Modulation of GAP-43 mRNA by GABA and glutamate in cultured cerebellar granule cells

Expression of GAP-43 in the cerebellum and selected regions of the brain has been shown to be developmentally regulated. Localization of GAP-43 mRNA within granule cells of the immature and mature rat cerebellum has been demonstrated by in situ hybridization. Higher levels are detected in the neonate compared to the adult. To determine if the cerebellar neurotransmitters, GABA (γ-amino-butyric acid) and glutamate are involved in the modulation of GAP-43 expression, cultured cerebellar granule cells were exposed to these transmitters. Cultures were treated with glutamate, GABA, or the agonists/antagonists to their receptors in serum-free media for 5–7 days. Analysis of the levels of GAP-43 mRNA by in situ hybridization indicated that a 7-day exposure to GABA (25 and 50 μM) significantly lowered levels of granule cell GAP-43 mRNA. Specific agonists to the GABA_A (muscimol) and GABA_B (baclofen) receptors produced a decrease similar to that observed for GABA. Results from these studies also indicated that exposure to non-NMDA (CNQX) and NMDA (CPP, MK-801) glutamate receptor antagonists, and a metabotropic receptor glutamate agonist (ACPD), decreased the level of GAP-43 mRNA. The involvement of GABA and glutamate in the modulation of GAP-43 expression was corroborated by Northern hybridization. These studies revealed that a 5-day exposure to GABA decreased the cellular content of GAP-43 mRNA by 21% whereas exposure to glutamate resulted in a 37% increase. Findings from the studies reported here, using an in vitro cerebellar granule cell model, suggest that levels of GAP-43 mRNA, in vivo, are modulated by input from both excitatory glutamatergic mossy fibers and inhibitory GABAergic Golgi interneurons. Thus, modulation of GAP-43 mRNA by these neurotransmitters may influence granule cell maturation during development in the neonate and neuroplasticity in the adult, possibly at the parallel fiber–Purkinje cell synapse.     

Effect of benzodiazepines and pentobarbital on the GABA-induced depolarization in cultured astrocytes

We have previously shown that cultured astrocytes from neonatal rat cerebral cortex are depolarized by GABA. The underlying ionic mechanism, activation of a Cl- conductance and responses to an agonist and antagonists were found to be similar to those of the neuronal GABAA receptor (Kettenmann et al.: Brain Research 404:1-9, 1987; Kettenmann and Schachner: Journal of Neuroscience 5:3295-3301, 1985). To characterize further the pharmacological properties of the GABA receptor we have tested the influence of pentobarbital and benzodiazepines on the GABA response. Pentobarbital potentiated and prolonged the GABA-induced depolarization and enhanced the velocity of the depolarization. Agonists of the neuronal benzodiazepine receptor, flunitrazepam, diazepam, and midazolam, increased the GABA-induced depolarization. As in neurons, an antagonist of the benzodiazepine receptor, Ro 15-1788, blocked the flunitrazepam-induced enhancement of the GABA response. In contrast to their effects on neurons, the inverse agonists Ro 22-7497 and DMCM increased the GABA-induced depolarization. The ligand of the putative peripheral benzodiazepine binding site, Ro 5-4864, did not show consistent effects on the GABA response. These studies confirm that cultured astrocytes express GABAA receptors. This receptor is similar to the neuronal GABAA receptor with regard to Cl- conductance and its pharmacological responses to muscimol, bicuculline, picrotoxin, pentobarbital, and benzodiazepine agonists and an antagonist, but it is different in its responses to inverse agonists of the benzodiazepine site. The physiological role of the glial GABAA receptor is at present unknown.     

