76 research outputs found
The increased activity of TRPV4 channel in the astrocytes of the adult rat hippocampus after cerebral hypoxia/ischemia.
No full textThe polymodal transient receptor potential vanilloid 4 (TRPV4) channel, a member of the TRP channel family, is a calcium-permeable cationic channel that is gated by various stimuli such as cell swelling, low pH and high temperature. Therefore, TRPV4-mediated calcium entry may be involved in neuronal and glia pathophysiology associated with various disorders of the central nervous system, such as ischemia. The TRPV4 channel has been recently found in adult rat cortical and hippocampal astrocytes; however, its role in astrocyte pathophysiology is still not defined. In the present study, we examined the impact of cerebral hypoxia/ischemia (H/I) on the functional expression of astrocytic TRPV4 channels in the adult rat hippocampal CA1 region employing immunohistochemical analyses, the patch-clamp technique and microfluorimetric intracellular calcium imaging on astrocytes in slices as well as on those isolated from sham-operated or ischemic hippocampi. Hypoxia/ischemia was induced by a bilateral 15-minute occlusion of the common carotids combined with hypoxic conditions. Our immunohistochemical analyses revealed that 7 days after H/I, the expression of TRPV4 is markedly enhanced in hippocampal astrocytes of the CA1 region and that the increasing TRPV4 expression coincides with the development of astrogliosis. Additionally, adult hippocampal astrocytes in slices or cultured hippocampal astrocytes respond to the TRPV4 activator 4-alpha-phorbol-12,-13-didecanoate (4αPDD) by an increase in intracellular calcium and the activation of a cationic current, both of which are abolished by the removal of extracellular calcium or exposure to TRP antagonists, such as Ruthenium Red or RN1734. Following hypoxic/ischemic injury, the responses of astrocytes to 4αPDD are significantly augmented. Collectively, we show that TRPV4 channels are involved in ischemia-induced calcium entry in reactive astrocytes and thus, might participate in the pathogenic mechanisms of astroglial reactivity following ischemic insult
SHRiMP2: Sensitive yet Practical Short Read Mapping
No full textAbstract
Summary: We report on a major update (version 2) of the original SHort Read Mapping Program (SHRiMP). SHRiMP2 primarily targets mapping sensitivity, and is able to achieve high accuracy at a very reasonable speed. SHRiMP2 supports both letter space and color space (AB/SOLiD) reads, enables for direct alignment of paired reads and uses parallel computation to fully utilize multi-core architectures.
Availability: SHRiMP2 executables and source code are freely available at: http://compbio.cs.toronto.edu/shrimp/.
Contact: [email protected]
Supplementary information: Supplementary data are available at Bioinformatics online.</jats:p
Polydendrocytes display large lineage plasticity following focal cerebral ischemia.
No full textPolydendrocytes (also known as NG2 glial cells) constitute a fourth major glial cell type in the adult mammalian central nervous system (CNS) that is distinct from other cell types. Although much evidence suggests that these cells are multipotent in vitro, their differentiation potential in vivo under physiological or pathophysiological conditions is still controversial.To follow the fate of polydendrocytes after CNS pathology, permanent middle cerebral artery occlusion (MCAo), a commonly used model of focal cerebral ischemia, was carried out on adult NG2creBAC:ZEG double transgenic mice, in which enhanced green fluorescent protein (EGFP) is expressed in polydendrocytes and their progeny. The phenotype of the EGFP(+) cells was analyzed using immunohistochemistry and the patch-clamp technique 3, 7 and 14 days after MCAo. In sham-operated mice (control), EGFP(+) cells in the cortex expressed protein markers and displayed electrophysiological properties of polydendrocytes and oligodendrocytes. We did not detect any co-labeling of EGFP with neuronal, microglial or astroglial markers in this region, thus proving polydendrocyte unipotent differentiation potential under physiological conditions. Three days after MCAo the number of EGFP(+) cells in the gliotic tissue dramatically increased when compared to control animals, and these cells displayed properties of proliferating cells. However, in later phases after MCAo a large subpopulation of EGFP(+) cells expressed protein markers and electrophysiological properties of astrocytes that contribute to the formation of glial scar. Importantly, some EGFP(+) cells displayed membrane properties typical for neural precursor cells, and moreover these cells expressed doublecortin (DCX)--a marker of newly-derived neuronal cells. Taken together, our data indicate that polydendrocytes in the dorsal cortex display multipotent differentiation potential after focal ischemia
A comprehensive evaluation of alignment algorithms in the context of RNA-seq.
