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Journal of Pharmacological Sciences Mar 2017Decreased brain glutamate level has emerged as a new therapeutic approach for epilepsy. This study investigated the effect and mechanism of amiodarone, an...
Decreased brain glutamate level has emerged as a new therapeutic approach for epilepsy. This study investigated the effect and mechanism of amiodarone, an anti-arrhythmic drug with antiepileptic activity, on glutamate release in the rat hippocampus. In a synaptosomal preparation, amiodarone reduced 4-aminopyridine-evoked Ca-dependent glutamate release and cytosolic Ca concentration elevation. Amiodarone did not affect the 4-aminopyridine-evoked depolarization of the synaptosomal membrane potential or the Na channel activator veratridine-evoked glutamate release, indicating that the amiodarone-mediated inhibition of glutamate release is not caused by a decrease in synaptosomal excitability. The inhibitory effect of amiodarone on 4-aminopyridine-evoked glutamate release was markedly decreased in synaptosomes pretreated with the Ca2.2 (N-type) and Ca2.1 (P/Q-type) channel blocker ω-conotoxin MVIIC, the calmodulin antagonists W7 and calmidazolium, or the protein kinase A inhibitors H89 and KT5720. However, the intracellular Ca-release inhibitors dantrolene and CGP37157 had no effect on the amiodarone-mediated inhibition of glutamate release. Furthermore, amiodarone reduced the frequency of miniature excitatory postsynaptic currents without affecting their amplitude in hippocampal slices. Our data suggest that amiodarone reduces Ca influx through N- and P/Q-type Ca channels, subsequently reducing the Ca-calmodulin/protein kinase A cascade to inhibit the evoked glutamate release from rat hippocampal nerve terminals.
Topics: 4-Aminopyridine; Amiodarone; Animals; Anti-Arrhythmia Agents; Aspartic Acid; Calcium; Calcium Channel Blockers; Calmodulin; Capsaicin; Carbazoles; Glutamic Acid; Hippocampus; Imidazoles; Isoquinolines; Macrolides; Male; Membrane Potentials; Protein Kinase Inhibitors; Pyrroles; Rats, Sprague-Dawley; Sulfonamides; Synaptosomes; omega-Conotoxins
PubMed: 28330759
DOI: 10.1016/j.jphs.2017.02.014 -
Mitochondrion Nov 2023Mitochondrial function at synapses can be assessed in isolated nerve terminals. Synaptosomes are structures obtained in vitro by detaching the nerve endings from...
Mitochondrial function at synapses can be assessed in isolated nerve terminals. Synaptosomes are structures obtained in vitro by detaching the nerve endings from neuronal bodies under controlled homogenization conditions. Several protocols have been described for the preparation of intact synaptosomal fractions. Herein a fast and economical method to obtain synaptosomes with optimal intrasynaptic mitochondria functionality was described. Synaptosomal fractions were obtained from mouse brain cortex by differential centrifugation followed by centrifugation in a Ficoll gradient. The characteristics of the subcellular particles obtained were analyzed by flow cytometry employing specific tools. Integrity and specificity of the obtained organelles were evaluated by calcein and SNAP-25 probes. The proportion of positive events of the synaptosomal preparation was 75 ± 2 % and 48 ± 7% for calcein and Synaptosomal-Associated Protein of 25 kDa (SNAP-25), respectively. Mitochondrial integrity was evaluated by flow cytometric analysis of cardiolipin content, which indicated that 73 ± 1% of the total events were 10 N-nonylacridine orange (NAO)-positive. Oxygen consumption, ATP production and mitochondrial membrane potential determinations showed that mitochondria inside synaptosomes remained functional after the isolation procedure. Mitochondrial and synaptosomal enrichment were determined by measuring synaptosomes/ homogenate ratio of specific markers. Functionality of synaptosomes was verified by nitric oxide detection after glutamate addition. As compared with other methods, the present protocol can be performed briefly, does not imply high economic costs, and provides an useful tool for the isolation of a synaptosomal preparation with high mitochondrial respiratory capacity and an adequate integrity and function of intraterminal mitochondria.
Topics: Mice; Animals; Synaptosomes; Mitochondria; Energy Metabolism; Brain; Cerebral Cortex
PubMed: 37944836
DOI: 10.1016/j.mito.2023.10.002 -
Neuroscience Jul 2019Astrocytes regulate extracellular glutamate homeostasis in the central nervous system through the Na-dependent glutamate transporters glutamate transporter-1 (GLT-1) and...
