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Slice Cultures (slice + culture)
Kinds of Slice Cultures Terms modified by Slice Cultures Selected AbstractsInduction of Innate Immune Gene Expression Cascades in Brain Slice Cultures by Ethanol: Key Role of NF-,B and Proinflammatory CytokinesALCOHOLISM, Issue 5 2010Jian Zou Background:, Postmortem human alcoholic brain has increased expression of proinflammatory cytokines (He and Crews, 2007). Nuclear factor ,B (NF-,B) is a transcription factor known to induce proinflammatory cytokine expression. Ethanol exposure increases NF-,B,DNA binding in rat brain (Crews et al., 2006) and in brain slice cultures in vitro (Zou and Crews, 2006). Using hippocampal-entorhinal cortex (HEC) brain slice cultures, we explored the effect of ethanol on NF-,B,DNA binding, proinflammatory gene expression, and sensitivity to glutamate neurotoxicity. Methods:, The HEC brain slice cultures are prepared from rats on P7 and used after 2 weeks in culture. NF-,B,DNA binding is determined by EMSA, NF-,B subunit,DNA binding by ELISA and mRNA by RT-PCR. Multiple antibody immunohistochemistry and confocal microscopy are used to characterize cell types expressing ethanol-induced genes. Results:, Ethanol treatment results in a progressive increase in NF-,B,DNA binding that includes large increases in NF-,B subunit p50 protein,DNA binding. The expression of NF-,B proinflammatory target genes progressively increased with time of ethanol treatment. Ethanol induces proinflammatory cytokines TNF,, MCP-1, and IL-1,, proinflammatory proteases TACE, and tissue plasminogen activator (tPA) as well as inducible nitric oxide synthase. Blockade of NF-,B by using NF-,B p65 siRNA and BHT reduces ethanol induction of proinflammatory genes. Neutralizing antibody to proinflammatory cytokine TNF, reduces ethanol induction of proinflammatory genes, suggesting cytokine propagation of proinflammatory gene induction. Furthermore, neutralizing antibodies to proinflammatory cytokines and protease tPA inhibitors blunt ethanol sensitization to glutamate neurotoxicity. Conclusions:, These findings indicate that ethanol treatment increases NF-,B,DNA binding and proinflammatory gene expression in brain slices. Ethanol-induced innate immune proinflammatory gene induction alters neurotransmission and likely contributes to alcoholic neurodegeneration. [source] Polyamines Contribute to Ethanol Withdrawal-Induced Neurotoxicity in Rat Hippocampal Slice Cultures Through Interactions With the NMDA ReceptorALCOHOLISM, Issue 7 2003D. Alex Gibson Background: Several reports demonstrate that withdrawal from long-term ethanol exposure is associated with significant central nervous system neurotoxicity, produced at least in part by increased activity of N -methyl-d-aspartate receptors (NMDARs). Recent evidence suggests that elevations in the synthesis and release of the polyamines spermidine and spermine, which are known modulators of NMDARs, contribute to the increased activity of the receptor during ethanol withdrawal. Therefore, the goal of this investigation was to examine what role, if any, spermidine and spermine have in the generation of ethanol withdrawal-induced neurotoxicity. Methods: Neurotoxicity (measured as fluorescence of the cell death indicator propidium iodide, PI), glutamate release (measured by high-performance liquid chromatography analysis), and polyamine concentrations (by high-performance liquid chromatography) were measured in rat hippocampal slice cultures undergoing withdrawal from chronic (10 day) ethanol exposure (100 mM). In addition, the effects of the polyamine synthesis inhibitor di-fluoro-methyl-ornithine (DFMO, 0.1,100 nM) and NMDAR polyamine-site antagonists ifenprodil, arcaine, and agmatine (1 nM-100 ,M) on ethanol withdrawal- and NMDA-induced neurotoxicity were measured. Results: Ethanol withdrawal significantly increased glutamate release (peaking at 18 hr with a 53% increase), increased concentrations of putrescine and spermidine (136% and 139% increases, respectively, at 18 hr), and produced significant cytotoxicity in the CA1 hippocampal region (56% increase in PI staining relative to controls) of the cultures. The cell death produced by ethanol withdrawal was significantly inhibited by ifenprodil (IC50= 14.9 nM), arcaine (IC50= 37.9 nM), agmatine (IC50= 41.5 nM), and DFMO (IC50= 0.6 nM). NMDA (5 ,M) significantly increased PI staining in the CA1 region of the hippocampal cultures (365% relative to controls), but ifenprodil, arcaine, agmatine, and DFMO all failed to significantly affect this type of toxicity. Conclusions: These data implicate a role for polyamines in ethanol withdrawal-induced neurotoxicity and suggest that inhibiting the actions of polyamines on NMDARs may be neuroprotective under these conditions. [source] Three-dimensional slice cultures from murine fetal gut for investigations of the enteric nervous systemDEVELOPMENTAL DYNAMICS, Issue 1 2007Marco Metzger Abstract Three-dimensional intestinal cultures offer new possibilities for the examination of growth potential, analysis of time specific gene expression, and spatial cellular arrangement of enteric nervous system in an organotypical environment. We present an easy to produce in vitro model of the enteric nervous system for analysis and manipulation of cellular differentiation processes. Slice cultures of murine fetal colon were cultured on membrane inserts for up to 2 weeks without loss of autonomous contractility. After slice preparation, cultured tissue reorganized within the first days in vitro. Afterward, the culture possessed more than 35 cell layers, including high prismatic epithelial cells, smooth muscle cells, glial cells, and neurons analyzed by immunohistochemistry. The contraction frequency of intestinal slice culture could be modulated by the neurotransmitter serotonin and the sodium channel blocker tetrodotoxin. Coculture experiments with cultured neurospheres isolated from enhanced green fluorescent protein (eGFP) transgenic mice demonstrated that differentiating eGFP-positive neurons were integrated into the intestinal tissue culture. This slice culture model of enteric nervous system proved to be useful for studying cell,cell interactions, cellular signaling, and cell differentiation processes in a three-dimensional cell arrangement. Developmental Dynamics 236:128,133, 2007. © 2006 Wiley-Liss, Inc. [source] The patterns of spontaneous Ca2+ signals generated by ventral spinal neurons in vitro show time-dependent refinementEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 8 2009Sara Sibilla Abstract Embryonic spinal neurons maintained in organotypic slice culture are known to mimic certain