Glutamine transport in cerebellar granule cells in culture

In the present study, uptake of glutamine by rat cerebellar granule cells, a predominantly glutamatergic nerve cell population, has been investigated. Glutamine is taken up by granule cells via at least three transport systems, A, ASC and L. The L-type low affinity system (K_m=2.6 mM) is the major transport system in the absence of Na⁺. The systems A and ASC represent the Na⁺-dependent transport routes, both with almost identical high affinity for glutamine (K_m=0.26 mM). Similar transport systems for glutamine are also found in cerebral cortical neurons, a predominantly GABAergic nerve cell population, and cerebral cortical astrocytes. The glutamine transport properties in granule cells, however, show a series of differences from that of cortical neurons and astrocytes: (1) uptake of glutamine by granule cells is primarily mediated by system A (54%), while contributions by system A in cortical neurons and astrocytes are less than 30%; (2) granule cells exhibit strikingly higher transport efficiency for glutamine (V_max/K_m=20 min⁻¹ for system A as compared to the V_max/K_m ratio of 5 min⁻¹ in cortical neurons and astrocytes), and (3) the initial uptake rates and the steady-state accumulation levels of glutamine are two- to threefold higher in granule cells than that of cortical neurons and astrocytes. These results taken together suggest that in accordance with the important need to replenish the neurotransmitter pool of glutamate, glutamatergic neurons exhibit highly efficient transport systems to accumulate glutamine, one of the major precursors of glutamate.     

Sodium-dependent high-affinity uptake of taurine in cultured cerebellar granule cells and astrocytes

Taurine uptake in cultured cerebellar granule cells and astrocytes consisted of a saturable high-affinity component and nonsaturable diffusion. The transport constant (Km) was significantly lower and the maximal velocity (V) higher in granule cells than in astrocytes. The uptakes were strictly sodium dependent and also moderately decreased in potassium-free medium. The specificity profile of taurine uptake was similar in both cell types, hypotaurine, beta-alanine, and guanidinoethanesulphonic acid being the most potent inhibitors, followed by GABA and homotaurine. Glutamate inhibited taurine uptake more in astrocytes than in granule cells. In principle, the uptake systems were similar in granule cells and astrocytes, exhibiting features characteristic of uptake of a neurotransmitter or -modulator.     

Characterization of cytosolic glutathione S-transferase in cultured astrocytes

To elucidate the contribution of glutathione S-transferase (GST) and glutathione peroxidase (GPx) to the protection against oxidative stress in rat brain, we prepared GST and GPx from newborn rat liver, brain and cultured astrocytes, and investigated the characteristics and kinetics of the enzymes. The activity of cytosolic GST of the cultured astrocytes toward 1-chloro-2,4-dinitrobenzene (CDNB) was much higher than that of GPx toward peroxides. The GST activity toward 4-hydroxy-2-nonenal (4HNE) was almost the same as the GPx activity. GST isozymes were purified from the cytosolic fraction of the liver and astrocytes. In the case of the astrocytes, a major GST isozyme with an isoelectric point (pI) of 9.02 accounted for approximately 40% of total GST activity toward CDNB, while hepatic GST isozymes showed seven peaks in the basic region. Each of astrocytes and liver showed a single GST peak with high activity toward 4HNE, namely AVIII and LVIII, respectively, and both of them had a similar pI value of about 6.7. The kinetic parameters of AVIII and LVIII were found to be similar to each other. These data suggest that the same types of GST isozymes are expressed in the astrocytes and liver, and take part mainly in the detoxification of 4HNE.     

Identification of cell types from action potential waveforms: cerebellar granule cells

We have recorded 404 single units extracellularly in the cerebellar cortex of the rat with tungsten microelectrodes. Waveforms of action potentials were analyzed in order to develop criteria for on-line identification of cell types. Two of the four most frequently recorded waveforms were simple and complex spikes from Purkinje cells. The other two originated from granule cells and glomeruli. Presumed granule cells showed biphasic action potentials with half-widths (0.78 ± 0.14ms, n = 51) broader than those of the simple spikes of Purkinje cells (0.22 ± 0.06ms, n = 54), whereas presumed glomerular potentials had complex action potentials with narrower half-widths (0.14 ± 0.05ms, n = 35). The mean inter-spike interval of presumed granule cells (333.3 ± 195.4ms, n = 53) was longer than that of Purkinje cells (47.3 ± 31.8ms, n = 59) and the presumed glomerular potentials (77.7 ± 50.8ms, n = 20). Results were virtually identical from 17 cerebellar units recorded extracellularly in the cat. Intracellular recording and staining of 20 granule cells with HRP-filled microelectrodes provided further support for our assessment. These results suggest that action potentials from granule cells may be identified on-line by waveform.     

Properties of single potassium channels in cultured primary astrocytes

The patch-clamp technique was used to characterize the single channel ion currents in primary cultures of rat astrocytes. The most dominant channel type, which was found in over half of the inside-out membrane patches, was a potassium channel. The measured reversal potential was -67 mV, which is close to the calculated Nernst potential for potassium ions (-80 mV). These potassium channels activated with bursts of very brief openings. Once activated the channels did not inactivate. The measured probabilities of the channels to be closed showed at least 3 different modes of channel behaviour: one voltage-independent and two voltage-dependent modes. During each activity-mode a 'main' conductance level plus two other conductance levels were observed. In some recordings a pronounced outward rectification could also be seen.     

