ABSTRACT: Using long-oligo beadschip arrays, we examined the global gene expression profile in cultured hippocampal slices under the simulated ischemic condition with and without the presence of DIDS . Control-ACSF: cultured hippocampal slices were incubated with ACSF for 2 hours and were analysed on whole-genome expression chips to determine the gene expression profile. This group was used as controls for IS-treated groups and ACSF+DIDS-treated grous. IS: cultured hippocampal slices were treated wtih IS for 2 hours and the gene expression was analyzed and compared with the control group. IS+DIDS: cultured slices were treated with both IS and DIDS for 2 hours and the gene expression profile was determined. Differentially expression genes were identified by comparing with IS group. ACSF+DIDS: cultured slices were incubated with both ACSF and DIDS for 2 hours and gene expression profile was determined. This group was used as a control for IS+DIDS group. ACSF: artificial cerebral spinal fluid DIDS: 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid IS: ischemic solution
Project description:Using long-oligo beadschip arrays, we examined the global gene expression profile in cultured hippocampal slices under the simulated ischemic condition with and without the presence of DIDS .
Project description:RNF10 is a synapse-to-nucleus protein messsenger regulating NMDAR-dependent gene trascription. We have charaterized the impact of the absence of RNF10 on structural synaptic plasticity. In this dataset we included expression data from homogenates of DIV14 rat organotypic hippocampal slices lentivirally infected with shRNF10 or scramble construct 8 total samples were analyzed (4 shRNF10 and 4 scramble).
Project description:Effect of different chemical stimuli (NMDA(N-methyl-D-aspartic acid), forskolin/rolipram/0 Mg, bicuculline) on gene expression after 1 h of stimulation in hippocampal slices
Project description:Adult cardiac tissue undergoes a rapid process of dedifferentiation when cultured in vitro. The aim of this experiment was to compare the transcriptome of freshly prepared rat myocardial slices with slices cultured in unloaded conditions (no electromechanical stimulation) for 24 hours, electromechanical stimulation @ SL=1.8 for 24 hours & electromechanical stimulation @ SL=2.2 for 24 hours.
Project description:Brain slice cultures offer advantages over other in vitro methods, as they mimic numerous in vivo aspects. For most purposes, slices of the developing brain, termed organotypic slice cultures , preserve a high degree of cellular differentiation and tissue organization. The entorhino-hippocampal connection (EHP) is the main entrance of information to the hippocampus proper and the dentate gyrus. Also it has some specific features that make them particularly interesting in studies of axonal regeneration: (i) the culture method obviates the need for extensive animal surgery and requires less time than other in vivo approaches; (ii) the EHP is reproduced easily in vitro in cultures with a degree of laminar specificity similar to that found in vivo; (iii) the EHP is myelinated both in vitro and in vivo; and (iv) most of the cellular and molecular barriers to axon regeneration are present after the axotomy of the EHP in vitro. Altogether this model is useful to evaluate axon regeneration and putative estrategies to promote axonal growth. Keywords: organotypic slice cultures; axonal lession; gene expression To evaluate the genes whose transcription was regulated after 1, 3 and 7 days after EHP (Entorhino-Hippocampal Pathway) axotomy, RNA samples were analyzed with Agilent whole genome rat long oligonucleotide (44 K base) probe based microarrays.
Project description:The neurite outgrowth inhibitory myelin protein Nogo-A has been well studied in the context of central nervous system (CNS) injury and disease. We studied the effects of the application of neutralizing anti-Nogo-A antibodies (11C7 and 7B12) in intact CNS tissue in vitro using rat organotypic hippocampal slice cultures. This study had the purpose of elucidating the role of Nogo-A in the adult intact CNS and determining the consequences of its neutralization through antibody application. In vitro cultures treated with anti-Nogo-A antibody showed an elicited growth response. The results also gave indications that hippocampal circuitry might be altered due to the regulation at the synaptic and neurotransmission level. Experiment Overall Design: Nogo-A function in the intact CNS tissue is not well known, but its neutralization in vivo produced a transitory growth response of Purkinje axons and of the corticospinal tract in intact adult rats (Buffo et al., 2000; Bareyre et al., 2002; Gianola et al., 2003). Nogo-A is relatively highly expressed in oligodendrocytes and some neurons of the hippocampus (Huber et al., 2002; Meier et al., 2003; Gil et al., 2006; Trifunovski et al., 2006). Organotypic hippocampal slice cultures are a good in vitro model to study hippocampal function and structure (Stoppini et al., 1991; 1993; Bahr, 1995; Gahwiler et al., 1997; Hakkoum et al., 2006). They mature in vitro and retain many in vivo features from a structural and functional perspective. We chose this model to study the effects of acute Nogo-A neutralization, using two function blocking monoclonal antibodies, 11C7 and 7B12 (Oertle et al., 2003; Wiessner et al., 2003; Liebscher et al., 2005), exclusively targeted against the Nogo-A specific region. Hippocampal slices from P7 Wistar rats were cultured for 21 DIV. Control untreated cultures where cultured for additional 5days for a total of 26DIV, while control IgG, and 11C7 and 7B12 were added to 21DIV cultures which were then further cultured for 5days, changing medium every 2 days. All the conditions were repeated in triplicates with separate cultures from different animals. For each condition and experimental replicate 24 cultures were pooled together before being processed for RNA extraction. Data analysis was performed by GeneSpring 7.2 (Silicon Genetics, Agilent, CA, US) comparing 11C7 and 7B12 treated samples versus Not treated and IgG treated, as controls. A present call filter (2 out of 3 present calls in at least one out of the 3 experimental replicates) was applied. Normalization was run per chip as well as per gene to the median of the control replicates. Data were statistical restricted through a 1-way Anova (pâ?¤0.05). A final threshold of â?¥1.2 fold of increase or decrease in the expression level of each single transcript was applied. Regulated transcripts have been assigned to functional categories according to GeneOntology as well as literature and database mining (Pubmed; Bioinformatics Harvester EMBL Heidelberg; Rat Genome Database).