maturation-dependent signalling changes. With such a model we investigated, in embryonic mouse spinal segments, the age-dependent spatio-temporal control of intracellular Ca2+ signalling generated by neuronal populations in ventral circuits and its relation with electrical activity. We used Ca2+ imaging to monitor areas located within the ventral spinal horn at 1 and 2 weeks of in vitro growth. Primitive patterns of spontaneous neuronal Ca2+ transients (detected at 1 week) were typically synchronous. Remarkably, such transients originated from widespread propagating waves that became organized into large-scale rhythmic bursts. These activities were associated with the generation of synaptically mediated inward currents under whole-cell patch-clamp. Such patterns disappeared during longer culture of spinal segments: at 2 weeks in culture, only a subset of ventral neurons displayed spontaneous, asynchronous and repetitive Ca2+ oscillations dissociated from background synaptic activity. We observed that the emergence of oscillations was a restricted phenomenon arising together with the transformation of ventral network electrophysiological bursting into asynchronous synaptic discharges. This change was accompanied by the appearance of discrete calbindin immunoreactivity against an unchanged background of calretinin-positive cells. It is attractive to assume that periodic oscillations of Ca2+ confer a summative ability to these cells to shape the plasticity of local circuits through different changes (phasic or tonic) in intracellular Ca2+. [source] Intrinsic and spontaneous neurogenesis in the postnatal slice culture of rat hippocampusEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 10 2004Maki Kamada Abstract Organotypic slice culture preserves the morphological and physiological features of the hippocampus of live animals for a certain time. The hippocampus is one of exceptional regions where neurons are generated intrinsically and spontaneously throughout postnatal life. We investigated the possibility that neurons are generated continuously at the dentate granule cell layer (GCL) in slice culture of the rat hippocampus. Using 5-bromodeoxyuridine (BrdU) labelling and retrovirus vector transduction methods, the phenotypes of the newly generated cells were identified immunohistochemically. At 4 weeks after BrdU exposure, BrdU-labelled cells were found in the GCL and were immunoreactive with a neuronal marker, anti-NeuN. There were fibrils immunoreactive with anti-glial fibrillary acidic protein (GFAP), an astrocyte marker, in the layer covering the GCL and occasionally encapsulated BrdU-labelled nuclei. When the newly divided cells were marked with the enhanced green fluorescent protein (EGFP) using a retrovirus vector, these cells had proliferative abilities throughout the following 4-week cultivation period. Four weeks after the inoculation, the EGFP-expressing cells consisted of various phenotypes of both early and late stages of differentiation; some were NeuN-positive cells with appearances of neurons in the GCL and some were immunoreactive with anti-Tuj1, a marker of immature neurons. Some EGFP-expressing cells were immunoreactive with anti-GFAP or anti-nestin, a marker of neural progenitors. The present study suggests that slice cultures intrinsically retain spontaneous neurogenic abilities for their cultivation period. The combination of slice culture and retrovirus transduction methods enable the newly divided cells to be followed up for a long period. [source] Nitric oxide-producing microglia mediate thrombin-induced degeneration of dopaminergic neurons in rat midbrain slice cultureJOURNAL OF NEUROCHEMISTRY, Issue 5 2006Hiroshi Katsuki Abstract Activated microglia are considered to play important roles in degenerative processes of midbrain dopaminergic neurons. Here we examined mechanisms of neurotoxicity of thrombin, a protease known to trigger microglial activation, in organotypic midbrain slice cultures. Thrombin induced a progressive decline in the number of dopaminergic neurons, an increase in nitric oxide (NO) production, and whole tissue injury indicated by lactate dehydrogenase release and propidium iodide uptake. Microglia expressed inducible NO synthase (iNOS) in response to thrombin, and inhibition of iNOS rescued dopaminergic neurons without affecting whole tissue injury. Inhibitors of mitogen-activated protein kinases (MAPKs) such as extracellular signal-regulated kinase (ERK), p38 MAPK and c-Jun N-terminal kinase (JNK) attenuated thrombin-induced iNOS induction and dopaminergic cell death. Whole tissue injury was also attenuated by inhibition of ERK and p38 MAPK. Moreover, depletion of resident microglia from midbrain slices abrogated thrombin-induced NO production and dopaminergic cell death, but did not inhibit tissue injury. Finally, antioxidative drugs prevented thrombin-induced dopaminergic cell death without affecting whole tissue injury. Hence, NO production resulting from MAPK-dependent microglial iNOS induction is a crucial event in thrombin-induced dopaminergic neurodegeneration, whereas damage of other midbrain cells is MAPK-dependent but is NO-independent. [source] Inhibition of neural activity depletes orexin from rat hypothalamic slice cultureJOURNAL OF NEUROSCIENCE RESEARCH, Issue 1 2010Shotaro Michinaga Abstract Orexins (hypocretins) are neuropeptides produced by a small population of hypothalamic neurons whose dysregulation may lead to narcolepsy, a neurological disorder characterized by disorganization of sleep and wakefulness. Excessive stimulation of the N-methyl-D-aspartate (NMDA) subtype of glutamate receptors causes preferential loss of orexin neurons in the hypothalamus, whereas an adequate level of neuronal excitatory activities is generally known to be important for the maintenance of central neurons. By examining the effect of manipulation of neural activity, we found that 24,72 hr application of tetrodotoxin (TTX) caused a substantial decrease in the number of orexin-immunoreactive neurons, but not of melanin-concentrating hormone-immunoreactive neurons, in hypothalamic slice culture. Similar results were obtained when neural activity was arrested by added extracellular Mg2+. Reduction of orexin expression by TTX and Mg2+ was also observed at mRNA level. The decrease of orexin-immunoreactive neurons was attributable to depletion of orexin, because it was reversible after washout of TTX or elevated extracellular Mg2+ and was not associated with induction of cell death. Blockers of voltage-dependent Ca2+ channels as well as of NMDA receptors also induced a significant and selective decrease of orexin-immunoreactive neurons. Moreover, TTX-induced