No full textTranscriptome sequencing (RNA-Seq) overcomes limitations of previously used RNA quantification methods and provides one experimental framework for both high-throughput characterization and quantification of transcripts at the nucleotide level. The first step and a major challenge in the analysis of such experiments is the mapping of sequencing reads to a transcriptomic origin including the identification of splicing events. In recent years, a large number of such mapping algorithms have been developed, all of which have in common that they require algorithms for aligning a vast number of reads to genomic or transcriptomic sequences. Although the FM-index based aligner Bowtie has become a de facto standard within mapping pipelines, a much larger number of possible alignment algorithms have been developed also including other variants of FM-index based aligners. Accordingly, developers and users of RNA-seq mapping pipelines have the choice among a large number of available alignment algorithms. To provide guidance in the choice of alignment algorithms for these purposes, we evaluated the performance of 14 widely used alignment programs from three different algorithmic classes: algorithms using either hashing of the reference transcriptome, hashing of reads, or a compressed FM-index representation of the genome. Here, special emphasis was placed on both precision and recall and the performance for different read lengths and numbers of mismatches and indels in a read. Our results clearly showed the significant reduction in memory footprint and runtime provided by FM-index based aligners at a precision and recall comparable to the best hash table based aligners. Furthermore, the recently developed Bowtie 2 alignment algorithm shows a remarkable tolerance to both sequencing errors and indels, thus, essentially making hash-based aligners obsolete
Primary and secondary antibodies used for immunohistochemistry.
No full text<p>Aldh1, aldehyde dehydrogenase–1; APC adenomatous polyposis coli; CD11b, integrin alpha M; DCX, doublecortin; GFAP, glial fibrillary acidic protein; GFP, green fluorescent protein; GLAST, glutamate/aspartate transporter; NG2, chondroitin sulfate proteoglycan; PCNA, proliferating cell nuclear antigen; PDGFαR, platelet-derived growth factor receptor alpha; PDGFβR, platelet-derived growth factor receptor beta; S100β, β-subunit of S100 calcium binding protein; GAR 594/660, goat anti-rabbit IgG conjugated with Alexa Fluor 594 or 660; GAM 594/660, goat anti-mouse IgG conjugated with Alexa Fluor 594 or 660 (all secondary antibodies from Invitrogen).</p
Polydendrocytes differentiate into cells with distinct current patterns after MCAo.
No full text<p><b><i>A</i></b>, A typical current pattern of polydendrocytes measured 3 days after MCAo in response to cell membrane depolarization from a holding potential of −70 mV to +40 mV and hyperpolarization to −160 mV (see the inset, bottom). Polydendrocytes showed a current pattern with K<sub>DR</sub>, K<sub>A</sub>, small K<sub>IR</sub> currents and fast activating Na<sup>+</sup> inward currents in a few cells 3 days after MCAo. Zero current is marked by the dashed line. <b><i>a1–a4</i></b>, Polydendrocyte expressing enhanced green fluorescent protein (EGFP) 3 days after MCAo (<b><i>a2</i></b>) loaded with Alexa Fluor 594 hydrazid during patch-clamp measurement (<b><i>a3</i></b>) subsequently stained with an antibody directed against nestin (<b><i>a4</i></b>). White arrow indicates the recorded cell. Scale bars, 50 µm. <b><i>B</i></b>, Current pattern of EGFP<sup>+</sup> astrocytes 7 days after MCAo recorded using the same protocol as in (<b><i>A</i></b>). EGFP<sup>+</sup> astrocytes showed a passive current pattern, i.e., time- and voltage-independent non-decaying K<sup>+</sup> currents, with only small voltage activated K<sup>+</sup> currents. Zero current is marked by the dashed line. <b><i>b1–b4</i></b>, Astrocytes expressing EGFP 7 days after MCAo (<b><i>b2</i></b>) loaded with Alexa Fluor 594 hydrazid during patch-clamp measurement (<b><i>b3</i></b>), subsequently stained with an antibody directed against glial fibrillary acidic protein (GFAP, <b><i>b4</i></b>), a marker of astrocytes. Note dye coupling (b3) between EGFP<sup>+</sup> cells with a passive current pattern compared to EFGP<sup>+</sup> cells with a complex current pattern in A (a3). Scale bars, 50 µm. <b><i>C</i></b>, Current pattern of EGFP<sup>+</sup> neuronal precursor cells 7 days after MCAo measured with the same protocol as in (<b><i>A</i></b>). EGFP<sup>+</sup> neuronal precursor cells displayed outwardly rectifying K<sup>+</sup> currents (K<sub>DR</sub>, K<sub>A</sub> currents) together with higher Na<sup>+</sup> currents when compared to polydendrocytes. <b><i>c1–c4</i></b>, Neuronal precursor cells expressing EGFP 7 days after MCAo (<b><i>c2</i></b>), loaded with Alexa Fluor 594 hydrazid during patch-clamp measurement (<b><i>c3</i></b>), subsequently stained with an antibody directed against doublecortin (DCX, <b><i>c4</i></b>), a marker of newly-derived neuronal precursor cells. Scale bars, 50 µm. <b><i>D</i></b>, Current pattern of EGFP<sup>+</sup> oligodendrocytes 7 days after MCAo measured with the same protocol as in (<b><i>A</i></b>). EGFP<sup>+</sup> oligodendrocytes displayed time- and voltage-independent K<sup>+</sup> currents decaying during the duration of the voltage pulse. <b><i>d1–d4</i></b>, Oligodendrocytes expressing EGFP 7 days after MCAo (<b><i>d2</i></b>) loaded with Alexa Fluor 594 hydrazid during patch-clamp measurement (<b><i>d3</i></b>), subsequently stained with an antibody directed against Olig2 (<b><i>d4</i></b>). <b>E</b>, Graph showing the percentage of EGFP<sup>+</sup> cell with distinct current patterns. Asterisks indicate significant differences between control cortex and post-ischemic cortex; **, p<0.01; ***, p<0,001.</p
Quantification of the changes in the number of enhanced green fluorescent protein positive (EGFP<sup>+</sup>) cells and in the expression of markers typical for glia, neurons and proliferating cells evoked by MCAo.
