Dye Destaining Research Articles

Mast Cell Changes the Phenotype of Microglia via Histamine and ATP

Microglia are the dynamic motile phagocytes of the brain considered the first line of defense against threats or disturbances to the Central Nervous System (CNS). Microglia help orchestrate the immunological response by interacting with others immune cells. Mast cells (MCs) are effector cells of the innate immune system distributed in all organs and vascularized tissues, brain included. Several molecular mechanisms for potential interactions between MCs and microglia have been determined. However, the effect of MCs on regulated exocytosis and phagocytic clearance in microglia has not been explored. Cocktails of MCs mediators (MCM) obtained at 37°C and 53°C were used to induce microglia activation. Changes in intracellular calcium [Ca2+]i and ATP release were studied by calcium and quinacrine fluorescence imaging. Fluorescent latex beads were used to assay phagocytosis in microglia after MCM treatment and compared to that measured in the presence of histamine, ATP and lipopolysaccharide (LPS). Iba-1 expression and area were quantified by immunofluorescence and histamine levels evaluated by ELISA techniques. Local application onto microglia of the MC mediator cocktail elicited Ca2+ transients and exocytotic release associated with quinacrine dye de-staining. Ca2+ signals were mimicked by histamine and blocked by the H1 receptor (H1R) antagonist, cetirizine. Hydrolysis of ATP by apyrase also affected Ca2+ transients to a lesser extent. Iba-1 fluorescence, cell area and phagocytosis were enhanced by histamine through H1R. However, ATP prevented iba-1 expression and microglial phagocytosis. MCM showed combined effects of histamine and ATP, increasing the number of internalized microbeads per cell and area without raising iba1 expression. Our results highlight the relevance of MC-derived histamine and ATP in the modulation of secretory and phagocytic activities that would explain the heterogeneity of microglial responses in different pathological contexts.

Neopestalotiopsis species presenting wide dye destaining activity: report of a mycelium-associated laccase

Wastewaters from textile dyeing industries represent an ecological concern, notably due to the known toxicity of azo dyes to the local microbiome and human health. Although physicochemical approaches are the rule for the treatment of industrial effluents, biological strategies such as enzyme-mediated dye destaining is a promising alternative. Notwithstanding a broad range of microorganisms, including fungi, algae, yeast, and bacteria, display dye-destaining properties, most of the literature has focused in ligninolytic fungi, leaving other classes of organisms somehow ignored. In this study, six endophytic strains isolated from Maytenus ilicifolia were studied for their destaining activity. The phylogenetic and morphological analysis allowed the identification of strain LGMF1504 as Neopestalotiopsis sp. LGMF1504 that decolorized several commercial dyes as the result of a mycelium-associated laccase. The enzyme expression was modulated by carbon and nitrogen content in the culture medium, it was weakly affected by the presence of aromatic compounds and metal ions while some common laccase mediators improved the destaining activity onto dye substrates. The best culture condition observed for laccase activity was a basic culture medium containing 5 g L−1 starch and 15 g L−1 ammonium tartrate. The laccase activity showed low substrate specificity and almost unaltered performance in a wide range of pH values and NaCl concentrations, suggesting the potential of Neopestalotiopsis sp. LGMF1504 for biodegradation approaches.

Proteoglycans involved in bidirectional communication between mast cells and hippocampal neurons

BackgroundMast cells (MCs) in the brain can respond to environmental cues and relay signals to neurons that may directly influence neuronal electrical activity, calcium signaling, and neurotransmission. MCs also express receptors for neurotransmitters and consequently can be activated by them. Here, we developed a coculture model of peritoneal MCs, incubated together with dissociated hippocampal neurons for the study of cellular mechanisms involved in the mast cell-neuron interactions.MethodsCalcium imaging was used to simultaneously record changes in intracellular calcium [Ca2+]i in neurons and MCs. To provide insight into the contribution of MCs on neurotransmitter release in rat hippocampal neurons, we used analysis of FM dye release, evoked by a cocktail of mediators from MCs stimulated by heat.ResultsBidirectional communication is set up between MCs and hippocampal neurons. Neuronal depolarization caused intracellular calcium [Ca2+]i oscillations in MCs that produced a quick response in neurons. Furthermore, activation of MCs with antigen or the secretagogue compound 48/80 also resulted in a neuronal [Ca2+]i response. Moreover, local application onto neurons of the MC mediator cocktail elicited Ca2+ transients and a synaptic release associated with FM dye destaining. Neuronal response was partially blocked by D-APV, a N-methyl-D-aspartate receptor (NMDAR) antagonist, and was inhibited when the cocktail was pre-digested with chondroitinase ABC, which induces enzymatic removal of proteoglycans of chondroitin sulfate (CS).ConclusionsMC-hippocampal neuron interaction affects neuronal [Ca2+]i and exocytosis signaling through a NMDAR-dependent mechanism.