decrease of orexin immunoreactivity was largely abrogated by concurrent application of a moderate concentration of NMDA. These results suggest that Ca2+ entry associated with nontoxic levels of spontaneous activity of glutamatergic inputs plays an important role in the maintenance of orexin neurons in a tissue culture model. © 2009 Wiley-Liss, Inc. [source] Environmental neurotoxin-induced progressive model of parkinsonism in ratsANNALS OF NEUROLOGY, Issue 1 2010Wei-Bin Shen PhD Objective Exposure to a number of drugs, chemicals, or environmental factors can cause parkinsonism. Epidemiologic evidence supports a causal link between the consumption of flour made from the washed seeds of the plant Cycas micronesica by the Chamorro population of Guam and the development of amyotrophic lateral sclerosis/parkinsonism dementia complex. Methods We now report that consumption of washed cycad flour pellets by Sprague-Dawley male rats induces progressive parkinsonism. Results Cycad-fed rats displayed motor abnormalities after 2 to 3 months of feeding such as spontaneous unilateral rotation, shuffling gait, and stereotypy. Histological and biochemical examination of brains from cycad-fed rats revealed an initial decrease in the levels of dopamine and its metabolites in the striatum (STR), followed by neurodegeneration of dopaminergic (DAergic) cell bodies in the substantia nigra (SN) pars compacta (SNc). ,-Synuclein (,-syn; proteinase K-resistant) and ubiquitin aggregates were found in the DAergic neurons of the SNc and neurites in the STR. In addition, we identified ,-syn aggregates in neurons of the locus coeruleus and cingulate cortex. No loss of motor neurons in the spinal cord was found after chronic consumption of cycad flour. In an organotypic slice culture of the rat SN and the striatum, an organic extract of cycad causes a selective loss of dopamine neurons and ,-syn aggregates in the SN. Interpretation Cycad-fed rats exhibit progressive behavioral, biochemical, and histological hallmarks of parkinsonism, coupled with a lack of fatality. ANN NEUROL 2010;68:70,80 [source] Three-dimensional slice cultures from murine fetal gut for investigations of the enteric nervous systemDEVELOPMENTAL DYNAMICS, Issue 1 2007Marco Metzger Abstract Three-dimensional intestinal cultures offer new possibilities for the examination of growth potential, analysis of time specific gene expression, and spatial cellular arrangement of enteric nervous system in an organotypical environment. We present an easy to produce in vitro model of the enteric nervous system for analysis and manipulation of cellular differentiation processes. Slice cultures of murine fetal colon were cultured on membrane inserts for up to 2 weeks without loss of autonomous contractility. After slice preparation, cultured tissue reorganized within the first days in vitro. Afterward, the culture possessed more than 35 cell layers, including high prismatic epithelial cells, smooth muscle cells, glial cells, and neurons analyzed by immunohistochemistry. The contraction frequency of intestinal slice culture could be modulated by the neurotransmitter serotonin and the sodium channel blocker tetrodotoxin. Coculture experiments with cultured neurospheres isolated from enhanced green fluorescent protein (eGFP) transgenic mice demonstrated that differentiating eGFP-positive neurons were integrated into the intestinal tissue culture. This slice culture model of enteric nervous system proved to be useful for studying cell,cell interactions, cellular signaling, and cell differentiation processes in a three-dimensional cell arrangement. Developmental Dynamics 236:128,133, 2007. © 2006 Wiley-Liss, Inc. [source] Inverse relationship between seizure expression and extrasynaptic NMDAR function following chronic NMDAR inhibitionEPILEPSIA, Issue 2010Suzanne B. Bausch Summary We showed previously that electrographic seizures involving dentate granule cells in organotypic hippocampal slice cultures were dramatically reduced following chronic treatment with the NR2B-selective antagonist, Ro25,6981, but were increased following chronic treatment with the high-affinity competitive antagonist, D(-)-2-amino-5-phosphonopentanoic acid (D-APV). To begin to investigate the potential mechanisms underlying the differential effects of N -methyl- d -aspartate receptor (NMDAR) antagonists on seizures, electrophysiologic experiments were conducted in dentate granule cells in hippocampal slice cultures treated for the entire 17,21 day culture period with vehicle, Ro25,6981 or D-APV. Initial experiments revealed a lack of an association between miniature excitatory postsynaptic current (mEPSC) measures and seizures suggesting that shifts in mEPSC were unlikely to account for the differential effects of D-APV and Ro25,6981 on seizures. However, the amplitude of tonic NMDAR-mediated currents was reduced in cultures treated chronically with D-APV and dramatically enhanced in cultures treated chronically with Ro25,6981. Because tonic NMDAR currents are mediated primarily by extrasynaptic NMDAR, these data show an inverse relationship between changes in extrasynaptic NMDAR function and alterations in seizure expression. [source] Neutralization of the membrane protein Nogo-A enhances growth and reactive sprouting in established organotypic hippocampal slice culturesEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 9 2008Luis M. Craveiro Abstract The reduced ability of central axons to regenerate after injury is significantly influenced by the presence of several molecules that inhibit axonal growth. Nogo-A is one of the most studied and most potent of the myelin-associated growth inhibitory molecules. Its neutralization, as well as interference with its signalling, allows for enhanced axonal sprouting and growth following injury. Using differentiated rat organotypic hippocampal slice cultures treated for 5 days with either of two different function-blocking anti-Nogo-A antibodies, we show an increase in CA3 fibre regeneration after lesion. In intact slices, 5 days of anti-Nogo-A antibody treatment led to increased sprouting of intact CA3 fibres that are positive for neurofilament 68. A transcriptomic approach confirmed the occurrence of a growth response on the molecular level upon Nogo-A neutralization in intact cultures. Our results demonstrate that Nogo-A neutralization for 5 days is sufficient for the induction of growth in mature CNS tissue without the prerequisite of an injury. Nogo-A may therefore act as a tonic growth suppressor/stabilizer in the adult intact hippocampus. [source] Recruiting new neurons from the subventricular zone to the rat postnatal cortex: an organotypic slice culture modelEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 5 2008A. G. Dayer Abstract The neurogenic subventricular zone (SVZ) of the lateral ventricle is a potential source for neuronal replacement in the postnatal or adult neocortex after injury. Here we present a novel model system to directly explore the cellular mechanisms of this process. In order to visualize directed migration from the SVZ towards the cortex, we transplanted green fluorescent protein-labeled progenitor/stem cells into the SVZ of newborn rats. At 2 days after transplantation, we generated organotypic slice cultures and applied fluorescent time-lapse imaging to explore directly the migration and integration of donor cells into the host tissue for up to 2 weeks. Our studies revealed that subventricular grafts provide a significant number of immature neurons to neocortical regions. In the cortex, immature neurons first migrate radially towards the pial surface and then differentiate into GABAergic interneurons. We conclude that our model system presents a novel and effective experimental paradigm to evaluate the recruitment of SVZ-derived neurons into the postnatal cortex, a phenomenon that may represent a potential route for cortical repair. [source] Oxidative stress on EAAC1 is involved in MPTP-induced glutathione depletion and motor dysfunctionEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 1 2008Koji Aoyama Abstract Excitatory amino acid carrier 1 (EAAC1) is a glutamate transporter expressed on mature neurons in the CNS, and is the primary route for uptake of the neuronal cysteine needed to produce glutathione (GSH). Parkinson's disease (PD) is a neurodegenerative disorder pathogenically related to oxidative stress and shows GSH depletion in the substantia nigra (SN). Herein, we report that 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated mice, an experimental model of PD, showed reduced motor activity, reduced GSH contents, EAAC1 translocation to the membrane and increased levels of nitrated EAAC1. These changes were reversed by pre-administration of n-acetylcysteine (NAC), a membrane-permeable cysteine precursor. Pretreatment with 7-nitroindazole, a specific neuronal nitric oxide synthase inhibitor, also prevented both GSH depletion and nitrotyrosine formation induced by MPTP. Pretreatment with hydrogen peroxide, l -aspartic acid ,-hydroxamate or 1-methyl-4-phenylpyridinium reduced the subsequent cysteine increase in midbrain slice cultures. Studies with chloromethylfluorescein diacetate, a GSH marker, demonstrated dopaminergic neurons in the SN to have increased GSH levels after NAC treatment. These findings suggest that oxidative stress induced by MPTP may reduce neuronal cysteine uptake, via EAAC1 dysfunction, leading to impaired GSH synthesis, and that NAC would exert a protective effect against MPTP neurotoxicity by maintaining GSH levels in dopaminergic neurons. [source] Activation of class I metabotropic glutamate receptors limits dendritic growth of Purkinje cells in organotypic slice culturesEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 11 2006Alexandra Sirzen-Zelenskaya Abstract The development of the dendritic tree of a neuron is a complex process which is thought to be regulated strongly by signals from afferent fibers. We showed previously that the blockade of glutamatergic excitatory neurotransmission has little effect on Purkinje cell dendritic development. We have now studied the effects of glutamate receptor agonists on the development of Purkinje cell dendrites in mouse organotypic slice cultures. The activation of N -methyl- d -aspartate receptors had no major effect on Purkinje cell dendrites and the activation of (RS)-alpha-amino-3-hydroxy-5-methyl-4-isoxazole proprionic acid receptors was strongly excitotoxic so that no analysis of its effects on dendritic development was possible. The activation of metabotropic glutamate receptors led to a very strong inhibition of dendritic growth, resulting in Purkinje cells with very small stubby dendrites. This effect was specific for the activation of class I metabotropic glutamate receptors and could not be reduced by blocking synaptic transmission in the cultures, indicating that it was mediated by receptors present on Purkinje cells. Pharmacological experiments suggest that the signaling pathway involved does not require activation of phospholipase C or protein kinase C. The inhibition of dendritic growth by activation of class I metabotropic glutamate receptor could be a useful negative feedback mechanism for limiting the size of the dendritic tree of Purkinje cells after the establishment of a sufficient number of parallel fiber contacts. This developmental mechanism could protect Purkinje cells from excitotoxic death through excessive release of glutamate from an overload of parallel fiber contacts. [source] Long-term depression activates transcription of immediate early transcription factor genes: involvement of serum response factor/Elk-1EUROPEAN JOURNAL OF NEUROSCIENCE, Issue 2 2006Antje Lindecke Abstract Long-term depression (LTD) is one of the paradigms used in vivo or ex vivo for studying memory formation. In order to identify genes with potential relevance for memory formation we used mouse organotypic hippocampal slice cultures in which chemical LTD was induced by applications of 3,5-dihydroxyphenylglycine (DHPG). The induction of chemical LTD was robust, as monitored electrophysiologically. Gene expression analysis after chemical LTD induction was performed using cDNA microarrays containing >7000 probes. The DHPG-induced expression of immediate early genes (c-fos, junB, egr1 and nr4a1) was subsequently verified by TaqMan polymerase chain reaction. Bioinformatic analysis suggested a common regulator element [serum response factor (SRF)/Elk-1 binding sites] within the promoter region of these genes. Indeed, here we could show a DHPG-dependent binding of SRF at the SRF response element (SRE) site within the promoter region of c-fos and junB. However, SRF binding to egr1 promoter sites was constitutive. The phosphorylation of the ternary complex factor Elk-1 and its localization in the nucleus of hippocampal neurones after DHPG treatment was shown by immunofluorescence using a phosphospecific antibody. We suggest that LTD leads to SRF/Elk-1-regulated gene expression of immediate early transcription factors, which could in turn promote a second broader wave of gene expression. [source] Quantitative effects produced by modifications of neuronal activity on the size of GABAA receptor clusters in hippocampal slice culturesEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 2 2004Serge Marty Abstract The number and strength of