Biomedical subjects

Gary P Schools

Publications and source records attributed to Gary P Schools.

5 recordsLinked to original sources

PubMed2006

Development of gap junctions in hippocampal astrocytes: evidence that whole cell electrophysiological phenotype is an intrinsic property of the individual cell.

Gap junction communication between astrocytes is prevalent and has been proposed to be involved in several astrocyte functions. One such proposal involves gap junctions in potassium spatial buffering. However, little is known about the developmental time course of gap junction coupling and how much the syncytium affects whole cell measurements of ion currents. Our previous work described three types of hippocampal astrocyte, each with a distinct electrophysiological profile when recorded in whole cell voltage-clamp mode. In the current study we correlated post-whole cell recording immunohistochemistry for GLAST and the spread of injected dye from the recorded cell with the measured electrophysiological phenotype to quantify cell coupling of astrocytes and the type of astrocyte coupled, in the rat hippocampus. We found that passive astrocytes, which predominate after 3 wk postnatally, have much lower membrane resistances (Rm) and are more frequently dye coupled and to more cells, than outwardly and variably rectifying astrocytes that predominate in early postnatal development. Dye coupling in GLAST(+) cells was first detected in the first postnatal week and the degree of coupling peaked before the complete transition to the low Rm, passive electrophysiological type. Also, the degree of dye coupling did not correlate with the passive electrophysiological phenotype. Passive cells were also detected after pretreatment with a gap junction inhibitor. Further evidence that cell coupling does not contribute to the mature astrocyte electrophysiological phenotype came from recording of excised membrane patches, which predominantly corresponded to the ion channel expression profiles of their cells of origin. These findings imply that in the hippocampus, interastrocyte cell coupling likely contributes little to the overall whole cell current profile of diverse glia, and the electrophysiological passivity reflects the intrinsic ion channel expression of the mature astrocyte.

Animals↗

PubMed2006

Pharmacological comparison of swelling-activated excitatory amino acid release and Cl- currents in cultured rat astrocytes.

Ubiquitously expressed volume-regulated anion channels (VRACs) are chloride channels which are permeable to a variety of small organic anions, including the excitatory amino acids (EAAs) glutamate and aspartate. Broad spectrum anion channel blockers strongly reduce EAA release in cerebral ischaemia and other pathological states associated with prominent astrocytic swelling. However, it is uncertain whether VRAC serves as a major pathway for EAA release from swollen cells. In the present study, we measured swelling-activated release of EAAs as D-[3H]aspartate efflux, and VRAC-mediated Cl- currents by whole-cell patch clamp in cultured rat astrocytes. We compared the pharmacological profiles of the swelling-activated EAA release pathway and Cl- currents. The expression of candidate Cl- channels was confirmed by RT-PCR. The maxi Cl- channel (p-VDAC) blocker Gd3+, the ClC-2 inhibitor Cd2+, and the MDR-1 blocker verapamil did not affect EAA release or VRAC currents. An antagonist of calcium-sensitive Cl- channels (CaCC), niflumic acid, had little effect on EAA release and only partially inhibited swelling-activated Cl- currents. The phorbol ester PDBu, which blocks ClC-3-mediated Cl- currents, had no effect on VRAC currents and up-regulated EAA release. In contrast, DCPIB, which selectively inhibits VRACs, potently suppressed both EAA release and VRAC currents. Two other relatively selective VRAC inhibitors, tamoxifen and phloretin, also blocked the VRAC currents and strongly reduced EAA release. Taken together, our data suggest that (i) astrocytic volume-dependent EAA release is largely mediated by the VRAC, and (ii) the ClC-2, ClC-3, ClC-4, ClC-5, VDAC, CaCC, MDR-1 and CFTR gene products do not contribute to EAA permeability.

Animals↗

PubMed2005

Development of GLAST(+) astrocytes and NG2(+) glia in rat hippocampus CA1: mature astrocytes are electrophysiologically passive.