Astrocytes regulate extracellular glutamate homeostasis in the central nervous system through the Na-dependent glutamate transporters glutamate transporter-1 (GLT-1) and glutamate aspartate transporter (GLAST). Impaired astrocyte glutamate uptake could contribute to the development of epilepsy but the regulation of glutamate transporters in epilepsy is not well understood. In this study, we investigate the expression of GLT-1 and GLAST in the mouse intrahippocampal kainic acid (IHKA) model of temporal lobe epilepsy (TLE). We used immunohistochemistry, synaptosomal fractionation and Western blot analysis at 1, 3, 7 and 30 days post-IHKA induced status epilepticus (SE) to examine changes in GLT-1 and GLAST immunoreactivity and synaptosomal expression during the development of epilepsy. We found a significant upregulation in GLT-1 immunoreactivity at 1 and 3 days post-IHKA in the ipsilateral dorsal hippocampus. However, GLT-1 immunoreactivity and synaptosomal protein levels were significantly downregulated at 7 days post-IHKA in the ipsilateral hippocampus, a time point corresponding to the onset of spontaneous seizures in this model. GLAST immunoreactivity was increased in specific layers at 1 and 3 days post-IHKA in the ipsilateral hippocampus. GLAST synaptosomal protein levels were significantly elevated at 30 days compared to 7 days post-IHKA in the ipsilateral hippocampus. Our findings suggest that astrocytic glutamate transporter dysregulation could contribute to the development of epilepsy.
Topics: Animals; Astrocytes; Disease Models, Animal; Epilepsy, Temporal Lobe; Excitatory Amino Acid Transporter 1; Excitatory Amino Acid Transporter 2; Hippocampus; Kainic Acid; Mice; Seizures; Synaptosomes
PubMed: 31158434
DOI: 10.1016/j.neuroscience.2019.05.048 -
Oxidative Medicine and Cellular... 2016Disruption of cellular redox homeostasis is implicated in a wide variety of pathologic conditions and aging. A fundamental factor that dictates such balance is the ratio... (Comparative Study)
Comparative Study
Disruption of cellular redox homeostasis is implicated in a wide variety of pathologic conditions and aging. A fundamental factor that dictates such balance is the ratio between mitochondria-mediated complete oxygen reduction into water and incomplete reduction into superoxide radical by mitochondria and NADPH oxidase (NOX) enzymatic activity. Here we determined mitochondrial as well as NOX-dependent rates of oxygen consumption in parallel with HO generation in freshly isolated synaptosomes using high resolution respirometry combined with fluorescence or electrochemical sensory. Our results indicate that although synaptic mitochondria exhibit substantially higher respiratory activities (8-82-fold greater than NOX oxygen consumption depending on mitochondrial respiratory state), NADPH-dependent oxygen consumption is associated with greater HO production (6-7-fold higher NOX-HO). We also show that, in terms of the consumed oxygen, while synaptic mitochondria "leaked" 0.71% ± 0.12 HO during NAD-linked resting, 0.21% ± 0.04 during NAD-linked active respiration, and 0.07% ± 0.02 during FAD-linked active respiration, NOX converted 38% ± 13 of O into HO. Our results indicate that NOX rather than mitochondria is the major source of synaptic HO. The present approach may assist in the identification of redox-modulating synaptic factors that underlie a variety of physiological and pathological processes in neurons.
Topics: Animals; Brain; Fluorometry; Hydrogen Peroxide; Kinetics; Male; Mice, Inbred C57BL; Mitochondria; NADPH Oxidases; Oxidation-Reduction; Oxygen Consumption; Synapses; Synaptosomes
PubMed: 28003863
DOI: 10.1155/2016/1089364 -
International Journal of Molecular... Feb 2022The neurotransmitter glutamate plays an essential role in excitatory neurotransmission; however, excessive amounts of glutamate lead to excitotoxicity, which is the most...
The neurotransmitter glutamate plays an essential role in excitatory neurotransmission; however, excessive amounts of glutamate lead to excitotoxicity, which is the most common pathogenic feature of numerous brain disorders. This study aimed to investigate the role of butyl 2-[2-(2-fluorophenyl)acetamido]benzoate (HFP034), a synthesized anthranilate derivative, in the central glutamatergic system. We used rat cerebro-cortical synaptosomes to examine the effect of HFP034 on glutamate release. In addition, we used a rat model of kainic acid (KA)-induced glutamate excitotoxicity to evaluate the neuroprotective potential of HFP034. We showed that HFP034 inhibits 4-aminopyridine (4-AP)-induced glutamate release from synaptosomes, and this inhibition was absent in the absence of extracellular calcium. HFP034-mediated inhibition of glutamate release was associated with decreased 4-AP-evoked Ca level elevation and had no effect on synaptosomal membrane potential. The inhibitory effect of HFP034 on evoked glutamate release was suppressed by blocking P/Q-type Ca channels and protein kinase C (PKC). Furthermore, HFP034 inhibited the phosphorylation of PKC and its substrate, myristoylated alanine-rich C kinase substrate (MARCKS) in synaptosomes. We also observed that HFP034 pretreatment reduced neuronal death, glutamate concentration, glial activation, and the levels of endoplasmic reticulum stress-related proteins, calpains, glucose-regulated protein 78 (GRP 78), C/EBP homologous protein (CHOP), and caspase-12 in the hippocampus of KA-injected rats. We conclude that HFP034 is a neuroprotective agent that prevents glutamate excitotoxicity, and we suggest that this effect involves inhibition of presynaptic glutamate release through the suppression of P/Q-type Ca channels and PKC/MARCKS pathways.