GABAergic synapses needs to be precisely adjusted for adequate control of excitatory activity. We investigated to what extent the size of GABAA receptor clusters at inhibitory synapses is under the regulation of neuronal activity. Slices from P7 rat hippocampus were cultured for 13 days in the presence of bicuculline or 4-aminopyridine (4-AP) to increase neuronal activity, or DNQX to decrease activity. The changes provoked by these treatments on clusters immunoreactive for the ,1 and ,2 subunits of the GABAA receptor or gephyrin were quantitatively evaluated. While an increase in activity augmented the density of these clusters, a decrease in activity provoked, in contrast, a decrease in their density. An inverse regulation was observed for the size of individual clusters. Bicuculline and 4-AP decreased whilst DNQX increased the mean size of the clusters. When the pharmacological treatments were applied for 2 days instead of 2 weeks, no effects on the size of the clusters were observed. The variations in the mean size of individual clusters were mainly due to changes in the number of small clusters. Finally, a regulation of the size of GABAA receptor clusters occurred during development in vivo, with a decrease of the mean size of the clusters between P7 and P21. This physiological change was also the result of an increase in the number of small clusters. These results indicate that neuronal activity regulates the mean size of GABAA receptor- and gephyrin-immunoreactive clusters by modifying specifically the number of synapses with small clusters of receptors. [source] 7-Hydroxylated epiandrosterone (7-OH-EPIA) reduces ischaemia-induced neuronal damage both in vivo and in vitroEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 1 2003Ashley K. Pringle Abstract Recent evidence suggests that steroids such as oestradiol reduce ischaemia-induced neurodegeneration in both in vitro and in vivo models. A cytochrome P450 enzyme termed cyp7b that 7-hydroxylates many steroids is expressed at high levels in brain, although the role of 7-hydroxylated steroids is unknown. We have tested the hypothesis that the steroid-mediated neuroprotection is dependent on the formation of 7-hydroxy metabolites. Organotypic hippocampal slice cultures were prepared from Wistar rat pups and maintained in vitro for 14 days. Cultures were then exposed to 3 h hypoxia and neuronal damage assessed 24 h later using propidium iodide fluorescence as a marker of cell damage. Neurodegeneration occurred primarily in the CA1 pyramidal cell layer. The steroids oestradiol, dehydroepiandrosterone and epiandrosterone (EPIA) were devoid of neuroprotective efficacy when present at 100 nm pre-, during and post-hypoxia. The 7-hydroxy metabolites of EPIA, 7,-OH-EPIA and 7,-OH-EPIA significantly reduced neurotoxicity at 100 nm and 10 nm. 7,-OH-EPIA was also neuroprotective in two in vivo rat models of cerebral ischaemia: 0.1 mg/kg 7,-OH-EPIA significantly reduced hippocampal cell loss in a model of global forebrain ischaemia, whereas 0.03 mg/kg was neuroprotective in a model of focal ischaemia even when administration was delayed until 6 h after the onset of ischaemia. Taken together, these data demonstrate that 7-hydroxylation of steroids confers neuroprotective efficacy, and that 7,-OH-epiandrosterone represents a novel class of neuroprotective compounds with potential for use in acute neurodegenerative diseases. [source] Glutamatergic input governs periodicity and synchronization of bursting activity in oxytocin neurons in hypothalamic organotypic culturesEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 12 2003Jean-Marc Israel Abstract During suckling, oxytocin (OT) neurons display a bursting electrical activity, consisting of a brief burst of action potentials which is synchronized throughout the OT neuron population and which periodically occurs just before each milk ejection in the lactating rat. To investigate the basis of such synchronization, we performed simultaneous intracellular recordings from pairs of OT neurons identified retrospectively by intracellular fluorescent labelling and immunocytochemistry in organotypic slice cultures derived from postnatal rat hypothalamus. A spontaneous bursting activity was recorded in 65% of OT neurons; the remaining showed only a slow, irregular activity. Application of OT triggered bursts in nonbursting neurons and accelerated bursting activity in spontaneously bursting cells. These cultures included rare vasopressinergic neurons showing no bursting activity and no reaction to OT. Bursts occurred simultaneously in all pairs of bursting OT neurons but, as in vivo, there were differences in burst onset, amplitude and duration. Coordination of firing was not due to electrotonic coupling because depolarizing one neuron in a pair had no effect on the membrane potential of its partner and halothane and proprionate did not desynchronize activity. On the other hand, bursting activity was superimposed on volleys of excitatory postsynaptic potentials (EPSPs) which occurred simultaneously in pairs of neurons. EPSPs, and consequently action potentials, were reversibly blocked by the non-NMDA glutamatergic receptor antagonist CNQX. Taken together, these data, obtained from organotypic cultures, strongly suggest that a local hypothalamic network governs synchronization of bursting firing in OT neurons through synchronous afferent volleys of EPSPs originating from intrahypothalamic glutamatergic inputs. [source] Activity-dependent modulation of GABAergic synapses in developing rat spinal networks in vitroEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 11 2002Marcelo Rosato-Siri Abstract The role of activity-dependent plasticity in modulating inhibitory synapses was investigated in embryonic rat spinal cord slice cultures, by chronic exposure to non-NMDA receptor blockers. GABAergic synaptic efficacy in control and chronic-treated cultures was investigated by patch-recordings from visually identified spinal interneurons. In both culture groups proximal stimulation induced the appearance of postsynaptic currents (PSCs), which were fully antagonized by 20 µM bicuculline application and reverse polarity at potential values close to those reported for spontaneous GABAergic PSCs. In chronically treated cells GABAergic evoked PSCs displayed a larger failure rate and a smaller coefficient of variation of mean PSC amplitude, when compared to controls. As opposed to controls, chronic GABAergic evoked PSCs did not facilitate upon paired-pulse stimulation. Facilitation at chronic synapses was observed when extracellular calcium levels were decreased below physiological values (< 2 mM). Kainate was used to disclose any