No full text<p><b><i>A</i></b>, Brain sections showing the increasing number and different distribution of EGFP<sup>+</sup> cells 3, 7 and 14 days after MCAo (<b><i>A2, A3, A4</i></b>) when compared to controls (<b><i>A1</i></b>). Borders of the ischemic tissue are highlighted with the dashed lines. Scale bars, 50 µm. <b><i>B</i></b>, Schematic figure of coronal slices of the ipsilateral and contralateral hemispheres depicting the size and localization of tissue injury 7 days after MCAo (grey region), together with the highlighted regions used for immunohistochemical quantification (yellow squares) in both hemispheres; Cx, cortex; Cc, corpus callosum. <b><i>C</i></b>, Bar graph showing the quantification of EGFP<sup>+</sup> cell numbers per 1 mm<sup>3</sup> in the ipsilateral and contralateral hemispheres in control mice and after MCAo. <b><i>D</i></b>, Graph showing the percentage of EGFP<sup>+</sup> cells expressing markers of polydendrocytes (NG2), proliferating cells (Ki-67, nestin), astrocytes (glial fibrillary acidic protein, GFAP) and neuronal precursor cells (doublecortin, DCX) in controls and after MCAo. Asterisks indicate significant differences between control cortex and post-ischemic cortex; *, p<0.05; ***, p<0.001.</p
Electrophysiological properties of EGFP<sup>+</sup> neuronal precursor cells.
No full text<p>RMP, resting membrane potential; Rm, membrane resistance; Cm, membrane capacitance; K<sub>IR</sub>, current density of inwardly-rectifying K<sup>+</sup> currents; K<sub>DR</sub>, current density of voltage-gated delayed outwardly-rectifying K<sup>+</sup> currents; K<sub>A</sub>, current density of the fast activating and inactivating outwardly-rectifying K<sup>+</sup> currents; Na, current density of Na<sup>+</sup> currents; n, number of measured cells; %, percentage of the cell type.</p
Enhanced green fluorescent protein positive (EGFP<sup>+</sup>) cells express markers of astrocytes and neuronal precursor cells in the dorsal cortex after ischemia.
No full text<p><b><i>A</i></b>, EGFP<sup>+</sup> cells in sham-operated mice (control) expressed a high level of NG2 proteoglycan in the majority of processes. White arrows highlight several examples. <b><i>B</i></b>, Virtually no EGFP<sup>+</sup> cells in the dorsal control cortex were positive for glial fibrillary acidic protein (GFAP). <b><i>C</i></b>, Low density of EGFP<sup>+</sup> cells in the subventricular zone (SVZ). EGFP<sup>+</sup> cells were never labeled for doublecortin (DCX) in the SVZ of control brains or those after ischemia, which indicates the absence of polydendrocytes from the process of adult neurogenesis; LV, lateral ventricle. <b><i>D</i></b>, Three days after MCAo the number of EGFP<sup>+</sup> cells is significantly increased when compared to controls. Moreover, a large number of EGFP<sup>+</sup> cells expressed markers of proliferating cells – nestin (<b><i>E</i></b>) and Ki-67 (<b><i>F</i></b>). <b><i>G</i></b>, EGFP<sup>+</sup> cells were present at high density seven days after MCAo; however, only a small subpopulation of them expressed NG2 proteoglycan. <b><i>H</i></b>, Some EGFP<sup>+</sup> cells were positive for the astrocytic marker GFAP. <b><i>I</i></b>, Starting 7 days after MCAo some scattered cells expressed DCX in their cell bodies and processes. <b><i>J</i></b>, Fourteen days after MCAo only a small subpopulation of EGFP<sup>+</sup> cells expressed NG2 proteoglycan. <b><i>K</i></b>, EGFP<sup>+</sup> cells expressing glutamate/aspartate transporter (GLAST), a marker of mature astrocytes. <b><i>L</i></b>, A subpopulation of EGFP<sup>+</sup> cells displaying polarized and highly developed processes, which were positive for DCX. Scale bars, 50 µm.</p
Electrophysiological properties of EGFP<sup>+</sup> cells with a passive current pattern.
No full text<p>RMP, resting membrane potential; Rm, membrane resistance; Cm, membrane capacitance; K<sub>IR</sub>, current density of inwardly-rectifying K<sup>+</sup> currents; K<sub>DR</sub>, current density of voltage-gated delayed outwardly-rectifying K<sup>+</sup> currents; K<sub>A</sub>, current density of the fast activating and inactivating outwardly-rectifying K<sup>+</sup> currents; Na, current density of Na<sup>+</sup> currents; n, number of measured cells; %, percentage of the cell type.</p
- …