BDNF enhances spontaneous and activity-dependent neurotransmitter release at excitatory terminals but not at inhibitory terminals in hippocampal neurons.

Brain-derived neurotrophic factor (BDNF) is widely reported to enhance synaptic vesicle (SV) exocytosis and neurotransmitter release. But it is still unclear whether BDNF enhances SV recycling at excitatory terminals only, or at both excitatory and inhibitory terminals. In the present study, in a direct comparison using cultured rat hippocampal neurons, we demonstrate that BDNF enhances both spontaneous and activity-dependent neurotransmitter release from excitatory terminals, but not from inhibitory terminals. BDNF treatment for 5 min or 48 h increased both spontaneous and activity-induced anti-synaptotagmin1 (SYT1) antibody uptake at excitatory terminals marked with vGluT1. Conversely, BDNF treatment did not enhance spontaneous or activity-induced uptake of anti-SYT1 antibodies in inhibitory terminals marked with vGAT. Time-lapse imaging of FM1-43 dye destaining in excitatory and inhibitory terminals visualized by post-hoc immunostaining of vGluT1 and vGAT also showed the same result: The rate of spontaneous and activity-induced destaining was increased by BDNF at excitatory synapses, but not at inhibitory synapses. These data demonstrate that BDNF enhances SV exocytosis in excitatory but not inhibitory terminals. Moreover, BDNF enhanced evoked SV exocytosis, even if vesicles were loaded under spontaneous vesicle recycling conditions. Thus, BDNF enhances both spontaneous and activity-dependent neurotransmitter release on both short and long time-scales, by the same mechanism.

Multiple Targets of μ-Opioid Receptor-Mediated Presynaptic Inhibition at Primary Afferent Aδ- and C-Fibers

Agonists at μ-opioid receptors (MORs) represent the gold standard for the treatment of severe pain. A key element of opioid analgesia is the depression of nociceptive information at the first synaptic relay in spinal pain pathways. The underlying mechanisms are, however, largely unknown. In spinal cord slices with dorsal roots attached prepared from young rats, we determined the inhibitory effect of the selective MOR agonist [d-Ala(2), N-Me-Phe(4), Gly(5)-ol]-enkephalin (DAMGO) on monosynaptic Aδ- and C-fiber-evoked EPSCs in lamina I neurons. DAMGO depressed presynaptically Aδ- and C-fiber-mediated responses, indicating that MORs are expressed on central terminals of both fiber types. We next addressed the mechanisms of presynaptic inhibition. The effect of DAMGO at both Aδ- and C-fiber terminals was mainly mediated by an inhibition of N-type voltage-dependent Ca(2+) channels (VDCCs), and to a lesser extent of P/Q-type VDCCs. Inhibition by DAMGO was not reduced by K(+) channel blockers. The rate of miniature EPSCs was reduced by DAMGO in a dose-dependent manner. The opioid also reduced Ca(2+)-dependent, ionomycin-induced EPSCs downstream of VDCCs. DAMGO had no effect on the kinetics of vesicle exocytosis in C-fiber terminals, but decreased the rate of unloading of Aδ-fiber boutons moderately, as revealed by two-photon imaging of styryl dye destaining. Together, these results suggest that binding of opioids to MORs reduces nociceptive signal transmission at central Aδ- and C-fiber synapses mainly by inhibition of presynaptic N-type VDCCs. P/Q-type VDCCs and the transmitter release machinery are targets of opioid action as well.