functional differences between control and treated slices. In accordance with the presynaptic action of kainate, the application of this drug along with GYKI, an AMPA receptor selective antagonist, changed, with analogous potency, short-term plasticity of GABAergic synapses from control and treated cultures. Nevertheless, in chronic cultures, the downstream effects of such activation unmasked short-term depression. Ultrastructural analysis of synapses in chronically treated cultures showed a reduction both in symmetric synapses and in the number of vesicles at symmetric terminals. Thus, based on electrophysiological and ultrastructural data, it could be suggested that during the development of spinal circuits, GABAergic synapses are modulated by glutamatergic transmission, and thus implying that excitatory transmission regulates the strength of GABAergic synapses. [source] The generation of rhythmic activity in dissociated cultures of rat spinal cordEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 2 2001Jürg Streit Abstract Locomotion in vertebrates is controlled by central pattern generators in the spinal cord. The roles of specific network architecture and neuronal properties in rhythm generation by such spinal networks are not fully understood. We have used multisite recording from dissociated cultures of embryonic rat spinal cord grown on multielectrode arrays to investigate the patterns of spontaneous activity in randomised spinal networks. We were able to induce similar patterns of rhythmic activity in dissociated cultures as in slice cultures, although not with the same reliability and not always with the same protocols. The most reliable rhythmic activity was induced when a partial disinhibition of the network was combined with an increase in neuronal excitability, suggesting that both recurrent synaptic excitation and neuronal excitability contribute to rhythmogenesis. During rhythmic activity, bursts started at several sites and propagated in variable ways. However, the predominant propagation patterns were independent of the protocol used to induce rhythmic activity. When synaptic transmission was blocked by CNQX, APV, strychnine and bicuculline, asynchronous low-rate activity persisted at ,,50% of the electrodes and ,,70% of the sites of burst initiation. Following the bursts, the activity in the interval was transiently suppressed below the level of intrinsic activity. The degree of suppression was proportional to the amount of activity in the preceding burst. From these findings we conclude that rhythmic activity in spinal cultures is controlled by the interplay of intrinsic neuronal activity and recurrent excitation in neuronal networks without the need for a specific architecture. [source] Astrocytic factors protect neuronal integrity and reduce microglial activation in an in vitro model of N -methyl- d -aspartate-induced excitotoxic injury in organotypic hippocampal slice culturesEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 2 2001Nils P. Hailer Abstract Acute CNS lesions lead to neuronal injury and a parallel glial activation that is accompanied by the release of neurotoxic substances. The extent of the original neuronal damage can therefore be potentiated in a process called secondary damage. As astrocytes are known to secrete immunomodulatory and neuroprotective substances, we investigated whether astrocytic factors can attenuate the amount of neuronal injury as well as the degree of microglial activation in a model of excitotoxic neurodegeneration. Treatment of organotypic hippocampal slice cultures with N-methyl- d -aspartate (NMDA) resulted in a reproducible loss of viable granule cells, partial destruction of the regular hippocampal cytoarchitecture and a concomitant accumulation of amoeboid microglial cells at sites of neuronal damage. Astrocyte-conditioned media reduced the amount of NMDA-induced neuronal injury by 45.3%, diminished the degree of microglial activation and resulted in an improved preservation of the hippocampal cytoarchitecture. Transforming growth factor (TGF)-, failed to act as a neuroprotectant and even enhanced the amount of neuronal injury by 52.5%. Direct effects of astrocytic factors on isolated microglial cells consisted of increased microglial ramification and down-regulated expression of intercellular adhesion molecule-1, whereas incubation with TGF-, had no such effects. In summary, our findings show that hitherto unidentified astrocyte-derived factors that are probably not identical with TGF-, can substantially enhance neuronal survival, either by eliciting direct neuroprotective effects or by modulating the microglial response to neuronal injury. [source] Reducing conditions significantly attenuate the neuroprotective efficacy of competitive, but not other NMDA receptor antagonists in vitroEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 11 2000Ashley K. Pringle Abstract Inappropriate activation of NMDA receptors during a period of cerebral ischaemia is a crucial event in the pathway leading to neuronal degeneration. However, significant research has failed to deliver a clinically active NMDA receptor antagonist, and competitive NMDA antagonists are ineffective in many experimental models of ischaemia. The NMDA receptor itself has a number of modulatory sites which may affect receptor function under ischaemic conditions. Using rat organotypic hippocampal slice cultures we have investigated whether the redox modulatory site affects the neuroprotective efficacy of NMDA receptor antagonists against excitotoxicity and experimental ischaemia (OGD). NMDA toxicity was significantly enhanced in cultures pretreated with a reducing agent. The noncompetitive antagonist MK-801 and a glycine-site blocker were equally neuroprotective in both normal and reduced conditions, but there was a significant rightward shift in the dose,response curves of the competitive antagonists APV and CPP and the uncompetitive antagonist memantine. OGD produced neuronal damage predominantly in the CA1 region, which was prevented by MK-801 and memantine, but not by APV or CPP. Inclusion of an oxidizing agent during the period of OGD had no effect alone, but significantly enhanced the neuroprotective potency of the competitive antagonists. These data clearly demonstrate that chemical reduction of the redox modulatory site of the NMDA receptor decreases the ability of competitive antagonists to block NMDA receptor-mediated neuronal damage, and that the reducing conditions which occur during simulated ischaemia are sufficient to produce a similar effect. This may have important implications for the design of future neuroprotective agents. [source] PDGF stimulates