Topics: 4-Aminopyridine; Animals; Calcium; Cerebral Cortex; Glutamic Acid; Kainic Acid; Protein Kinase C; Rats; Rats, Sprague-Dawley; Synaptosomes; ortho-Aminobenzoates
PubMed: 35269784
DOI: 10.3390/ijms23052641 -
ENeuro 2019Dendritic spines are the postsynaptic targets of excitatory synaptic inputs that undergo extensive proliferation and maturation during the first postnatal month in mice....
Dendritic spines are the postsynaptic targets of excitatory synaptic inputs that undergo extensive proliferation and maturation during the first postnatal month in mice. However, our understanding of the molecular mechanisms that regulate spines during this critical period is limited. Previous work has shown that pannexin 1 (Panx1) regulates neurite growth and synaptic plasticity. We therefore investigated the impact of global Panx1 KO on spontaneous cortical neuron activity using Ca imaging and network analysis. Panx1 KO increased both the number and size of spontaneous co-active cortical neuron network ensembles. To understand the basis for these findings, we investigated Panx1 expression in postnatal synaptosome preparations from early postnatal mouse cortex. Between 2 and 4 postnatal weeks, we observed a precipitous drop in cortical synaptosome protein levels of Panx1, suggesting it regulates synapse proliferation and/or maturation. At the same time points, we observed significant enrichment of the excitatory postsynaptic density proteins PSD-95, GluA1, and GluN2a in cortical synaptosomes from global Panx1 knock-out mice. analysis of pyramidal neuron structure in somatosensory cortex revealed a consistent increase in dendritic spine densities in both male and female Panx1 KO mice. Similar findings were observed in an excitatory neuron-specific Panx1 KO line (Emx1-Cre driven; Panx1 cKO) and in primary Panx1 KO cortical neurons cultured Altogether, our study suggests that Panx1 negatively regulates cortical dendritic spine development.
Topics: Animals; Calcium Signaling; Cerebral Cortex; Connexins; Dendritic Spines; Disks Large Homolog 4 Protein; Female; Male; Mice, Inbred C57BL; Mice, Knockout; Nerve Tissue Proteins; Neural Pathways; Optical Imaging; Synaptosomes
PubMed: 31118206
DOI: 10.1523/ENEURO.0503-18.2019 -
Developmental Neuroscience 2023Alterations in the expression of genes encoding proteins involved in synapse formation, maturation, and function are a hallmark of many neurodevelopmental and...
Alterations in the expression of genes encoding proteins involved in synapse formation, maturation, and function are a hallmark of many neurodevelopmental and psychiatric disorders. For example, there is reduced neocortical expression of the MET receptor tyrosine kinase (MET) transcript and protein in Autism Spectrum Disorder (ASD) and Rett syndrome. Preclinical in vivo and in vitro models manipulating MET signaling reveal that the receptor modulates excitatory synapse development and maturation in select forebrain circuits. The molecular adaptations underlying the altered synaptic development remain unknown. We performed a comparative mass spectrometry analysis of synaptosomes generated from the neocortex of wild type and Met null mice during the peak of synaptogenesis (postnatal day 14; data are available from ProteomeXchange with identifier PXD033204). The analyses revealed broad disruption of the developing synaptic proteome in the absence of MET, consistent with the localization of MET protein in pre- and postsynaptic compartments, including proteins associated with the neocortical synaptic MET interactome and those encoded by syndromic and ASD risk genes. In addition to an overrepresentation of altered proteins associated with the SNARE complex, multiple proteins in the ubiquitin-proteasome system and associated with the synaptic vesicle, as well as proteins that regulate actin filament organization and synaptic vesicle exocytosis/endocytosis, were disrupted. Taken together, the proteomic changes are consistent with structural and functional changes observed following alterations in MET signaling. We hypothesize that the molecular adaptations following Met deletion may reflect a general mechanism that produces circuit-specific molecular changes due to loss or reduction of synaptic signaling proteins.
Topics: Mice; Animals; Synaptosomes; Neocortex; Proteome; Autism Spectrum Disorder; Proteomics; Synapses
PubMed: 36882009
DOI: 10.1159/000529981 -
PloS One 2022Ionotropic glutamate receptors (iGluRs) at postsynaptic terminals mediate the majority of fast excitatory neurotransmission in response to release of glutamate from the...
