMPK activation in the BS and CE leads to a significant reduction in neuroinflammation in these regions. AMPK was able to restore the changes in fatty acid composition and pro-inflammatory cytokines caused by 3-AP, suggesting that the action of AMPK seems to result in a possible neuroprotection on the cerebellar ataxia model.

The mannose receptor is expressed by olfactory ensheathing cells in the rat olfactory bulb.

Complex carbohydrate structures are essential molecules of infectious bacteria, parasites, and host cells and are involved in cell signaling associated with immune responses, glycoprotein homeostasis, and cell migration. The uptake of mannose-tailed glycans is usually carried out by professional phagocytes to trigger MHC class I- and MHC class II-restricted antigen presentation or, alternatively, to end inflammation. We have detected the mannose receptor (MR) in cultured olfactory ensheathing cells (OECs), so we investigated by flow cytometry whether recently dissociated cells of the olfactory bulb (OB) nerve fiber layer (ONL) could bind a mannosylated ligand (fluorescein conjugate of mannosyl bovine serum albumin; Man/BSA-FITC) in a specific manner. In addition, we estimated the relative proportion of ONL OECs, microglia, and astrocytes, tagged by 2'3'-cyclic nucleotide 3'-phosphodiesterase (CNPase), by the B4 isolectin of Griffonia simplicifonia (IB4), and by glial fibrillary acidic protein (GFAP), respectively, that were Man/BSA-FITC(+) . We also determined by histochemistry and/or immunohistochemistry whether Man/BSA-FITC or an anti-MR antibody (anti-C-terminal MR peptide; anti-cMR) labeled OECs and/or parenchymal microglia. In addition, we confirmed by Western blot with the K1K2 (against the entire MR molecule) antibody that a band of about 180 kDA is expressed in the OB. Our findings are compatible with a prospective sentinel role of OECs against pathogens of the upper airways and/or damage-associated glycidic patterns as well as with homeostasis of OB mannosylated glycoproteins.

Activation of cannabinoid type 1 receptor (CB1) modulates oligodendroglial process branching complexity in rat hippocampal cultures stimulated by olfactory ensheathing glia-conditioned medium.

The endocannabinoid system (ECS) refers to a complex cell-signaling system highly conserved among species formed by numerous receptors, lipid mediators (endocannabinoids) and synthetic and degradative enzymes. It is widely distributed throughout the body including the CNS, where it participates in synaptic signaling, plasticity and neurodevelopment. Besides, the olfactory ensheathing glia (OEG) present in the olfactory system is also known to play an important role in the promotion of axonal growth and/or myelination. Therefore, both OEG and the ECS promote neurogenesis and oligodendrogenesis in the CNS. Here, we investigated if the ECS is expressed in cultured OEG, by assessing the main markers of the ECS through immunofluorescence, western blotting and qRT-PCR and quantifying the content of endocannabinoids in the conditioned medium of these cells. After that, we investigated whether the production and release of endocannabinoids regulate the differentiation of oligodendrocytes co-cultured with hippocampal neurons, through Sholl analysis in oligodendrocytes expressing O4 and MBP markers. Additionally, we evaluated through western blotting the modulation of downstream pathways such as PI3K/Akt/mTOR and ERK/MAPK, being known to be involved in the proliferation and differentiation of oligodendrocytes and activated by CB1, which is the major endocannabinoid responsive receptor in the brain. Our data show that OEG expresses key genes of the ECS, including the CB1 receptor, FAAH and MAGL. Besides, we were able to identify AEA, 2-AG and AEA related mediators palmitoylethanolamide (PEA) and oleoylethanolamide (OEA), in the conditioned medium of OEG cultures. These cultures were also treated with URB597 10-9 M, a FAAH selective inhibitor, or JZL184 10-9 M, a MAGL selective inhibitor, which led to the increase in the concentrations of OEA and 2-AG in the conditioned medium. Moreover, we found that the addition of OEG conditioned medium (OEGCM) enhanced the complexity of oligodendrocyte process branching in hippocampal mixed cell cultures and that this effect was inhibited by AM251 10-6 M, a CB1 receptor antagonist. However, treatment with the conditioned medium enriched with OEA or 2-AG did not alter the process branching complexity of premyelinating oligodendrocytes, while decreased the branching complexity in mature oligodendrocytes. We also observed no change in the phosphorylation of Akt and ERK 44/42 in any of the conditions used. In conclusion, our data show that the ECS modulates the number and maturation of oligodendrocytes in hippocampal mixed cell cultures.

Norepinephrine depresses the nitric oxide production in the ascidian hemocytes.

Norepinephrine (NE) is a neuro-hormone released by vertebrates and invertebrates during acute stress, and can influence their immune function. We found that NE depressed the production of nitric oxide (NO) by the hemocytes of ascidians. Our results with a fluorescent indicator for NO in assays using both NE and either α or ß-antagonist revealed that NE down-regulated NO production by the ascidian hemocytes. Our data suggest that NE may be acting via specific hemocyte receptors to induce a decrease in immune function.