Sodium channel currents in rat hippocampal NG2 glia: characterization and contribution to resting membrane potential.

We have recently reported that most of NG2 glycoprotein expressing glial cells, or NG2 glia, in rat hippocampus persistently express sodium channel currents (I(Na)) during development, but little is known about its function. We report here that hippocampal NG2 glia recorded in either acute slices or freshly isolated preparations from postnatal days (P) 7-21 rats express low density I(Na) (9.5-15.7 pA/pF) that is characterized by a fast activation and rapid inactivation kinetics with a tetrodotoxin (TTX) IC(50) value of 39.3 nM. The I(Na) expression correlated with a approximately 25 mV more depolarized resting membrane potential (RMP) as compared with non-I(Na)-expressing GLAST(+) astrocytes in situ at the same age. In the presence of the sodium channel blocker TTX (0.1 microM), these depolarized RMPs were negatively shifted by an average of 19 mV and 16 mV for I(Na)-expressing glia recordings from in situ and freshly isolated preparations, respectively. The I(Na) expressing glia actually showed a positive RMP (+12 mV) in the absence of potassium conductance that was inhibited to 0 mV by 0.1 microM TTX. Analysis of the I(Na) activation/inactivation curves yields an I(Na) "window current" at -40+/-20 mV, implying a persistent I(Na) component being active around the NG2 glia RMP of approximately -45 mV. According to the constant-field equation analysis, this active I(Na) component leads to a pNa/pK ratio of 0.14 at RMP which is approximately threefold higher than astrocytes (0.05). These results indicate that a TTX sensitive I(Na) component in NG2 glia contributes significantly to the depolarized NG2 glia RMP in the developing brain.

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 < or =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 Ca(2+) 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.

Acutely isolated astrocytes as models to probe astrocyte functions.

Neuroscientists have become increasingly aware and accepting of the concept that astrocytes likely have many important functions in the CNS. One limitation in establishing these functions is the usual problem of what constitutes suitable experimental approaches. A major experimental step for functional studies of astrocytes has been the widespread use of primary astrocyte cultures, an approach that Leif Hertz pioneered. However, it is now becoming clear that, building on this work, an experimental paradigm shift is now needed. Namely, to increasingly study preparations corresponding to in situ conditions, such as slices. An alternative experimental system where the cells have some of the technical advantages of primary astrocyte cultures is freshly isolated astrocytes. Recent experiments from our laboratory have shown metabotropic glutamate receptor expression by such cells. Examples are given of how functional receptor studies and channel activity measured by patch clamp electrophysiology can be combined with single cell RT-PCR to define further the receptor or channel type are described to illustrate the uses of such preparations.

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.

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 microM 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 Ca(2+) 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.

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.

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.

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.

Oxidant-inducible adapt 15 RNA is associated with growth arrest- and DNA damage-inducible gadd153 and gadd45.

We have recently described a novel RNA, designated adapt15, which is strongly induced by a pretreatment concentration of hydrogen peroxide in hamster HA-1 fibroblasts under conditions where a protective "adaptive response" occurs. adapt15 may therefore be involved in protecting cells against the damaging effects of oxidative stress. Since two other known sequences, gadd45 and gadd153m were also found to be induced under our pretreatment conditions and are known to be growth arrest and DNA damage inducible, we decided to also assess the possible association of adapt15 with growth arrest and DNA damage. We found that, like gadd45 and gadd153, the levels of adapt15 RNA were low during proliferation, but high during density saturation- and low serum (G0)-growth arrests. Exposure of HA-1 cells to DNA-damaging agents revealed significant induction of adapt15 RNA by methylmeth- anesulfonate and cis-platinum but not X-irradiation. Near identical responses were also observed for gadd45 and gadd153 RNAs, suggesting coordinate regulation of adapt15, gadd45, and gadd153. All three RNAs were also increased relative to control following heat shock, a nongenotoxic treatment. Finally, the induction of adapt15 by hydrogen peroxide was strongly dependent upon calcium, a hallmark of gadd153 induction. The coordinate inductions of adapt15, gadd45, and gadd153 by multiple agents, and their induction by an adaptive- but not by a nonadaptive-response level of hydrogen peroxide, suggest these RNAs may act in concert to protect cells against the damaging effects of oxidative stress.

Oxidative stress induces the levels of a MafG homolog in hamster HA-1 cells.

The Maf family encodes nuclear proteins that recognize AP-1-like response elements. MafB, MafK, MafF, and MafG, are all able to heterodimerize with each other, c-fos, and erythroid cell-specific transcription factor NF-E2, to affect the transcription of target genes. Using the technique of differential display, we recently identified a new oxidant-inducible mRNA, designated adapt66, in HA-1 hamster fibroblasts. Cloning, partial sequencing, and GenBank analysis of adapt66 revealed strong homology to chicken mafG, a newly identified member of the maf oncogene family. The mafG homolog/adapt66 mRNA induction appeared to be dependent upon calcium; occurred as early as 90 minutes following exposure of HA-1 cells to hydrogen peroxide; and peaked between 5 and 10 hours after peroxide treatment. It has previously been demonstrated that several cellular transcription factors, including Fos, can be induced by oxidative stress. The induction of the DNA binding sequence mafG homolog/adapt66 by hydrogen peroxide, and it's known interaction with c-fos, may represent important mechanisms by which oxidative stress can modulate gene expression.

Hydrogen peroxide induces the expression of adapt15, a novel RNA associated with polysomes in hamster HA-1 cells.

In mammalian cells, hydrogen peroxide is known to induce the expression of several genes thought to be important in protection against oxidative stress. Using mRNA differential display, we have identified a novel RNA in hamster HA1 cells that is strongly induced by hydrogen peroxide under conditions where a protective adaptive response occurs. This induction was observed between 1 and 18 h after peroxide treatment, and at an H202 concentration that caused little or no cytotoxicity. Northern blot analysis revealed that this major inducible RNA species, termed adapt15, is 950 bases in size. Two versions of this RNA were found by sequence analysis, differing only by a short trinucleotide stretch. Despite polyadenylation, no large open reading frame was observed. Fractionation studies, however, indicate that adapt15 RNA is primarily located in the cytoplasm, and a significant percentage of it is associated with active translation. adapt15 RNA may act at the level of translation to protect cells against the damaging effect of oxidative stress.