Glia show marked heterogeneity in terms of electrophysiology in the developing brain, and two major types can be identified based on GFAP or NG2 expression. However, it remains to be determined if such an electrophysiological diversity holds for the adult brain and how GFAP and NG2 lineage glia are associated with different electrophysiological phenotypes during the course of development. To address these fundamental questions, we performed in situ whole cell recording from morphologically identified glia from the rat hippocampal CA1 region from postnatal (P) days 1-106 and double-stained postrecorded cells with GLAST and NG2 antibodies. We found glia express mostly voltage-gated outward K(+) currents and also have inward Na(+) currents in the newborn (P1-P3), but these are no longer present after P22. They consist equally of GLAST(+) and NG2(+) cells in the newborn, but are mainly NG2(+) in juvenile animals (P4-P21). Glia showing voltage-gated outward and inward K(+) currents are also present at P1, peak at P5 and decline to a stationary level of approximately 10% in the adult. They are GLAST(+) astrocytes from newborn to juvenile but NG2(+) glia in the adult. Electrophysiologically passive glia first appear at P4 and increase to 91% in adults, of which 85% are GLAST(+). These results indicate that glial electrophysiological diversity occurs predominantly in the developing brain. While most glia in the NG2 lineage preserve a certain amount of voltage-gated ion conductances, mature GLAST(+) astrocytes are electrophysiologically passive.

Action Potentials↗

PubMed2003

Electrophysiologically "complex" glial cells freshly isolated from the hippocampus are immunopositive for the chondroitin sulfate proteoglycan NG2.

We have recently described a subgroup of isolated glial fibrillary acidic protein-positive (GFAP+) hippocampal astrocytes that predominantly express outwardly rectifying currents (which we term "ORAs" for outwardly rectifying astrocytes), which are similar to the currents already described for hippocampal GFAP- "complex glia." We now report that post-recording staining of cells that were first selected as "complex" by morphology and then confirmed by their electrophysiological characteristics were NG2+ approximately 90% of the time. Also, the morphology of freshly isolated NG2+ cells differs from that of isolated GFAP+ ORAs in having a smaller and round cell body with thinner processes, which usually are collapsed back onto the soma. Upon detailed examination, NG2+ cells were found to differ quantitatively in some electrophysiological characteristics from GFAP+ ORAs. The outward, transient K+ currents (IKa) in the NG2+ cells showed a slower decay than the IKa in ORAs, and their density decreased in NG2+ cells from older animals. The other two major cation currents, the voltage-activated Na+ and outwardly delayed rectifier K+ currents, were similar in NG2+ cells and ORAs. To further distinguish isolated complex cells from outwardly rectifying GFAP+ astrocytes, we performed immunocytochemistry for glial markers in fixed, freshly isolated rat hippocampal glia. NG2+ cells were negative for GFAP and also for the astrocytic glutamate transporters GLT-1 and GLAST. Thus the isolated hippocampal NG2+ glial cells, though having an electrophysiological phenotype similar to that of ORAs, are an immunologically and morphologically distinct glial cell population and most likely represent NG2+ cells in situ.

Amino Acid Transport System X-AG↗

PubMed2002

Differential display: a critical analysis.

Differential display (DD) is a well-established analytical tool for measuring gene expression that is still popular due to its documented success and ability to identify novel genes not yet available for analysis by more powerful microarray hybridization. For a comprehensive analysis of all mRNAs in a given cell, it is statistically predicted that at least 240 different DD primer combinations are required. This prediction, however, has never been empirically tested. Using far more primer combinations than that predicted to evaluate 90% of the mRNAs in a cell, plus other modifications, we identified and confirmed the induction of five mRNAs by hydrogen peroxide in HA-1 hamster cells. However, five other known oxidant-inducible mRNAs were not identified by DD. Filter microarray hybridization did not result in the identification of any additional species modulated twofold or greater but previous two-dimensional protein gel electrophoresis identified 15 induced protein species. We conclude that the current statistical prediction for comprehensive analysis of all the mRNAs in a given cell is inaccurate, at least in our hands, and further conclude that DD is a useful but less than comprehensive method for assessing changes in mRNA levels.

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