the massive expansion of glial progenitors in the neonatal forebrainGLIA, Issue 16 2009M. C. Assanah Abstract Platelet-derived growth factor (PDGF) plays a major role in regulating migration, proliferation, and differentiation of glial progenitors during normal brain development and in the abnormal proliferation and dispersion that drives the formation of malignant gliomas. To further explore the relationship between PDGF's effects on normal glial progenitors and its role in the formation of gliomas, we infected progenitor cells in the subventricular zone (SVZ) of the lateral ventricle of neonatal rat pups with a retrovirus that expresses PDGF and green fluorescent protein (GFP). At 3 days post-injection (dpi), a proliferation of PDGFR,+ progenitors was seen in the SVZ and white matter around the injection site and by 10 dpi the animals had large diffusely infiltrating tumors that resembled glioblastomas. The tumors contained a massive proliferation of both infected and uninfected PDGFR,+ progenitors, suggesting that PDGF was driving tumor formation via both autocrine and paracrine signaling. Rats co-injected with two retroviruses (one that expresses PDGF-IRES-DSRED and one that expresses only GFP) formed tumors that contained a mixture of DSRED+ cells (PDGF producers) and GFP+ cells (recruited progenitors). Time-lapse microscopy of slice cultures confirmed that both DSRED+ and GFP+ cells were highly migratory and proliferative. Furthermore, adding exogenous PDGF to slice cultures generated from nontumor-bearing brains (injected with control GFP retrovirus only) stimulated the migration and proliferation of GFP+ progenitors. These findings reveal the inherent growth factor responsiveness and tumorigenic potential of PDGFR,+ progenitors and highlight the importance of paracrine signaling in stimulating glioma growth and infiltration. © 2009 Wiley-Liss, Inc. [source] Transplanted glioma cells migrate and proliferate on host brain vasculature: A dynamic analysisGLIA, Issue 8 2006Azadeh Farin Abstract Glioma cells have a remarkable capacity to infiltrate the brain and migrate long distances from the tumor, making complete surgical resection impossible. Yet, little is known about how glioma cells interact with the complex microenvironment of the brain. To investigate the patterns and dynamics of glioma cell infiltration and migration, we stereotactically injected eGFP and DsRed-2 labeled rat C6 glioma cells into neonatal rat forebrains and used time-lapse microscopy to observe glioma cell migration and proliferation in slice cultures generated from these brains. In this model, glioma cells extensively infiltrated the brain by migrating along the abluminal surface of blood vessels. Glioma cells intercalated their processes between the endothelial cells and the perivascular astrocyte end feet, but did not invade into the blood vessel lumen. Dynamic analysis revealed notable similarities between the migratory behavior of glioma cells and that previously observed for glial progenitor cells. Glioma cells had a characteristic leading process and migrated in a saltatory fashion, with bursts of migration separated by periods of immobility, and maximum speeds of over 100 ,m/h. Migrating glioma cells proliferated en route, pausing for as short as an hour to divide before the daughter cells resumed migrating. Remarkably, the majority of glioma cell divisions took place at or near vascular branch points, suggesting that mitosis is triggered by local environmental cues. This study provides the first dynamic analysis of glioma cell infiltration in living brain tissue and reveals that the migration and proliferation of transplanted glioma cells is directed by interactions with host brain vasculature. © 2006 Wiley-Liss, Inc. [source] Cholesterol-promoted synaptogenesis requires the conversion of cholesterol to estradiol in the hippocampusHIPPOCAMPUS, Issue 8 2009Lars Fester Abstract Cholesterol of glial origin promotes synaptogenesis (Mauch et al., (2001) Science 294:1354,1357). Because in the hippocampus local estradiol synthesis is essential for synaptogenesis, we addressed the question of whether cholesterol-promoted synapse formation results from the function of cholesterol as a precursor of estradiol synthesis in this brain area. To this end, we treated hippocampal cultures with cholesterol, estradiol, or with letrozole, a potent aromatase inhibitor. Cholesterol increased neuronal estradiol release into the medium, the number of spine synapses in hippocampal slice cultures, and immunoreactivity of synaptic proteins in dispersed cultures. Simultaneous application of cholesterol and letrozole or blockade of estrogen receptors by ICI 182 780 abolished cholesterol-induced synapse formation. As a further approach, we inhibited the access of cholesterol to the first enzyme of steroidogenesis by knock-down of steroidogenic acute regulatory protein, the rate-limiting step in steroidogenesis. A rescue of reduced synaptic protein expression in transfected cells was achieved by estradiol but not by cholesterol. Our data indicate that in the hippocampus cholesterol-promoted synapse formation requires the conversion of cholesterol to estradiol. © 2009 Wiley-Liss, Inc. [source] Distinct, but compensatory roles of PAK1 and PAK3 in spine morphogenesisHIPPOCAMPUS, Issue 9 2008Bernadett Boda Abstract PAK1 and PAK3 belong to a family of protein kinases that are effectors of small Rho GTPases. In humans, mutations of PAK3 have been associated with mental retardation and result in in vitro studies in defects of spine morphogenesis. The functional specificities of PAK1 and PAK3 remain, however, unclear. Here, we investigated using loss and gain of function experiments how PAK1 and PAK3 affect spine morphology in hippocampal slice cultures. We find that while knockdown of PAK3 is associated with an increase in thin, elongated, immature-type spines, downregulation of PAK1 does not alter spine morphology. Conversely, expression of a constitutively active form of PAK3 remains without effect, while expression of constitutively active PAK1 results in the formation of spines with smaller head diameters. Interestingly, expression of constitutively active PAK1 can rescue the long spine phenotype induced by suppression of PAK3. We conclude that while PAK1 and PAK3 share distinct roles in the regulation of spine morphogenesis, their activity may overlap allowing the compensation of the PAK3 deficit by PAK1. This result opens interesting perspectives in the context of reversing the spine defects associated with PAK3 mutations. © 2008 Wiley-Liss, Inc. [source] The immunosuppressant mycophenolate mofetil improves preservation of the perforant path in