Cortical glial cells contain both ionotropic and metabotropic glutamate receptors. Despite several efforts, a comprehensive analysis of the entire family of glutamate receptors and their subunits present in glial cells is still missing. Here, we provide an overall picture of the gene expression of ionotropic (AMPA, kainate, NMDA) and the main metabotropic glutamate receptors in cortical glial cells isolated from GFAP/EGFP mice before and after focal cerebral ischemia. Employing single-cell RT-qPCR, we detected the expression of genes encoding subunits of glutamate receptors in GFAP/EGFP-positive (GFAP/EGFP(+)) glial cells in the cortex of young adult mice. Most of the analyzed cells expressed mRNA for glutamate receptor subunits, the expression of which, in most cases, even increased after ischemic injury. Data analyses disclosed several classes of GFAP/EGFP(+) glial cells with respect to glutamate receptors and revealed in what manner their expression correlates with the expression of glial markers prior to and after ischemia. Furthermore, we also examined the protein expression and functional significance of NMDA receptors in glial cells. Immunohistochemical analyses of all seven NMDA receptor subunits provided direct evidence that the GluN3A subunit is present in GFAP/EGFP(+) glial cells and that its expression is increased after ischemia. In situ and in vitro Ca(2+) imaging revealed that Ca(2+) elevations evoked by the application of NMDA were diminished in GFAP/EGFP(+) glial cells following ischemia. Our results provide a comprehensive description of glutamate receptors in cortical GFAP/EGFP(+) glial cells and may serve as a basis for further research on glial cell physiology and pathophysiology.

• Klíčová slova
• Astrocytes, Calcium imaging, MCAo, NG2 glia, Single-cell RT-qPCR,
• MeSH
• gliový fibrilární kyselý protein analýza   biosyntéza   MeSH
• glutamátové receptory analýza   biosyntéza   MeSH
• ischemie mozku metabolismus   MeSH
• kultivované buňky MeSH
• lidé MeSH
• mozková kůra chemie   metabolismus   MeSH
• myši transgenní MeSH
• myši MeSH
• neuroglie chemie   metabolismus   MeSH
• receptory N-methyl-D-aspartátu analýza   biosyntéza   MeSH
• zelené fluorescenční proteiny analýza   biosyntéza   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• enhanced green fluorescent protein MeSH Prohlížeč
• glial fibrillary astrocytic protein, mouse MeSH Prohlížeč
• gliový fibrilární kyselý protein MeSH
• glutamátové receptory MeSH
• receptory N-methyl-D-aspartátu MeSH
• zelené fluorescenční proteiny MeSH

Two-Photon Processor (TPP) is a versatile, ready-to-use, and freely available software package in MATLAB to process data from in vivo two-photon calcium imaging. TPP includes routines to search for cell bodies in full-frame (Search for Neural Cells Accelerated; SeNeCA) and line-scan acquisition, routines for calcium signal calculations, filtering, spike-mining, and routines to construct parametric fields. Searching for somata in artificial in vivo data, our algorithm achieved better performance than human annotators. SeNeCA copes well with uneven background brightness and in-plane motion artifacts, the major problems in simple segmentation methods. In the fast mode, artificial in vivo images with a resolution of 256 × 256 pixels containing ≈ 100 neurons can be processed at a rate up to 175 frames per second (tested on Intel i7, 8 threads, magnetic hard disk drive). This speed of a segmentation algorithm could bring new possibilities into the field of in vivo optophysiology. With such a short latency (down to 5-6 ms on an ordinary personal computer) and using some contemporary optogenetic tools, it will allow experiments in which a control program can continuously evaluate the occurrence of a particular spatial pattern of activity (a possible correlate of memory or cognition) and subsequently inhibit/stimulate the entire area of the circuit or inhibit/stimulate a different part of the neuronal system. TPP will be freely available on our public web site. Similar all-in-one and freely available software has not yet been published.

• Klíčová slova
• algorithm, calcium imaging, processing, segmentation,
• MeSH
• algoritmy * MeSH
• aniliny analýza   MeSH
• fluoresceiny analýza   MeSH
• fluorescenční barviva MeSH
• mikroskopie fluorescenční multifotonová metody   MeSH
• mozková kůra chemie   MeSH
• myši inbrední C57BL MeSH
• myši MeSH
• software * MeSH
• vápník analýza   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• aniliny MeSH
• fluoresceiny MeSH
• fluorescenční barviva MeSH
• Oregon green 488 BAPTA-1 MeSH Prohlížeč
• vápník MeSH

