Gary P. Schools

TWIK-1 and TREK-1 Are Potassium Channels Contributing Significantly to Astrocyte Passive Conductance in Rat Hippocampal Slices

Expression of a linear current–voltage (I–V) relationship (passive) K+ membrane conductance is a hallmark of mature hippocampal astrocytes. However, the molecular identifications of the K+ channels underlying this passive conductance remain unknown. We provide the following evidence supporting significant contribution of the two-pore domain K+ channel (K2P) isoforms, TWIK-1 and TREK-1, to this conductance. First, both passive astrocytes and the cloned rat TWIK-1 and TREK-1 channels expressed in CHO cells conduct significant amounts of Cs+ currents, but vary in their relative PCs/PK permeability, 0.43, 0.10, and 0.05, respectively. Second, quinine, which potently inhibited TWIK-1 (IC50 = 85 μm) and TREK-1 (IC50 = 41 μm) currents, also inhibited astrocytic passive conductance by 58% at a concentration of 200 μm. Third, a moderate sensitivity of passive conductance to low extracellular pH (6.0) supports a combined expression of acid-insensitive TREK-1, and to a lesser extent, acid-sensitive TWIK-1. Fourth, the astrocyte passive conductance showed low sensitivity to extracellular Ba2+, and extracellular Ba2+ blocked TWIK-1 channels at an IC50 of 960 μm and had no effect on TREK-1 channels. Finally, an immunocytochemical study showed colocalization of TWIK-1 and TREK-1 proteins with the astrocytic markers GLAST and GFAP in rat hippocampal stratum radiatum. In contrast, another K2P isoform TASK-1 was mainly colocalized with the neuronal marker NeuN in hippocampal pyramidal neurons and was expressed at a much lower level in astrocytes. These results support TWIK-1 and TREK-1 as being the major components of the long-sought K+ channels underlying the passive conductance of mature hippocampal astrocytes.

MGLUR3 AND MGLUR5 ARE THE PREDOMINANT METABOTROPIC GLUTAMATE RECEPTOR MRNAS EXPRESSED IN HIPPOCAMPAL ASTROCYTES ACUTELY ISOLATED FROM YOUNG RATS

The metabotropic glutamate receptors (mGluRs) that are expressed and not expressed on astrocytes in the brain have not been defined. While immunohistochemistry and in situ mRNA hybridization have been used on a limited basis to address this question, they do not readily enable the proportion of astrocytes expressing a particular mRNA or protein to be determined. Also, for many receptors, expression by cultured astrocytes does not reflect in situ expression. In this study, therefore, we examined expression of mRNA for all the mGluRs except mGluR6 by single‐cell reverse transcriptase‐polymerase chain reaction (RT‐PCR) in freshly isolated hippocampal astrocytes from postnatal day P1–10 rats, as an additional approach to address the question of which mGluRs are expressed on astrocytes in situ. The astrocytic nature of the cells was supported by simultaneously measuring mRNA for the astrocytic marker glial fibrillary acidic protein (GFAP) from the same cells. In these studies, the percentage of cells showing GFAP mRNA expression was the same as the percentage of cells showing immunocytochemical staining for GFAP. We found that only mGluR3 and mGluR5 mRNAs were significantly present in GFAP mRNA(+) cells. The mGluR5 PCR products were primarily of the “a” splice variant. mGluR1, 2, 4, 7, and 8 were very rarely or never detected. mGluR6 mRNA level was too low in whole rat brain and hippocampus to warrant examination. These results show that interpretation of effects involving mGluR3 or 5 activation in the hippocampus of young rats needs to also consider effects due to astrocytes. J. Neurosci. Res. 58:533–543, 1999.

Down-regulation of Mammalian Mitochondrial RNAs During Oxidative Stress

We have identified an RNA species that appears to be induced by oxidative stress in hamster HA-1 fibroblasts using the differential display technique, but instead is found to be degraded when evaluated by Northern blot hybridization. Cloning and subsequent sequencing identified the partially degraded RNA as 16S ribosomal RNA (rRNA), a major component of mitochondrial ribosomes. Degradation, and associated decreases in the levels of the mature- and precursor-species of 16S rRNA, appear to be dependent upon calcium, but not cytoplasmic protein synthesis nor nuclear transcription. Other decreased mitochondrial RNAs were also identified, including 12S rRNA, NADH dehydrogenase subunit 6, ATPase subunit 6, and cytochrome oxidase subunits I and III. A significant part of many, if not all, of these RNA decreases was due to degradation. As compared with 16S rRNA, significantly less degradation was observed for cytoplasmic 28S/18S rRNAs, even at very high peroxide concentration. Analysis of 21 cytoplasmic mRNAs revealed little or no decrease in mature band signal in response to peroxide, and several cytoplasmic mRNAs were actually up-regulated. Thus, a preferential down-regulation of mitochondrial RNAs occurs in HA-1 fibroblasts in response to hydrogen peroxide. Subcellular fractionation analysis, using 16S rRNA degradation as a gauge, indicates that this down-regulation is specific to mitochondria. The down-regulation of mitochondrial RNAs may represent a general mechanism by which cells protect themselves against oxidative stress.