Project description:Transcriptional profiling of rat hippocampus 4.5h post intracranial self-stimulation (ICSS) show that this treatment, which improves learning and memory processes in a variety of paradigms of implicit and explicit memory, regulates gene expression of genes related to cAMP-mediated signaling and regulation of synaptic plasticity, among others. Rats were implanted with a monopolar stainless steel electrode aimed at the lateral hypothalamus in the right hemisphere. Hippocampal gene expression was analyzed 4.5h post treatment comparing rats subjected to acute self-stimulation (2500 stimulation trains; ICSS group) with sham-operated rats (with electrode placement but placed in the ICSS without stimulation during 40 min.; Sham group). Two-condition experiment, ICSS and Sham. Every sample consisted of pooled hippocampi ipsilateral to the electrode from three rats. Slices between Bregma levels -2 and -4 were used for dissections of the hippocampi. Common reference design, comparing every condition versus a brain pooled tissue consisting of pooled hippocampal tissue ipsilateral to the electrode of sham-operated (n=3) and stimulated (n=3) rats. Biological replicates: 4 DBS and 4 sham. One replicate per array.
Project description:Metabolic stress and the increased production of reactive oxygen species (ROS) are two main contributors to neuronal damage and synaptic plasticity in acute ischemic stroke (AIS). The superoxide scavenger MnTMPyP has been previously reported to have a neuroprotective effect in organotypic hippocampal slices and to modulate synaptic transmission after in vitro hypoxia and oxygen glucose deprivation (OGD). However, the mechanisms involved in the effect of this scavenger remain elusive. In this study we evaluated the concentration dependent effect of MnTMPyP on synaptic transmission during ischemia and post-ischemic synaptic potentiation. We also investigated the complex molecular changes supporting cellular adaptation to metabolic stress and how these are modulated by MnTMPyP. Electrophysiological data showed that MnTMPyP causes a concentration dependent decrease in baseline synaptic transmission and impairment of synaptic potentiation. Proteomic analysis performed on MnTMPyP treated tissue indicated an impairment in vesicular trafficking mechanisms including alteration of Secretory Carrier Membrane Protein 3 (SCAMP3) and RAB proteins and downregulation of mitochondrial-mediated apoptosis. Alterations of vesicular trafficking may lead to reduced probability of neurotransmitter release and AMPA receptor activity, resulting in the observed modulatory effect of MnTMPyP. In ischemia and glucose deprivation, protein enrichment analysis highlighted impairments in cell proliferation and differentiation, such as TGFβ1 and CDKN1B signalling, in addition to downregulation of actin signalling, cell stress and mitochondrial-mediated apoptosis. Taken together our results indicate modulation of neuronal sensitivity to the ischemic insult, and a complex role for MnTMPyP on synaptic transmission and plasticity and provide molecular insights into the mechanisms mediating the effects of MnTMPyP during ischemia
Project description:Paradoxical sleep function remains unknown although several studies indicate that it might play a role in learning and memory. To investigate what modifications paradoxical sleep may bring at the molecular level in neocortex and in hippocampal formation, we profiled gene expression in these structures in rats with different quantities of PS by cDNA microarrays approach. The first group of rats (n=12) was deprived of PS (PSD) by the multiple platform method during 78h, the second group of rats was allowed to recover from this deprivation (PSR) during 6 hours (n=12), the third group of rats remained in their home cage during all the protocol PSC (n=12). All animals were euthanasied at 16h. An equal mass amount of total RNA from the right neocortex and hippocampal formation was pooled from two sets of 6 rats from each experimental group (PSC, PSD, PSR). For the first set of animals, two independent pools of total RNA from both structures were made for each condition to control for the technical variability of the microarray method.