The perirhinal cortex (PRC) composed of areas 35 and 36 forms an important route for activity transfer between the hippocampus-entorhinal cortex and neocortex. Its function in memory formation and consolidation as well as in the initiation and spreading of epileptic activity was already partially elucidated. We studied the general pattern of calretinin (CR), parvalbumin (PV) and calbindin (CB) immunoreactivity and its corrected relative optical density (cROD) as well as morphological features and density of CR and PV immunoreactive (CR+, PV+) interneurons in the rat PRC. Neighboring neocortical association area Te3V was analyzed as well. The PRC differed from the Te3V in higher CR and lower PV overall immunoreactivity level. On CR immunostained sections, the difference between high cROD value in area 35 and low cROD value in area Te3V reached statistical significance (p<0.05). The pattern of CB immunoreactivity was similar to that of the neocortex. Vertically oriented bipolar neurons were the most common morphological type of CR+ neurons, multipolar neuronal morphology was typical among PV+ neurons and vertically oriented bipolar neurons and multipolar neurons were approximately equally frequent among CB+ neurons. The density of CR+ and PV+ neurons was stereologically measured. While the density of PV+ neurons was not significantly different in PRC when compared to Te3V, density of CR+ neurons in area 35 was significantly higher by comparison with Te3V (p<0.05). Further, the overall neuronal density was measured on Nissl stained sections and the proportion of CR+ and PV+ interneurons was expressed as a percentage of the total neurons counts. The percentage of CR+ interneurons was higher in area 35 by comparison with area Te3 (p<0.05), while the percentage of PV+ interneurons did not significantly differ among the examined areas. In conclusion, the PRC possesses specific interneuronal equipment with unusually high proportion of CR+ interneurons, what might be of importance for the presumed gating function of PRC in normal and diseased states.

• MeSH
• interneurony chemie   metabolismus   MeSH
• kalbindin 2 MeSH
• kalbindiny MeSH
• krysa rodu Rattus MeSH
• mozková kůra chemie   metabolismus   MeSH
• neurony chemie   metabolismus   MeSH
• parvalbuminy analýza   metabolismus   MeSH
• S100 kalcium vázající protein G analýza   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• Calb2 protein, rat MeSH Prohlížeč
• kalbindin 2 MeSH
• kalbindiny MeSH
• parvalbuminy MeSH
• S100 kalcium vázající protein G MeSH

Immunohistochemical studies of the presence of lactosylceramide (LacCer) in lysosomal storage disorders (LSDs) were done using anti-LacCer monoclonal antibody of the CDw 17 type (clone MG-2). No sign of an association between LacCer and the lysosomal system in normal cells was observed, except for histiocytes active in phagocytosis. A comparative study of a group of LSDs showed a general tendency for LacCer to increase in storage cells in Niemann-Pick disease type C (NPC), and types A and B, GM1 gangliosidosis, acid lipase deficiency, glycogen storage disease type II and mucopolysaccharidoses. LacCer accumulated in storage cells despite normal activity of relevant lysosomal degrading enzymes. The accumulation of LacCer displayed variability within storage cell populations, and was mostly expressed in neurons in NPC. An absence of the increase in LacCer in storage cells above control levels was seen in neuronal ceroid lipofuscinoses (neurons and cardiocytes) and in Fabry disease. Gaucher and Krabbe cells showed significantly lower levels, or even the absence, of LacCer compared with control macrophages. Results of immunohistochemistry were corroborated by semiquantitative lipid thin-layer chromatography (TLC). It is suggested that different associations of LacCer with the lysosomal storage process may reflect differences in glycosphingolipid turnover induced by the storage-compromised lysosomal/endosomal system.

• MeSH
• biologické markery analýza   MeSH
• CD antigeny analýza   metabolismus   MeSH
• chromatografie na tenké vrstvě metody   MeSH
• dítě MeSH
• dospělí MeSH
• histiocyty chemie   metabolismus   patologie   MeSH
• imunohistochemie metody   MeSH
• játra chemie   metabolismus   patologie   MeSH
• laktosylceramidy analýza   metabolismus   MeSH
• lidé MeSH
• lyzozomální nemoci z ukládání klasifikace   metabolismus   patologie   MeSH
• makrofágy chemie   metabolismus   patologie   MeSH
• mozková kůra chemie   metabolismus   patologie   MeSH
• neurony chemie   metabolismus   patologie   MeSH
• slezina chemie   metabolismus   patologie   MeSH
• Check Tag
• dítě MeSH
• dospělí MeSH
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• srovnávací studie MeSH
• Názvy látek
• biologické markery MeSH
• CD antigeny MeSH
• CDw17 antigen MeSH Prohlížeč
• laktosylceramidy MeSH

A method is described for simultaneous assessment of dehydroepiandrosterone (DHEA), its sulfate (DHEAS), and their 7-hydroxylated metabolites in cortex and subcortex of the rat brain. The procedure for determination of unconjugated steroids and DHEAS involved diethyl ether extraction of the homogenized tissue, solvent partition of the dry extract, and final quantification by specific radioimmunoassays. In addition, determination of 7-hydroxy-dehydroepiandrosterone sulfates required solvolysis, followed by high-performance liquid chromatography for separation of 7-hydroxylated metabolites from their precursor. The losses during this process were monitored by measurement of spiked radioactivity of [(3)H]testosterone or [(3)H]dehydroepiandrosterone sulfate. The content of dehydroepiandrosterone sulfate in both brain tissues was of the order of ten(s) nmol/g tissue irrespective its type (cortex or subcortex), while concentrations of other steroids were about 10 times lower in both tissues. In contrast to the ratio of sulfated/unconjugated DHEA, the levels of unconjugated 7-hydroxylated metabolites and their sulfates were close to each other. The reproducibility of the method with respect to coefficients of variation varied from 12 to 25%. An age-related decrease of sulfated dehydroepiandrosterone in the cortex of animals was also observed.