Activation of substance P receptors leads to membrane potential responses in cultured astrocytes

Cultured astrocytes from rat cortex and spinal cord responded with different types of membrane potential changes upon brief (10 seconds) applications of the natural neurokinin agonists substance P and neurokinin A. The most prominent type of response was a long-lasting membrane depolarization. In some cells, an initial rapid depolarization followed by a partial repolarization preceded the slow depolarizing event. Few astrocytes responded with a hyperpolarization of the membrane. Selective agonists at the NK-1 receptive site, substance P-methyl ester (SP-OME) and septide, mimicked the response to the natural neurokinins as did DiMe-C7, a selective NK-3 receptor agonist. A putative neurokinin antagonist, (D-Arg1,D-Pro2,D-Trp7,9,Leu11)SP (DADPDT) partially blocked membrane potential responses induced by substance P, SP-OME, septide, DiMe-C7, and NKA. The authors conclude that astrocytes express NK-1 and NK-3 receptors, which upon activation affect the electrical properties of these cells.     

C6细胞和星形细胞对神经干细胞体外迁移分化的不同影响

目的观察C6细胞和星形细胞在体外对神经干细胞（NSCs）迁移和分化是否有不同影响，为进一步研究调节NSCs迁移、分化的因子打下基础。方法取处于指数生长期的C6细胞、星形细胞分别与NSCs限定区域培养，观察NSCs的形态变化和迁移方向。并用二者的无血清培养上清和无血清培养基分别加入“Transwell Inserts”细胞培养系统的下室，培养室的上室加NSCs悬液，共培养36h，光镜下计数位于培养系统中间膜上的细胞球数，并观察其形态变化。结果与C6细胞共培养的NSCs向C6细胞生长的方向迁移，而与星形细胞共培养的NSCs则呈分化现象。C6细胞的上清引起神经球迁移的数目明显多于星形细胞的上清和无血清培养基（P〈0．01），星形细胞的上清则明显引起NSCs突起生长。结论C6细胞主要引起NSCs迁移，而星形细胞主要引起NSCs分化。     

Ciliary neurotrophic factor stimulates nuclear hypertrophy and increases the GFAP content of cultured astrocytes

Studies using transgenic mice that overexpress ciliary neurotrophic factor (CNTF), direct injection of CNTF into brain parenchyma, and ectopic expression of CNTF by an adenoviral vector have demonstrated that CNTF activates astrocytes. Paradoxically, studies to date have failed to show an effect of CNTF on the expression of GFAP by cultured astrocytes. Therefore, the goal of this study was to use nuclear hypertrophy and GFAP expression as indices of glial activation to compare the responsiveness of forebrain type 1 and type 2 astrocytes to CNTF. As reported by others, CNTF did not increase GFAP in type 1 astrocytes; however, it rapidly increased their nuclear size by 20%. Nuclear hypertrophy was apparent within 4 h after CNTF exposure and persisted for at least 48 h. In contrast, type 2 astrocyte GFAP increased 2-fold over the course of 48 h of CNTF treatment. During this same treatment period type 2 astroglial nuclei enlarged by 25%. We conclude that CNTF stimulates both type 1 and type 2 astrocytes directly. Together with our in vivo studies (Levison et al., 1996: Exp. Neurol. 141: 256), these data support the concept that CNTF is responsible for many of the progressive astroglial changes that appear after CNS injury and disease.     

大鼠海马胶质细胞培养牵张损伤模型的建立

目的 建立大鼠海马胶质细胞培养牵张损伤模型.方法 提取出生时间24h到48h的SD大鼠海马组织行星形胶质细胞培养,采用CIC Ⅱ型细胞损伤装置、根据Ellis方法建立改良的大鼠海马胶质细胞体外牵张损伤模型,损伤程度分轻、中、重三级.对照组不予损伤.分别在2h和24h检测细胞乳酸脱氧酶释放量,并通过碘化丙啶荧光染色观察细胞损伤情况.结果 细胞培养液中乳酸脱氧酶释放量随着损伤程度加重而增高.PrI红染细胞数随着牵张损伤的程度增高而增加.结论 本实验建立的牵张损伤模型使用方便,可重复性好,适于进行神经细胞机械件体外损伤的研究.     

