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1.
Eur J Neurosci ; 49(1): 40-50, 2019 01.
Article in English | MEDLINE | ID: mdl-30367533

ABSTRACT

Dietary omega-3 fatty acids accumulate and are actively retained in central nervous system membranes, mainly in synapses, dendrites and photoreceptors. Despite this selective enrichment, their impact on synaptic function and plasticity has not been fully determined at the molecular level. In this study, we explored the impact of omega-3 fatty acid deficiency on synaptic function in the hippocampus. Dietary omega-3 fatty acid deficiency for 5 months after weaning led to a 65% reduction in the concentration of docosahexaenoic acid in whole brain synaptosomal phospholipids with no impact on global dopaminergic or serotonergic turnover. We observed reduced concentrations of glutamate receptor subunits, including GluA1, GluA2 and NR2B, and synaptic vesicle proteins synaptophysin and synaptotagmin 1 in hippocampal synaptosomes of omega-3 fatty acid-deficient mice as compared to the omega-3 fatty acid rich group. In contrast, an increased concentration of neuronal inositol 1,4,5-trisphosphate-receptor (IP3 -R) was observed in the deficient group. Furthermore, omega-3 fatty acid deficiency reduced the long-term potentiation (LTP) in stratum oriens of the hippocampal CA1 area, but not in stratum radiatum. Thus, omega-3 fatty acids seem to have specific effects in distinct subsets of glutamatergic synapses, suggesting specific molecular interactions in addition to altering plasma membrane properties on a more global scale.


Subject(s)
Fatty Acids, Omega-3/administration & dosage , Hippocampus/physiology , Long-Term Potentiation , Receptors, Glutamate/physiology , Synapses/physiology , Animals , Dopamine/metabolism , Excitatory Postsynaptic Potentials , Female , Hippocampus/drug effects , Hippocampus/metabolism , Long-Term Potentiation/drug effects , Male , Mice, Inbred C57BL , Serotonin/metabolism , Synapses/drug effects , Synaptosomes/metabolism
2.
Mol Cell Neurosci ; 92: 93-103, 2018 10.
Article in English | MEDLINE | ID: mdl-30064010

ABSTRACT

Excitotoxicity caused by excessive stimulation of glutamate receptors, resulting in pathologically increased Ca2+-concentrations, is a decisive factor in neurodegenerative diseases. We investigated long-term changes in synaptic contents of AMPA receptor subunits that play important roles in calcium regulation in chronic epilepsy. Such plastic changes may be either adaptive or detrimental. We used a kainic acid (KA)-based rat model of chronic temporal lobe epilepsy (TLE). Using hippocampal synaptosomes, we found significant reductions in the concentration of the AMPA receptor subunits GluA1 and GluA2, and the NMDA receptor subunit NR2B. The relative size of GluA1 and GluA2 reductions were almost identical, at 28% and 27%, respectively. In order to determine whether the synaptic reduction of the AMPA receptor subunits actually reflected the pool of receptors present along the postsynaptic density (PSD), as opposed to cytoplasmic or extrasynaptic pools, we performed postembedding immunogold electron microscopy (EM) of GluA1 and GluA2 in Schaffer collateral synapses in the hippocampal CA1 area. We found significant reductions, at 32% and 52% of GluA1 and GluA2 subunits, respectively, along the PSD, indicating that these synapses undergo lasting changes in glutamatergic neurotransmission during chronic TLE. When compared to the overall concentration and composition of AMPA receptors expressed in the brain, there was a relative increase in GluA2-lacking AMPA receptor subunits following chronic epilepsy. These changes in synaptic AMPA receptor subunits may possibly contribute to further aggravate the excitotoxic vulnerability of the neurons as well as have significant implications for hippocampal cognitive functions.


Subject(s)
Epilepsy, Temporal Lobe/metabolism , Receptors, AMPA/metabolism , Synapses/metabolism , Animals , Epilepsy, Temporal Lobe/etiology , Excitatory Postsynaptic Potentials , Hippocampus/metabolism , Kainic Acid/toxicity , Male , Rats , Rats, Sprague-Dawley , Receptors, AMPA/genetics , Synapses/physiology , Synapses/ultrastructure
3.
Hippocampus ; 27(11): 1168-1177, 2017 11.
Article in English | MEDLINE | ID: mdl-28686803

ABSTRACT

Synaptotagmin 1 is a presynaptic calcium sensor, regulating SNARE-mediated vesicle exocytosis of transmitter. Increasing evidence indicate roles of SNARE proteins in postsynaptic glutamate receptor trafficking. However, a possible postsynaptic expression of synaptotagmin 1 has not been demonstrated previously. Here, we used postembedding immunogold electron microscopy to determine the subsynaptic localization of synaptotagmin 1 in rat hippocampal CA1 Schaffer collateral synapses. We report for the first time that synaptotagmin 1 is present in rat hippocampal postsynaptic spines, both on cytoplasmic vesicles and at the postsynaptic density. We further investigated whether postsynaptic synaptotagmin 1 is regulated during synaptic plasticity. In a rat model of chronic temporal lobe epilepsy, we found that presynaptic and postsynaptic concentrations of the protein are reduced compared to control animals. This downregulation may possibly be an adaptive measure to decrease both presynaptic and postsynaptic calcium sensitivity in excitotoxic conditions.