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.

Metabotropic glutamate receptors in acutely isolated hippocampal astrocytes: Developmental changes of mGluR5 mRNA and functional expression

We previously found that 82% of glial fibrillary acidic protein (GFAP)‐positive hippocampal astrocytes acutely isolated from P1–10 rats responded to glutamate (Glu) with transient intracellular calcium increases via activation of a Group I metabotropic glutamate receptor (mGluR). Fewer cells responded to ATP and none to serotonin (5‐HT). In this study we asked the question whether hippocampal astrocytes in older animals retain this relative pattern of expression. We have found that 77% of GFAP (+) cells from P11–20 rats responded to 50 μM Glu, 43% to ATP, and none to 5‐HT. Thirty‐three percent of GFAP (+) cells from P25–35 rats responded to Glu, 12% to ATP and 3% to 5‐HT. In the case of the responses to Glu, pharmacological characterization and single‐cell RT‐PCR data confirmed that these responses were mediated by the mGluR5 subtype of group I mGluRs. Also, fewer (36%) GFAP mRNA (+) cells from P25–35 rats expressed detectable mGluR5 mRNA than those from P11–20 rats (77%). This number essentially corresponds to the number of GFAP(+) showing a Ca2+ response to Glu. Both mGluR5a and b were expressed with equal frequency in cells from P11–20 rats, but the b form predominated in cells from older animals. Overall, our studies show that expression of mGluR5 in hippocampal astrocytes decreases with increasing age and the “a” splice variant declines to a greater extent than the “b” splice variant, corresponding to the developmental changes shown in total tissue for mGluR5. GLIA 29:70–80, 2000.

16S Mitochondrial Ribosomal RNA Degradation Is Associated with Apoptosis

The use of mitochondrial RNA as an indicator of apoptosis was investigated. Exposure of HA-1 fibroblastic cells to 10 micromol H(2)O(2) per 10(7) cells induced nuclear fragmentation, cell shrinkage, and internucleosomal DNA fragmentation, all characteristics of apoptosis. RNA extracted from control and apoptotic cultures, and analyzed by Northern blot hybridization, revealed a significant increase in the degradation of mitochondrial 16S ribosomal RNA (rRNA) that was associated with apoptosis. Conversely, minimal, if any, degradation of glyceraldehyde-3-phosphate dehydrogenase or actin mRNAs was observed. Similar results were obtained for HA-1 cells treated with the protein kinase inhibitor staurosporine, and for HT-2 T-lymphocytes induced to undergo apoptosis by interleukin-2 withdrawal. In addition, 16S rRNA degradation was an early event that was discernable well before chromatin condensation in hydrogen peroxide-treated HA-1 cells. These observations suggest that degradation of mitochondrial 16S ribosomal RNA is a new marker of mammalian cell apoptosis.

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+ ∼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.

GFAP mRNA positive glia acutely isolated from rat hippocampus predominantly show complex current patterns.

Electrophysiologically complex glial cells have been widely identified from different regions of the central nervous system and constitute a dominant glial type in juvenile mice or rats. As these cells express several types of ion channels and neurotransmitter channels that were thought to be only present in neurons, this glial cell type has attracted considerable attention. However, the actual classification of these electrophysiologically complex glial cells remains unclear. They have been speculated to be an immature astrocyte because, although these cells show positive staining for the predominantly astrocytic marker S 100beta, it has not been possible to show staining for the commonly accepted mature astrocytic marker, glial fibrillary acidic protein (GFAP). To address the question of whether these cells might express GFAP at the transcript level, we combined patch-clamp electrophysiological recording with single cell RT-PCR for GFAP mRNA in glial cells acutely isolated from 4 to 12 postnatal day rats. In fresh cell suspensions from the CA1 region, complex glial cells were found to be the dominant cell type (65% total cells). We found that the majority of these electrophysiologically complex cells (74%) were positive for GFAP mRNA. We also showed that the complex cells responded to AMPA and GABA application, and these were also GFAP mRNA positive. We also fixed and stained the preparations for GFAP without electrophysiological recording to better preserve GFAP immunoreactively. In agreement with other studies, only 1.5% of these presumed electrophysiologically complex cells, based on morphology, showed immunoreactivity for GFAP. The expression of GFAP at the transcript level indicates GFAP (-)/GFAP mRNA (+) glial cells have an astrocytic identity. As single cell RT-PCR is able to detect both GFAP (-)/GFAP mRNA (+) and GFAP (+)/GFAP mRNA (+) astrocytic subtypes, the present study also suggests it is a feasible approach for astrocytic lineage studies.