Ionotropic glutamate receptors (iGluRs) at postsynaptic terminals mediate the majority of fast excitatory neurotransmission in response to release of glutamate from the presynaptic terminal. Obtaining structural information on the molecular organization of iGluRs in their native environment, along with other signaling and scaffolding proteins in the postsynaptic density (PSD), and associated proteins on the presynaptic terminal, would enhance understanding of the molecular basis for excitatory synaptic transmission in normal and in disease states. Cryo-electron tomography (ET) studies of synaptosomes is one attractive vehicle by which to study iGluR-containing excitatory synapses. Here we describe a workflow for the preparation of glutamatergic synaptosomes for cryo-ET studies. We describe the utilization of fluorescent markers for the facile detection of the pre and postsynaptic terminals of glutamatergic synaptosomes using cryo-laser scanning confocal microscope (cryo-LSM). We further provide the details for preparation of lamellae, between ~100 to 200 nm thick, of glutamatergic synaptosomes using cryo-focused ion-beam (FIB) milling. We monitor the lamella preparation using a scanning electron microscope (SEM) and following lamella production, we identify regions for subsequent cryo-ET studies by confocal fluorescent imaging, exploiting the pre and postsynaptic fluorophores.
Topics: Animals; Cryoelectron Microscopy; Electron Microscope Tomography; Lasers; Mice; Microscopy, Confocal; Synapses; Synaptosomes
PubMed: 35960737
DOI: 10.1371/journal.pone.0271799 -
Cytokine & Growth Factor Reviews Apr 2017Cytokines play crucial roles in the communication between brain cells including neurons and glia, as well as in the brain-periphery interactions. In the brain, cytokines...
Cytokines play crucial roles in the communication between brain cells including neurons and glia, as well as in the brain-periphery interactions. In the brain, cytokines modulate long-term potentiation (LTP), a cellular correlate of memory. Whether cytokines regulate LTP by direct effects on neurons or by indirect mechanisms mediated by non-neuronal cells is poorly understood. Elucidating neuron-specific effects of cytokines has been challenging because most brain cells express cytokine receptors. Moreover, cytokines commonly increase the expression of multiple cytokines in their target cells, thus increasing the complexity of brain cytokine networks even after single-cytokine challenges. Here, we review evidence on both direct and indirect-mediated modulation of LTP by cytokines. We also describe novel approaches based on neuron- and synaptosome-enriched systems to identify cytokines able to directly modulate LTP, by targeting neurons and synapses. These approaches can test multiple samples in parallel, thus allowing the study of multiple cytokines simultaneously. Hence, a cytokine networks perspective coupled with neuron-specific analysis may contribute to delineation of maps of the modulation of LTP by cytokines.
Topics: Animals; Cells, Cultured; Cytokines; Hippocampus; Humans; Inflammation; Learning; Long-Term Potentiation; Memory; Metabolic Networks and Pathways; Mice; Neurons; Receptors, Cytokine; Signal Transduction; Synapses; Synaptic Transmission; Synaptosomes
PubMed: 28377062
DOI: 10.1016/j.cytogfr.2017.03.005 -
Cells May 2021Synaptic plasticity events, including long-term potentiation (LTP), are often regarded as correlates of brain functions of memory and cognition. One of the central...
Synaptic plasticity events, including long-term potentiation (LTP), are often regarded as correlates of brain functions of memory and cognition. One of the central players in these plasticity-related phenomena is the α-amino-3-hydroxy-5-methylisoxazole-4-propionate receptor (AMPAR). Increased levels of AMPARs on postsynaptic membranes thus constitute a biochemical measure of LTP. Isolated synaptic terminals (synaptosomes) are an excellent ex vivo tool to monitor synaptic physiology in healthy and diseased brains, particularly in human research. We herein describe three protocols for chemically-induced LTP (cLTP) in synaptosomes from both rodent and human brain tissues. Two of these chemical stimulation protocols are described for the first time in synaptosomes. A pharmacological block of synaptosomal actin dynamics confirmed the efficiency of the cLTP protocols. Furthermore, the study prototypically evaluated the deficiency of cLTP in cortical synaptosomes obtained from human cases of early-onset Alzheimer's disease (EOAD) and frontotemporal lobar degeneration (FLTD), as well as an animal model that mimics FLTD.
Topics: Actins; Aged; Alzheimer Disease; Animals; Brain; Colforsin; Female; Frontal Lobe; Humans; Long-Term Potentiation; Male; Mice; Mice, Inbred C57BL; Mice, Transgenic; Middle Aged; Neuronal Plasticity; Rats; Rats, Sprague-Dawley; Receptors, Glutamate; Rolipram; Stimulation, Chemical; Synapses; Synaptosomes
PubMed: 34065927
DOI: 10.3390/cells10051174