Subject(s)
Cytoplasmic Vesicles/metabolism , Dendritic Spines/metabolism , Hippocampus/metabolism , Post-Synaptic Density/metabolism , Synaptotagmin I/metabolism , Animals , Cells, Cultured , Chronic Disease , Cytoplasmic Vesicles/ultrastructure , Dendritic Spines/ultrastructure , Disease Models, Animal , Down-Regulation , Epilepsy, Temporal Lobe/metabolism , Epilepsy, Temporal Lobe/pathology , Hippocampus/ultrastructure , Immunohistochemistry , Kainic Acid , Male , Mice, Knockout , Microscopy, Electron , Post-Synaptic Density/ultrastructure , Presynaptic Terminals/metabolism , Presynaptic Terminals/ultrastructure , Rats, Sprague-Dawley , Rats, Wistar , Synaptotagmin I/deficiency , Synaptotagmin I/genetics
4.
Tidsskr Nor Laegeforen ; 133(5): 524-7, 2013 Mar 05.
Article in Norwegian | MEDLINE | ID: mdl-23463064

ABSTRACT

BACKGROUND: Executive function deficit is a cognitive dysfunction resulting in a reduced ability to initiate, control and monitor targeted behaviour. Our clinical experience indicates that this often remains undiagnosed following stroke. METHOD: The article is based on literature searches using the search terms «Stroke¼ and «Executive function¼ via the search engine McMaster Plus, in the databases Cochrane Library and PubMed, coupled with the authors' own experience. RESULTS: Executive function deficit is a common form of stroke-related cognitive dysfunction which often accompanies emotional instability and depression. The condition is an important risk factor for loss of self-sufficiency and for reduced survival. Diagnosis is based on the patient's history and observation, supplemented by cognitive testing. Executive function deficits also occur in patients with no clinical signs of stroke, but who have image diagnostic signs of cerebral ischaemia, and with other cerebral diseases such as Parkinson's disease and dementia. Executive function is mainly located in the prefrontal cortex and the subcortical circuits, but executive function deficits are also seen in cases of lesions in other areas of the brain. The treatment of executive function deficits focuses on compensatory strategies and on recovery of lost function. INTERPRETATION: Executive function deficits are common with stroke-related cognitive impairment, and may affect the prognosis. There is a need for systematic testing and strategies for treatment and prevention.


Subject(s)
Cognition Disorders/physiopathology , Executive Function/physiology , Stroke/complications , Basal Ganglia/physiology , Cognition Disorders/diagnosis , Cognition Disorders/etiology , Cognition Disorders/therapy , Comprehension , Humans , Neuropsychological Tests , Prefrontal Cortex/physiology , Problem Solving , Prognosis , Stroke/diagnosis , Stroke/physiopathology , Stroke Rehabilitation , Thalamus/physiology
5.
Neuroreport ; 34(2): 75-80, 2023 02 01.
Article in English | MEDLINE | ID: mdl-36608162

ABSTRACT

OBJECTIVE: Perisynaptic astrocytic processes have been suggested as sites for the regulated release of neuroactive substances. However, very little is known about the molecular properties of regulated exocytosis in these processes. Soluble N-ethylmaleimide-sensitive-factor attachment protein receptor (SNARE) proteins mediate synaptic vesicle exocytosis from neuronal cells and might be candidates for regulated exocytosis also from astrocytic processes. The expression of SNARE proteins in astrocytes, however, is not clarified. Thus, we aimed to investigate the localization and relative concentrations of neuronal SNARE proteins syntaxin-1, synaptosomal nerve-associated protein 25 (SNAP-25), vesicle-associated membrane protein 2 (VAMP-2) (synaptobrevin-2) and calcium sensor synaptotagmin 1 in perisynaptic astrocytic processes compared to nerve terminals and dendrites. METHODS: We used quantitative immunogold electron microscopy of the rat hippocampus to investigate the localization and concentration of neuronal SNARE proteins. RESULTS: As expected, analysis of the immunogold data revealed a lower labeling density of SNARE proteins in the perisynaptic astrocytic processes than in presynaptic terminals. The same was also true when compared to dendrites. Contrary to VAMP-2, labeling intensities for syntaxin-1, SNAP-25 and synaptotagmin 1 were not distinguishable from background labeling in the processes. The relative concentration of VAMP-2 stands out, as the mean perisynaptic astrocytic process concentration of the protein was only 68 % lower than in presynaptic terminals and still 32 % higher than in dendrites. VAMP-2 was associated with small vesicles in the processes. Some gold particles were located over the astrocytic plasma membrane. CONCLUSION: VAMP-2 is expressed in perisynaptic astrocytic processes, with a concentration higher than in the dendrites. Our results are compatible with the role of VAMP-2 in exocytosis from perisynaptic astrocytic processes.