Functional analysis of calcium channel-mediated exocytosis in synaptic terminals by FM imaging technique

Presynaptic voltage-gated Ca(2+) channels mediate rapid Ca(2+) influx into the synaptic terminal which triggers synaptic vesicle exocytosis and neurotransmitter release. The FM 1-43 dye was firstly introduced as a fluorescence probe by Betz and his colleagues in 1992, and has been used to monitor exocytosis, endocytosis and endosomal traffic in a variety of cell types. The present study aims to investigate the feasibility of applying the FM 1-43 dye in the functional analysis of calcium channel-mediated exocytosis in synaptic terminals. The hippocampi were isolated from embryos of pregnant rats, and hippocampal neurons were then transfected with Ds-Red conjugated plasmid. The neurons were then loaded with 8 micromol/L FM 1-43 and 47 mmol/L KCl for 90 s after transfection. After that, 90 mmol/L KCl was employed to induce FM dye destaining, which was recorded by FM imaging system. The neuron synaptic terminals of rat hippocampus could be effectively stained by the FM 1-43 dye. Besides, the destaining of the labeled neuron terminals was in accordance with the transmitter release, which could be blocked by the application of nifedipine (inhibitor for L-type calcium channel). The FM imaging technique is an advanced and effective method for analyzing synaptic vesicle exocytosis and neurotransmitter release, and can be applied in various synaptic functional studies.

Frequency-Dependent Kinetics and Prevalence of Kiss-and-Run and Reuse at Hippocampal Synapses Studied with Novel Quenching Methods

The kinetics of exo-endocytotic recycling could restrict information transfer at central synapses if neurotransmission were entirely reliant on classical full-collapse fusion. Nonclassical fusion retrieval by kiss-and-run would be kinetically advantageous but remains controversial. We used a hydrophilic quencher, bromophenol blue (BPB), to help detect nonclassical events. Upon stimulation, extracellular BPB entered synaptic vesicles and quenched FM1-43 fluorescence, indicating retention of FM dye beyond first fusion. BPB also quenched fluorescence of VAMP (synaptobrevin-2)-EGFP, thus indicating the timing of first fusion of vesicles in the total recycling pool. Comparison with FM dye destaining revealed that kiss-and-run strongly prevailed over full-collapse fusion at low frequency, giving way to a near-even balance at high frequency. Quickening of kiss-and-run vesicle reuse was also observed at higher frequency in the average single vesicle fluorescence response. Kiss-and-run and reuse could enable hippocampal nerve terminals to conserve scarce vesicular resources when responding to widely varying input patterns.

Fast Vesicle Recycling Supports Neurotransmission during Sustained Stimulation at Hippocampal Synapses

High-frequency induced short-term synaptic depression is a common feature of central synapses in which synaptic responses rapidly decrease to a sustained level. A limitation in the availability of release-ready vesicles is thought to be a major factor underlying this phenomenon. Here, we studied the kinetics of vesicle reavailability and reuse during synaptic depression at hippocampal synapses. High-intensity stimulation of neurotransmitter release was induced by hyperosmolarity, high potassium, or action potential firing at 30 Hz to produce synaptic depression. Under these conditions, synaptic transmission rapidly depressed to a plateau level that was typically 10-40% of the initial response and persisted at this level for at least 5 min regardless of the developmental stage of synapses. This nondeclining phase of transmission was partly sustained by fast recycling and reuse of synaptic vesicles even after minutes of stimulation. Simultaneous electrical recording of postsynaptic responses and styryl dye destaining showed that after an initial round of exocytosis, vesicles were available for reuse with a delay between 1 and 3 sec during 30 Hz action potential or hypertonicity-induced stimulation. During these stimulation paradigms, there was a limited mobilization of vesicles from the reserve pool. During 10 Hz stimulation, however, the extent of vesicle reuse was minimal during the first 20 sec. These results suggest a role for fast vesicle recycling as a functional homeostatic mechanism that prevents vesicle depletion and maintains synaptic responses in the face of intense stimulation.