organotypic hippocampal slice cultures: A retrograde tracing studyHIPPOCAMPUS, Issue 5 2006Tilman M. Oest Abstract Previous studies with excitotoxically lesioned organotypic hippocampal slice cultures (OHSC) have revealed that the immunosuppressant mycophenolate mofetil (MMF) inhibits microglial activation and suppresses neuronal injury in the dentate gyrus. We here investigate whether MMF also has beneficial effects on axon survival in a long-range projection, the perforant path. Complex OHSC including the entorhinal cortex were obtained from Wistar rats (p8); the plane of section ensuring that perforant path integrity was preserved. These preparations were cultured for 9 days in vitro with or without MMF (100 ,g/ml). After fixation, the perforant path was retrogradely labeled by application of the fluorescent dye DiI (1,1,-dioctadecyl-3,3,3,,3,-tetramethylindo-carbocyanine) in the hilus of the dentate gyrus, and neuronal perikarya were immunohistochemically stained by the neuron-specific marker NeuN. Analysis of DiI-labeled and NeuN-stained OHSC by confocal laser scanning microscopy revealed double-labeled neurons in the entorhinal cortex, which projected to the dentate gyrus via the perforant path. Quantitative analysis showed that the number of these double-labeled neurons was 19-fold higher in OHSC treated with MMF than in control cultures (P < 0.05). Our findings indicate that MMF treatment improves preservation of the perforant path and encourage further studies on development and regeneration of long-range projections under the influence of immunosuppressants. © 2006 Wiley-Liss, Inc. [source] Nitric oxide-producing microglia mediate thrombin-induced degeneration of dopaminergic neurons in rat midbrain slice cultureJOURNAL OF NEUROCHEMISTRY, Issue 5 2006Hiroshi Katsuki Abstract Activated microglia are considered to play important roles in degenerative processes of midbrain dopaminergic neurons. Here we examined mechanisms of neurotoxicity of thrombin, a protease known to trigger microglial activation, in organotypic midbrain slice cultures. Thrombin induced a progressive decline in the number of dopaminergic neurons, an increase in nitric oxide (NO) production, and whole tissue injury indicated by lactate dehydrogenase release and propidium iodide uptake. Microglia expressed inducible NO synthase (iNOS) in response to thrombin, and inhibition of iNOS rescued dopaminergic neurons without affecting whole tissue injury. Inhibitors of mitogen-activated protein kinases (MAPKs) such as extracellular signal-regulated kinase (ERK), p38 MAPK and c-Jun N-terminal kinase (JNK) attenuated thrombin-induced iNOS induction and dopaminergic cell death. Whole tissue injury was also attenuated by inhibition of ERK and p38 MAPK. Moreover, depletion of resident microglia from midbrain slices abrogated thrombin-induced NO production and dopaminergic cell death, but did not inhibit tissue injury. Finally, antioxidative drugs prevented thrombin-induced dopaminergic cell death without affecting whole tissue injury. Hence, NO production resulting from MAPK-dependent microglial iNOS induction is a crucial event in thrombin-induced dopaminergic neurodegeneration, whereas damage of other midbrain cells is MAPK-dependent but is NO-independent. [source] Impaired postnatal development of hippocampal neurons and axon projections in the Emx2,/, mutantsJOURNAL OF NEUROCHEMISTRY, Issue 5 2002Nicolai E. Savaskan Abstract The specification and innervation of cerebral subregions is a complex layer-specific process, primed by region-specific transcription factor expression and axonal guidance cues. In Emx2,/, mice, the hippocampus fails to form a normal dentate gyrus as well as the normal layering of principal neurons in the hippocampus proper. Here, we analyzed the late embryonic and postnatal development of the hippocampal formation and its axonal projections in mice lacking Emx2 expression in vitro. As these mutants die perinatally, we used slice cultures of Emx2 mutant hippocampus to circumvent this problem. In late embryonic Emx2,/, cultivated hippocampi, both the perforant path as well as the distribution of calretinin-positive cells are affected. Traced entorhinal afferents in co-cultures with hippocampus from embryonic Emx2,/, mice terminate diffusely in the prospective dentate gyrus in contrast to the layer-specific termination of co-cultures from wild-type littermates. In addition, in brain slice cultures from null mutants the presumptive dentate gyrus failed to develop its normal cytoarchitecture and mature dentate granule cells, including the lack of their mossy fiber projection. Our data indicate that Emx2 is essential for the terminal differentiation of granular cells and the correct formation of extrinsic and intrinsic hippocampal connections. [source] Direct Inhibitory Effect of Glucocorticoids on Corticotrophin-Releasing Hormone Gene Expression in Neurones of the Paraventricular Nucleus in Rat Hypothalamic Organotypic CulturesJOURNAL OF NEUROENDOCRINOLOGY, Issue 9 2008B. Bali Corticotrophin-releasing hormone (CRH) in the parvocellular neurosecretory neurones of hypothalamic paraventricular nucleus governs neuroendocrine stress cascade and is the major target of the negative feedback effect of corticosteroids. To assess whether glucocorticoids exert their inhibitory effect on CRH expression directly on parvocellular neurones or indirectly through a complex neuronal circuit, we examined the effect of corticosterone (CORT) and dexamethasone (DEX) on CRH mRNA levels in slice explant cultures of the rat hypothalamus. Organotypic slice cultures were prepared from 6 days old rat pups and maintained in vitro for 14 days. CRH mRNA expression was measured by in situ hybridisation histochemistry. Under basal conditions, CRH mRNA expressing cells were exclusively revealed in the paraventricular region along the third ventricle. Inhibition of action potential spike activity by tetrodotoxin (TTX, 1 ,m) reduced CRH mRNA signal in the organotypic cultures. CORT (500 nm) or DEX (50 nm) treatment for 24 h significantly inhibited CRH expression in the parvocellular neurones and this effect of corticosteroids was not affected following blockade of voltage dependent sodium channels by TTX. Forskolin-stimulated CRH mRNA levels in the paraventricular nucleus were also inhibited by CORT or DEX in the presence and in the absence of TTX. These studies identify paraventricular CRH neurones as direct target of corticosteroid feedback. Type II corticosteroid receptor agonists act directly on paraventricular neurones to inhibit basal and forskolin-induced CRH mRNA expression in explant cultures of the rat hypothalamus. [source] | |||||||||||||