Subject(s)
Synaptotagmin I , Vesicle-Associated Membrane Protein 2 , Animals , Rats , Hippocampus , Qa-SNARE Proteins , R-SNARE Proteins , Synaptosomal-Associated Protein 25 , Syntaxin 1 , SNARE Proteins/metabolism
6.
Front Mol Neurosci ; 16: 1225533, 2023.
Article in English | MEDLINE | ID: mdl-38025262

ABSTRACT

The activity-regulated cytoskeleton-associated protein (Arc/Arg3.1) is a neuron-specific immediate early gene (IEG) product. The protein regulates synaptic strength through modulation of spine density and morphology, AMPA receptor endocytosis, and as being part of a retrovirus-like inter-cellular communication mechanism. However, little is known about the detailed subsynaptic localization of the protein, and especially its possible presynaptic localization. In the present study, we provide novel electron microscopical data of Arc localization at hippocampal Schaffer collateral synapses in the CA1 region. The protein was found in both pre-and postsynaptic cytoplasm in a majority of synapses, associated with small vesicles. We also observed multivesicular body-like structures positive for Arc. Furthermore, the protein was located over the presynaptic active zone and the postsynaptic density. The relative concentration of Arc was 25% higher in the postsynaptic spine than in the presynaptic terminal. Notably, small extracellular vesicles labeled for Arc were detected in the synaptic cleft or close to the synapse, supporting a possible transsynaptic transmission of the protein in the brain.

7.
Neurosci Res ; 191: 1-12, 2023 Jun.
Article in English | MEDLINE | ID: mdl-36535366

ABSTRACT

Expression of immediate early genes (IEGs) in the brain is important for synaptic plasticity, and probably also in neurodegenerative conditions. To understand the cellular mechanisms of the underlying neuropathophysiological processes in epilepsy, we need to pinpoint changes in concentration of synaptic plasticity-related proteins at subsynaptic levels. In this study, we examined changes in synaptic expression of Activity-regulated cytoskeleton-associated (Arc) and Brai Derived Neurotrophic Factor (BDNF) in a rat model of kainate-induced temporal lobe epilepsy (TLE). Western blotting showed reduced concentrations of Arc and increased concentrations of BDNF in hippocampal synaptosomes in chronic TLE rats. Then, using quantitative electron microscopy, we found corresponding changes in subsynaptic regions in the hippocampus. Specifically, we detected significant reductions in the concentrations of Arc in the presynaptic terminal of Schaffer collateral glutamatergic synapses in the stratum radiatum of the CA1 area in TLE, as well as in their adjacent postsynaptic spines. In CA3, there was a significant reduction of Arc only in the presynaptic terminal cytoplasm. Conversely, in CA3, there was a significant increase in the expression of BDNF in the presynaptic terminal, but not in the postsynaptic spine. Significant increase in BDNF concentration in the CA1 postsynaptic density was also obtained. We hypothesize that the observed changes in Arc and BDNF may contribute to both cognitive impairment and increased excitotoxic vulnerability in chronic epilepsy.


Subject(s)
Epilepsy, Temporal Lobe , Epilepsy , Rats , Animals , Brain-Derived Neurotrophic Factor/metabolism , Epilepsy, Temporal Lobe/chemically induced , Epilepsy, Temporal Lobe/metabolism , Hippocampus/metabolism , Synapses/physiology , Epilepsy/metabolism
8.
J Comp Neurol ; 529(12): 3194-3205, 2021 08.
Article in English | MEDLINE | ID: mdl-33843051

ABSTRACT

Major depressive disorder involves changes in synaptic structure and function, but the molecular underpinnings of these changes are still not established. In an initial pilot experiment, whole-brain synaptosome screening with quantitative western blotting was performed to identify synaptic proteins that may show concentration changes in a congenital rat learned helplessness model of depression. We found that the N-methyl-d-aspartate receptor (NMDAR) subunits GluN2A/GluN2B, activity-regulated cytoskeleton-associated protein (Arc) and syntaxin-1 showed significant concentration differences between congenitally learned helpless (LH) and nonlearned helpless (NLH) rats. Having identified these three proteins, we then performed more elaborate quantitative immunogold electron microscopic analyses of the proteins in a specific synapse type in the dorsal hippocampus: the Schaffer collateral synapse in the CA1 region. We expanded the setup to include also unstressed wild-type (WT) rats. The concentrations of the proteins in the LH and NLH groups were compared to WT animals. In this specific synapse, we found that the concentration of NMDARs was increased in postsynaptic spines in both LH and NLH rats. The concentration of Arc was significantly increased in postsynaptic densities in LH animals as well as in presynaptic cytoplasm of NLH rats. The concentration of syntaxin-1 was significantly increased in both presynaptic terminals and postsynaptic spines in LH animals, while pre- and postsynaptic syntaxin-1 concentrations were significantly decreased in NLH animals. These protein changes suggest pathways by which synaptic plasticity may be increased in dorsal hippocampal Schaffer collateral synapses during depression, corresponding to decreased synaptic stability.