Tamoxifen mediated estrogen receptor activation protects against early impairment of hippocampal neuron excitability in an oxygen/glucose deprivation brain slice ischemia model

Pretreatment of ovarectomized rats with estrogen shows long-term protection via activation of the estrogen receptor (ER). However, it remains unknown whether activation of the ER can provide protection against early neuronal damage when given acutely. We simulated ischemic conditions by applying oxygen and glucose deprived (OGD) solution to acute male rat hippocampal slices and examined the neuronal electrophysiological changes. Pyramidal neurons and interneurons showed a time-dependent membrane potential depolarization and reduction in evoked action potential frequency and amplitude over a 10 to 15 min OGD exposure. These changes were largely suppressed by 10 microM TAM. The TAM effect was neuron-specific as the OGD-induced astrocytic membrane potential depolarization was not altered. The TAM effect was mediated through ER activation because it could be simulated by 17beta-estradiol and was completely inhibited by the ER inhibitor ICI 182, 780, and is therefore an example of TAMs selective estrogen receptor modulator (SERM) action. We further show that TAMs effects on OGD-induced impairment of neuronal excitability was largely due to activation of neuroprotective BK channels, as the TAM effect was markedly attenuated by the BK channel inhibitor paxilline at 10 microM. TAM also significantly reduced the frequency and amplitude of AMPA receptor mediated spontaneous excitatory postsynaptic currents (sEPSCs) in pyramidal neurons which is an early consequence of OGD. Altogether, this study demonstrates that both 17beta-estradiol and TAM attenuate neuronal excitability impairment early on in a simulated ischemia model via ER activation mediated potentiation of BK K(+) channels and reduction in enhanced neuronal AMPA/NMDA receptor-mediated excitotoxicity.

Freshly isolated astrocyte (FIA) preparations: A useful single cell system for studying astrocyte properties

Astrocytes are cell constituents of the mammalian CNS whose intricate relationships with neurons, blood vessels and meninges in situ are well documented. These relationships and their complex morphologies imply numerous functions. Over the past quarter century or so, however, the main experimental basis for determining which roles are likely have been derived from studies on primary astrocyte cultures, usually prepared from neonatal rodent brains. We list a number of examples where these cultures have shown quantitative and qualitative differences from the properties exhibited by astrocytes in situ. The absence of an adequate reliable database makes proposals of likely hypotheses of astrocyte function difficult to formulate. In this article we describe representative studies from our laboratory showing that freshly isolated astrocytes (FIAs), can be used to determine the properties of astrocytes that seem more in concordance with the properties exhibited in situ. Although the cells are most easily isolated from ≤15 day old rat hippocampi they can be isolated from up to 30 day old rats. The examples we describe are that several different types of K+ currents can be determined by patch clamp electrophysiology, of all the mGluRs only mGluR3 and 5 were detected by single cell RT‐PCR, and that single cell Ca2+ imaging shows that the mGluR5 receptor is functional. It was found that the frequency of cells expressing mGluR5 declines with the age of the animal with the mGluR5b type splice variant replacing the mGluR5a type, as occurs in the intact brain. It is concluded that FIAs can be used to determine the individual characteristics of astrocytes and their properties without the problems of indirect effects inherent in a heterogeneous system such as the slice, and without the problem of cultures unpredictably reflecting the in situ state. The FIAs obviously cannot be used to study interactions of astrocytes with the other CNS components but we propose that they will provide a good database on which hypotheses regarding such interactions can be tested in slices. FIAs can also be isolated from brain slices or intact brain after various pharmacological or electrophysiological perturbations to determine the changes in astrocyte properties that correlate with the perturbations. J. Neurosci. Res. 61:577–587, 2000.