• MeSH
• alkany chemie   MeSH
• dehydroepiandrosteron analogy a deriváty   analýza   izolace a purifikace   MeSH
• dehydroepiandrosteronsulfát analýza   izolace a purifikace   MeSH
• ether chemie   MeSH
• krysa rodu Rattus MeSH
• methanol chemie   MeSH
• mozek - chemie MeSH
• mozková kůra chemie   MeSH
• potkani Wistar MeSH
• radioimunoanalýza metody   MeSH
• reprodukovatelnost výsledků MeSH
• tkáňové extrakty chemie   MeSH
• věkové faktory MeSH
• voda chemie   MeSH
• vysokoúčinná kapalinová chromatografie MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• mužské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• 7-hydroxydehydroepiandrosterone MeSH Prohlížeč
• alkany MeSH
• dehydroepiandrosteron MeSH
• dehydroepiandrosteronsulfát MeSH
• ether MeSH
• methanol MeSH
• naphtha MeSH Prohlížeč
• tkáňové extrakty MeSH
• voda MeSH

Bilateral intracerebroventricular infusion of dl-homocysteic acid (DL-HCA) (600 nmol on each side) to immature 12-day-old rats induced generalized clonic-tonic seizures, recurring frequently for at least 90 min, with a high rate of survival. Electrographic recordings from sensorimotor cortex, hippocampus, and striatum demonstrated isolated spikes in the hippocampus and/or striatum as the first sign of dl-HCA action. Generalization of epileptic activity occurred during generalized clonic-tonic seizures, but electroclinical correlation was very low; dissociation between EEG pattern and motor phenomena was common. Seizures were accompanied by large decreases of cortical glucose and glycogen and by approximately 7- to 10-fold accumulation of lactate. ATP and phosphocreatine (PCr) levels remained unchanged even during longlasting (3 h) convulsions. Metabolite levels became normalized during the recovery period (24 h). The examination of the effect of selected antagonists of NMDA [AP7 (18.5 and 37 mg/kg, respectively), MK-801 (0.5 mg/kg)] and non-NMDA [NBQX (10, 15 and 30 mg/kg, respectively)] receptors revealed that seizures could be attenuated or prevented (depending on the dose employed) by antagonists of both NMDA and non-NMDA receptors, as evaluated not only according to the suppression of behavioral manifestations of seizures, but also in terms of the protection of metabolite changes accompanying seizures. All antagonists employed, when given alone in the same doses as those used for seizure protection, did not influence metabolite levels, with the exception of increased glucose concentrations. Furthermore, the pronounced anticonvulsant effect could be achieved by the combined treatment with low subthreshold doses of NMDA (AP7) and non-NMDA (NBQX) receptor antagonists, which may be of potential significance for a new approach to the treatment of epilepsy.

• MeSH
• 2-amino-5-fosfonovalerát analogy a deriváty   farmakologie   MeSH
• antagonisté excitačních aminokyselin farmakologie   MeSH
• chinoxaliny farmakologie   MeSH
• dizocilpinmaleát farmakologie   MeSH
• elektroencefalografie MeSH
• energetický metabolismus účinky léků   fyziologie   MeSH
• epilepsie chemicky indukované   farmakoterapie   metabolismus   MeSH
• glukosa metabolismus   MeSH
• homocystein analogy a deriváty   MeSH
• injekce intraventrikulární MeSH
• krysa rodu Rattus MeSH
• modely nemocí na zvířatech MeSH
• mozková kůra chemie   metabolismus   patofyziologie   MeSH
• neuroprotektivní látky farmakologie   MeSH
• potkani Wistar MeSH
• receptory N-methyl-D-aspartátu fyziologie   MeSH
• věkové faktory MeSH
• záchvaty chemicky indukované   farmakoterapie   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• mužské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• 2-amino-5-fosfonovalerát MeSH
• 2-amino-7-phosphonoheptanoic acid MeSH Prohlížeč
• 2,3-dioxo-6-nitro-7-sulfamoylbenzo(f)quinoxaline MeSH Prohlížeč
• antagonisté excitačních aminokyselin MeSH
• chinoxaliny MeSH
• dizocilpinmaleát MeSH
• glukosa MeSH
• homocysteic acid MeSH Prohlížeč
• homocystein MeSH
• neuroprotektivní látky MeSH
• receptory N-methyl-D-aspartátu MeSH