Subject(s)
Cytoskeletal Proteins/biosynthesis , Depression/metabolism , Hippocampus/metabolism , Nerve Tissue Proteins/biosynthesis , Receptors, N-Methyl-D-Aspartate/biosynthesis , Synapses/metabolism , Syntaxin 1/biosynthesis , Animals , Cytoskeletal Proteins/analysis , Disease Models, Animal , Helplessness, Learned , Hippocampus/chemistry , Nerve Tissue Proteins/analysis , Rats , Receptors, N-Methyl-D-Aspartate/analysis , Synapses/chemistry , Syntaxin 1/analysis
9.
Cereb Cortex ; 19(5): 1092-106, 2009 May.
Article in English | MEDLINE | ID: mdl-18832333

ABSTRACT

Glutamate mediates several modes of neurotransmission in the central nervous system including recently discovered retrograde signaling from neuronal dendrites. We have previously identified the system N transporter SN1 as being responsible for glutamine efflux from astroglia and proposed a system A transporter (SAT) in subsequent transport of glutamine into neurons for neurotransmitter regeneration. Here, we demonstrate that SAT2 expression is primarily confined to glutamatergic neurons in many brain regions with SAT2 being predominantly targeted to the somatodendritic compartments in these neurons. SAT2 containing dendrites accumulate high levels of glutamine. Upon electrical stimulation in vivo and depolarization in vitro, glutamine is readily converted to glutamate in activated dendritic subsegments, suggesting that glutamine sustains release of the excitatory neurotransmitter via exocytosis from dendrites. The system A inhibitor MeAIB (alpha-methylamino-iso-butyric acid) reduces neuronal uptake of glutamine with concomitant reduction in intracellular glutamate concentrations, indicating that SAT2-mediated glutamine uptake can be a prerequisite for the formation of glutamate. Furthermore, MeAIB inhibited retrograde signaling from pyramidal cells in layer 2/3 of the neocortex by suppressing inhibitory inputs from fast-spiking interneurons. In summary, we demonstrate that SAT2 maintains a key metabolic glutamine/glutamate balance underpinning retrograde signaling by dendritic release of the neurotransmitter glutamate.


Subject(s)
Amino Acid Transport System A/metabolism , Dendrites/physiology , Glutamic Acid/metabolism , Neocortex/physiology , Neuronal Plasticity/physiology , Signal Transduction/physiology , Amino Acid Transport System A/immunology , Amino Acid Transport Systems/metabolism , Animals , Antibody Specificity , Cells, Cultured , Female , Glutamine/metabolism , Hippocampus/cytology , Hippocampus/physiology , Immunoenzyme Techniques , Male , Neocortex/cytology , Patch-Clamp Techniques , Pregnancy , Pyramidal Cells/physiology , Pyramidal Cells/ultrastructure , Rats , Rats, Sprague-Dawley , Rats, Wistar , Signal Transduction/drug effects , beta-Alanine/analogs & derivatives , beta-Alanine/pharmacology
10.
Percept Mot Skills ; 111(1): 291-306, 2010 Aug.
Article in English | MEDLINE | ID: mdl-21058608

ABSTRACT

Some of the most popular meditation practices emphasize a relaxed focus of attention in which thoughts, images, sensations, and emotions may emerge and pass freely without actively controlling or pursuing them. Several recent studies show that meditation activates frontal brain areas associated with attention focusing and physical relaxation. The objective of the present study was to assess whether brain activation during relaxed focusing on a meditation sound could be distinguished from similar, concentrative control tasks. Brain activation was measured with functional magnetic resonance imaging (fMRI) in experienced practitioners of Acem meditation. Bilateral areas of the inferior frontal gyrus (BA47) were significantly more activated during repetition of a meditation sound than during concentrative meditation-like cognitive tasks. Meditation-specific brain activation did not habituate over time, but increased in strength with continuous meditation bouts. These observations suggest that meditation with a relaxed focus of attention may activate distinct areas of the prefrontal cortex, with implications for the understanding of neurobiological correlates of meditation.


Subject(s)
Magnetic Resonance Imaging , Meditation , Prefrontal Cortex/physiology , Adult , Attention/physiology , Dominance, Cerebral/physiology , Humans , Male , Meditation/psychology , Middle Aged , Relaxation/physiology , Relaxation/psychology
11.
Brain Res ; 1706: 125-134, 2019 03 01.
Article in English | MEDLINE | ID: mdl-30408477

ABSTRACT

The inositol 1,4,5-trisphosphate receptor (IP3R) subtype IP3R1 is highly enriched in the brain, including hippocampal neurons. It plays an important function in regulating intracellular calcium concentrations. Residing on the smooth endoplasmic reticulum (sER), the IP3R1 mobilizes calcium into the cytosol upon binding the intracellular signaling molecule IP3, whose concentration is increased by stimulating certain metabotropic glutamate receptors. Increased calcium may mediate synaptic changes occurring during long-term plasticity, which includes molecular mechanisms underlying memory encoding. The exact synaptic localization of IP3R1 in the central nervous system (CNS) remains unclear. We hypothesized that IP3R1, in addition to its known expression in soma and dendritic shafts of hippocampal CA1 pyramidal neurons, also may be present in postsynaptic spines. Moreover, we hypothesized that IP3R1 may be present in presynaptic terminals as well, given the importance of calcium in regulating presynaptic neurotransmitter exocytosis. To test these two hypotheses, we used IP3R1 immunocytochemistry at the light and electron microscopical levels in the CA1 area of the hippocampus. Furthermore, we hypothesized that induction of long-term potentiation (LTP) would be accompanied by an increase in synaptic IP3R1 concentrations, thereby facilitating synaptic mechanisms of long term plasticity. To investigate this, we used quantitative immunogold electron microscopy to determine possible changes in IP3R1 concentration in sub-synaptic compartments before and five minutes after high frequency tetanizations. Firstly, our data confirm localization of IP3R1 in both presynaptic terminals and postsynaptic spines. Secondly, the concentration of IP3R1 after tetanization was significantly increased in the presynaptic compartment, suggesting a presynaptic role of IP3R1 in early phases of synaptic plasticity. It is therefore possible that IP3R1 is involved in modulating neurotransmitter release by regulating calcium homeostasis presynaptically.


Subject(s)
CA1 Region, Hippocampal/metabolism , Inositol 1,4,5-Trisphosphate Receptors/metabolism , Neuronal Plasticity/physiology , Animals , Endoplasmic Reticulum/metabolism , Hippocampus/metabolism , Long-Term Potentiation/physiology , Male , Memory/physiology , Mice , Mice, Inbred Strains , Neurons/metabolism , Neurotransmitter Agents/metabolism , Presynaptic Terminals/metabolism , Pyramidal Cells/metabolism , Rats , Rats, Inbred WKY , Receptors, Metabotropic Glutamate/metabolism , Receptors, N-Methyl-D-Aspartate/metabolism , Synaptic Transmission/physiology
12.
Exp Brain Res ; 184(4): 479-92, 2008 Feb.
Article in English | MEDLINE | ID: mdl-17851655

ABSTRACT

Glutamate, the main excitatory neurotransmitter in the brain, may cause excitotoxic damage through excessive release during a number of pathological conditions. We have developed an immunocytochemical assay to investigate the mechanisms and regulation of glutamate release from intact, cultured neurons. Our results indicate that cultured hippocampal neurons have a large surplus of glutamate available for release upon chemically induced depolarization. Long incubations with high K(+)-concentrations, and induction of repetitive action potentials with the K(+)-channel blocker 4-aminopyridine (4-AP), caused a significant reduction in glutamate labeling in a subset of boutons, demonstrating that transmitter release exceeded the capacity for replenishment. The number of boutons where release exceeded replenishment increased continuously with time of stimulation. This depletion was Ca(2+)-dependent and sensitive to bafilomycin A1 (baf), indicating that it was dominated by vesicular release mechanisms. The depletion of glutamate from cell bodies and dendrites was also Ca(2+)-dependent. Thus, under the present conditions, cytosolic glutamate is taken up in vesicles prior to release, and the main escape route for the amino acid is through vesicular exocytosis. Depolarization with lower concentrations of K(+) caused sustainable release of glutamate, i.e., without full depletion.


Subject(s)
Glutamic Acid/metabolism , Hippocampus/cytology , Neurons/metabolism , Presynaptic Terminals/metabolism , Synaptic Vesicles/metabolism , 4-Aminopyridine/pharmacology , Animals , Calcium/pharmacology , Cell Compartmentation/drug effects , Cell Compartmentation/physiology , Cells, Cultured , Cytoplasm/metabolism , Dendrites/metabolism , Dendrites/ultrastructure , Enzyme Inhibitors/pharmacology , Exocytosis/drug effects , Exocytosis/physiology , Image Processing, Computer-Assisted , Immunohistochemistry , Macrolides/pharmacology , Microscopy, Electron , Neurons/drug effects , Neurons/ultrastructure , Potassium/pharmacology , Potassium Channel Blockers/pharmacology , Presynaptic Terminals/drug effects , Presynaptic Terminals/ultrastructure , Rats , Rats, Wistar , Synaptic Vesicles/drug effects , Synaptic Vesicles/ultrastructure
13.
Mol Biol Cell ; 14(2): 698-720, 2003 Feb.
Article in English | MEDLINE | ID: mdl-12589064

ABSTRACT

SNAREs are required for specific membrane fusion throughout the endomembrane system. Here we report the characterization of rat ykt6, a prenylated SNARE selectively expressed in brain neurons. Immunofluorescence microscopy in neuronal and neuroendocrine cell lines revealed that membrane-associated ykt6 did not colocalize significantly with any conventional markers of endosomes, lysosomes, or the secretory pathway. However, ykt6-containing membranes displayed very minor overlaps with lysosomes and dense-core secretory granules and were similar to lysosomes in buoyant density. Thus, ykt6 appears to be specialized for the trafficking of a unique membrane compartment, perhaps related to lysosomes, involved in aspects of neuronal function. Targeting of this SNARE to the ykt6 compartment was mediated by its profilin-like amino-terminal domain, even in the absence of protein prenylation. Although several other R-SNAREs contain related amino-terminal domains, only the ykt6 version was able to confer the specialized localization. Rat ykt6, which contains an arginine in its SNARE motif zero-layer, was found to behave like other R-SNAREs in its SNARE assembly properties. Interestingly, cytosolic ykt6, constituting more than half of the total cellular pool, appeared to be conformationally inactive for SNARE complex assembly, perhaps indicative of a regulatory mechanism that prevents promiscuous and potentially deleterious SNARE interactions.


Subject(s)
Contractile Proteins , Membrane Proteins/metabolism , Membrane Proteins/physiology , Microfilament Proteins/chemistry , Vesicular Transport Proteins , Amino Acid Motifs , Animals , Arginine/chemistry , Brain/metabolism , Cell Line , Cell Membrane/metabolism , Chickens , Cytoskeleton/metabolism , Cytosol/metabolism , DNA, Complementary/metabolism , Electrophoresis, Polyacrylamide Gel , Glutathione Transferase/metabolism , Immunohistochemistry , Lysosomes/metabolism , Microscopy, Fluorescence , Neurons/metabolism , Neurons/pathology , PC12 Cells , Peptides/chemistry , Profilins , Protein Binding , Protein Conformation , Protein Structure, Tertiary , R-SNARE Proteins , Rats , SNARE Proteins , Subcellular Fractions , Tissue Distribution
14.
Epilepsy Res ; 137: 25-32, 2017 11.
Article in English | MEDLINE | ID: mdl-28888867

ABSTRACT

Overstimulation of glutamate receptors resulting in excessive intracellular calcium concentrations is a major cause of neuronal cell death in epilepsy. The main source of increased calcium concentration during this excitotoxicity is an influx through NMDA subtype of glutamate receptors. The GluR2 (GluA2) hypothesis states that following a neurological insult such as an epileptic seizure, the AMPA receptor subunit GluR2 protein is downregulated. This increases the likelihood of the formation of GluR2-lacking, calcium-permeable AMPA receptor which might further enhance the toxicity of the neurotransmitter, glutamate. The cytosolic protein, PICK1, facilitates the removal of GluA2 subunits from the synaptic plasma membrane. High calcium concentrations may cause PICK1 to bind to the GluA2 subunit of calcium-impermeable AMPARs, leading to an increased internalization of these receptor subunits and a relative increase of GluA2-lacking, calcium-permeable AMPARs. This further escalates the cytosolic calcium concentration. In order to test this hypothesis, we have used kainic acid to induce epilepsy in rats. Using semi-quantitative western blotting combined with univariate and multivariate statistical analyses, we found that both GluA2 and PICK1 were down-regulated in kainate-treated rats for as long as eight weeks after induction of epilepsy. An interesting finding was that statistical analysis indicates that the functional role of PICK1 in our material is to increase GluA2 concentrations in the cells. The observed reduction in PICK1 concentration may thus be an independent contributor to the observed GluA2 reduction. This reduction may possibly be an adaptive mechanism, serving to prevent further loss of GluA2 from the synapses.


Subject(s)
Carrier Proteins/metabolism , Epilepsy/metabolism , Hippocampus/metabolism , Nuclear Proteins/metabolism , Receptors, AMPA/metabolism , Animals , Blotting, Western , Chronic Disease , Cytoskeletal Proteins , Disease Models, Animal , Down-Regulation , Kainic Acid , Male , Multivariate Analysis , Neurons/metabolism , Rats, Wistar , Regression Analysis , Tubulin/metabolism
15.
Front Mol Neurosci ; 9: 10, 2016.
Article in English | MEDLINE | ID: mdl-26903802

ABSTRACT

Syntaxins are a family of membrane-integrated proteins that are instrumental in exocytosis of vesicles. Syntaxin-1 is an essential component of the presynaptic exocytotic fusion machinery in the brain and interacts with several other proteins. Syntaxin-1 forms a four-helical bundle complex with proteins SNAP-25 and VAMP2 that drives fusion of vesicles with the plasma membrane in the active zone (AZ). Little is known, however, about the ultrastructural localization of syntaxin-1 at the synapse. We have analyzed the intrasynaptic expression of syntaxin-1 in glutamatergic hippocampal synapses in detail by using quantitative postembedding immunogold labeling. Syntaxin-1 was present in highest concentrations at the presynaptic AZ, supporting its role in transmitter release. Presynaptic plasma membrane lateral to the AZ, as well as presynaptic cytoplasmic (PreCy) vesicles were also labeled. However, syntaxin-1 was also significantly expressed in postsynaptic spines, where it was localized at the postsynaptic density (PSD), at postsynaptic lateral membranes and in postsynaptic cytoplasm. Postsynaptically, syntaxin-1 colocalized in the nanometer range with the N-methyl-D-aspartate (NMDA) receptor subunit NR2B, but only weakly with the AMPA receptor subunits GluA2/3. This observation points to the possibility that syntaxin-1 may be involved with NR2B vesicular trafficking from cytoplasmic stores to the postsynaptic plasma membrane, thus facilitating synaptic plasticity. Confocal immunofluorescence double labeling with PSD-95 and ultrastructural fractionation of synaptosomes also confirm localization of syntaxin-1 at the PSD.

16.
PLoS One ; 10(10): e0140868, 2015.
Article in English | MEDLINE | ID: mdl-26488171

ABSTRACT

Vertebrate organisms adapt to a continuously changing environment by regulating the strength of synaptic connections between brain cells. Excitatory synapses are believed to increase their strength by vesicular insertion of transmitter glutamate receptors into the postsynaptic plasma membrane. These vesicles, however, have never been demonstrated or characterized. For the first time, we show the presence of small vesicles in postsynaptic spines, often closely adjacent to the plasma membrane and PSD (postsynaptic density). We demonstrate that they harbor vesicle-associated membrane protein 2 (VAMP2/synaptobrevin-2) and glutamate receptor subunit 1 (GluA1). Disrupting VAMP2 by tetanus toxin treatment reduces the concentration of GluA1 in the postsynaptic plasma membrane. GluA1/VAMP2-containing vesicles, but not GluA2/VAMP2-vesicles, are concentrated in postsynaptic spines relative to dendrites. Our results indicate that small postsynaptic vesicles containing GluA1 are inserted directly into the spine plasma membrane through a VAMP2-dependent mechanism.


Subject(s)
Excitatory Postsynaptic Potentials/physiology , Receptors, AMPA/metabolism , Synaptic Vesicles/physiology , Vesicle-Associated Membrane Protein 2/metabolism , Animals , Antibodies/immunology , Cells, Cultured , Dendrites/metabolism , Enzyme Inhibitors/pharmacology , Hippocampus/cytology , Macrolides/pharmacology , Male , Mice , Mice, Knockout , Neurons/metabolism , Neurotoxins/pharmacology , Rats , Rats, Wistar , Receptors, Glutamate/metabolism , Synapses/physiology , Tetanus Toxin/pharmacology , Vesicle-Associated Membrane Protein 2/genetics , Vesicle-Associated Membrane Protein 2/immunology
17.
Front Hum Neurosci ; 8: 86, 2014.
Article in English | MEDLINE | ID: mdl-24616684

ABSTRACT

Nondirective meditation techniques are practiced with a relaxed focus of attention that permits spontaneously occurring thoughts, images, sensations, memories, and emotions to emerge and pass freely, without any expectation that mind wandering should abate. These techniques are thought to facilitate mental processing of emotional experiences, thereby contributing to wellness and stress management. The present study assessed brain activity by functional magnetic resonance imaging (fMRI) in 14 experienced practitioners of Acem meditation in two experimental conditions. In the first, nondirective meditation was compared to rest. Significantly increased activity was detected in areas associated with attention, mind wandering, retrieval of episodic memories, and emotional processing. In the second condition, participants carried out concentrative practicing of the same meditation technique, actively trying to avoid mind wandering. The contrast nondirective meditation > concentrative practicing was characterized by higher activity in the right medial temporal lobe (parahippocampal gyrus and amygdala). In conclusion, the present results support the notion that nondirective meditation, which permits mind wandering, involves more extensive activation of brain areas associated with episodic memories and emotional processing, than during concentrative practicing or regular rest.

18.
Physiol Rep ; 2(11)2014 Nov 01.
Article in English | MEDLINE | ID: mdl-25413318

ABSTRACT

Muscle lipid stores and insulin sensitivity have a recognized association although the mechanism remains unclear. We investigated how a 12-week supervised combined endurance and strength exercise intervention influenced muscle lipid stores in sedentary overweight dysglycemic subjects and normal weight control subjects (n = 18). Muscle lipid stores were measured by magnetic resonance spectroscopy (MRS), electron microscopy (EM) point counting, and direct EM lipid droplet measurements of subsarcolemmal (SS) and intramyofibrillar (IMF) regions, and indirectly, by deep sequencing and real-time PCR of mRNA of lipid droplet-associated proteins. Insulin sensitivity and VO2max increased significantly in both groups after 12 weeks of training. Muscle lipid stores were reduced according to MRS at baseline before and after the intervention, whereas EM point counting showed no change in LD stores post exercise, indicating a reduction in muscle adipocytes. Large-scale EM quantification of LD parameters of the subsarcolemmal LD population demonstrated reductions in LD density and LD diameters. Lipid droplet volume in the subsarcolemmal LD population was reduced by ~80%, in both groups, while IMF LD volume was unchanged. Interestingly, the lipid droplet diameter (n = 10 958) distribution was skewed, with a lack of small diameter lipid droplets (smaller than ~200 nm), both in the SS and IMF regions. Our results show that the SS LD lipid store was sensitive to training, whereas the dominant IMF LD lipid store was not. Thus, net muscle lipid stores can be an insufficient measure for the effects of training.

19.
Eur J Prev Cardiol ; 19(4): 773-80, 2012 Aug.
Article in English | MEDLINE | ID: mdl-21693507

ABSTRACT

PURPOSE: Meditation practices are in use for relaxation and stress reduction. Some studies indicate beneficial cardiovascular health effects of meditation. The effects on the autonomous nervous system seem to vary among techniques. The purpose of the present study was to identify autonomic nerve activity changes during nondirective meditation. MATERIALS AND METHODS: Heart rate variability (HRV), blood pressure variability (BPV), and baroreflex sensitivity (BRS) were monitored in 27 middle-aged healthy participants of both genders, first during 20 min regular rest with eyes closed, thereafter practising Acem meditation for 20 min. Haemodynamic and autonomic data were collected continuously (beat-to-beat) and non-invasively. HRV and BPV parameters were estimated by power spectral analyses, computed by an autoregressive model. Spontaneous activity of baroreceptors were determined by the sequence method. Primary outcomes were changes in HRV, BPV, and BRS between rest and meditation. RESULTS: HRV increased in the low-frequency (LF) and high-frequency (HF) bands during meditation, compared with rest (p = 0.014, 0.013, respectively). Power spectral density of the RR-intervals increased as well (p = 0.012). LF/HF ratio decreased non-significantly, and a reduction of LF-BPV power was observed during meditation (p < 0.001). There was no significant difference in BRS. Respiration and heart rates remained unchanged. Blood pressure increased slightly during meditation. CONCLUSION: There is an increased parasympathetic and reduced sympathetic nerve activity and increased overall HRV, while practising the technique. Hence, nondirective meditation by the middle aged may contribute towards a reduction of cardiovascular risk.


Subject(s)
Heart Rate , Heart/innervation , Meditation/methods , Parasympathetic Nervous System/physiology , Sympathetic Nervous System/physiology , Adult , Baroreflex , Blood Pressure , Cardiography, Impedance , Electrocardiography , Female , Humans , Male , Middle Aged , Norway , Pressoreceptors/physiology , Time Factors
20.
J Neurosci Methods ; 193(1): 39-46, 2010 Oct 30.
Article in English | MEDLINE | ID: mdl-20727911

ABSTRACT

Transfection is a useful tool for studying molecular signalling pathways. However, neurons have proven hard to transfect. In the present paper we have optimized a new electroporation procedure using the Cellaxess(®) system for transient transfection of adherent primary neurons from chicken (Gallus gallus) and compared it to a liposome based procedure using Metafectene(®) Pro. In order to evaluate the two methods, glucocorticoid receptor (GR) function was chosen as a test. GRs are expressed in high amounts in the cerebellum. GR is regulated by another nuclear receptor (NGFI-B, the first member found in the NR4A family). We first showed that forskolin and phorbol ester activated an NR4A-dependent reporter gene indicating that members of the NR4A nuclear receptor family are present endogenously and upregulated by external stimuli. Then, transfected NGFI-B was shown to antagonize the dexamethasone-activated transcriptional activation by endogenous GR, leading to the conclusion that NR4A-family members are important modulators of GR mediated regulatory processes in the cerebellum, as in other cell types. Both transfection methods proved useful. While the electroporation technique yielded small rings with many transfected cells optimal for microscopy studies, the liposome based method resulted in transfected cells evenly distributed in the dish rendering this method well suited for biochemical studies.


Subject(s)
Cerebellum/metabolism , Electroporation/methods , Neurons/metabolism , Nuclear Receptor Subfamily 4, Group A, Member 1/genetics , Receptors, Glucocorticoid/genetics , Transfection/methods , Analysis of Variance , Animals , Cells, Cultured , Cerebellum/cytology , Chickens , Neurons/cytology , Nuclear Receptor Subfamily 4, Group A, Member 1/metabolism , Receptors, Glucocorticoid/metabolism
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