Laminin promotes cerebellar granule cells migration in vitro and is synthesized by cultured astrocytes

Newborn rat cerebellum microexplants have been used as a model to study neuronal migration. Laminin in a substrate-bound form modifies extensively the migratory behavior of the neurones, an effect which is blocked if antilaminin antibodies are present during the assay. 35S methionine incorporation followed by SDS-PAGE electrophoresis, fluorography and immunoprecipitation with antilaminin antibodies allowed to demonstrate that laminin is synthesized and secreted by cultured newborn rat cerebellum as well as by cultured newborn rat cerebral cortex astrocytes.     

Modification of taurine and hypotaurine uptake systems in cultured primary astrocytes by serum-free medium and dibutyryl cyclic AMP treatment

The characteristics of taurine and hypotaurine uptake were studied during astrocyte maturation in cultures grown in normal, serum-containing medium or in serum-free medium in the presence of 0.1 mmol/l dibutyryl cyclic AMP (dBcAMP). The uptake of both amino acids consisted of one saturable high-affinity component in both control and treated cultures. The dBcAMP treatment produced no marked modification of the transport systems. In the treated cultures the kinetic parameters of taurine and hypotaurine uptake remained unaltered during maturation. In the control cells the transport constant and maximal velocity of taurine uptake were greater in 21-day-old than in 16-day-old cultures, while the changes in hypotaurine transport were the opposite. The uptakes were strictly sodium-dependent and also considerably decreased when potassium ions were omitted from incubation medium. The uptake of both amino acids was affected more by potassium omission in the dBcAMP-treated than in the control cultures. Also the results with metabolic poisons suggest that physiological ion gradients sustained by an active Na⁺, K⁺-pump are essential for the normal uptake of taurine and hypotaurine.     

Role of Clast1 in development of cerebellar granule cells

We have identified the murine Clast1/LR8 gene by subtraction of cDNA derived from CD40 ligand-activated and naive B cells. The Clast1 gene is ubiquitously expressed in various organs of adult mice. However, its physiological function was largely unknown. To study a role of Clast1, we established Clast1-deficient (Clast1-KO) mice. Here, we reveal that approximately 65% of Clast1-KO mice showed severe ataxia. The Clast1-KO cerebellum with ataxia is small in size and revealed a severely aberrant lobulation, loss of the internal granule cell layer, and the disorganized Purkinje cells. Clast1 mRNA is expressed in the cerebellar granule cells of normal adult mice. Developmentally, Clast1 mRNA is also detected in the external germinal layer of the embryonic cerebellum, indicating its expression in granule cell precursors. Histopathological analysis of the developing Clast1-KO cerebellum demonstrated the reduced number of cells in the external germinal layer. Thus, Clast1 is required for development of cerebellar granule cells.     

NPPB对星形胶质细胞增殖的抑制作用

目的观察体积调节性阴离子通道阻滞剂NPPB对体外培养星形胶质细胞增殖的影响。方法采用原代培养的大鼠大脑皮层星形胶质细胞,实验分为对照组(正常培养基培养)与NPPB干预组;在不同时间点(0、6、12、24、48h),应用流式细胞技术以及免疫细胞荧光双标法(Brdu/DAPI)检测各实验组星形胶质细胞增殖及细胞周期进展的情况。结果与对照组相比,NPPB干预组在12、24h时星形胶质细胞增殖率较正常对照组降低(P<0.05),同时处于S期细胞的百分率亦相对正常对照组明显下降(P<0.01)。结论体积调节性氯离子通道阻滞剂NPPB可以显著抑制体外培养星形胶质细胞增殖及细胞周期进展,提示VRAC通道参与了体外培养星形胶质细胞的增殖。     

Adenosine and ADP prevent apoptosis in cultured rat cerebellar granule cells

Cerebellar granule cells (CGCs) explanted in vitro undergo death via apoptosis when the concentration of potassium is shifted from 25 mM to 5 mM. We report that adenosine and ADP, which act as neurotransmitters and neuromodulators in the brain, exert in cultured cerebellar granule cells a specific and marked antiapoptotic action with half-maximal effect in the 10–100 μM range. The action of adenosine is partly inhibited by the A1AR antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) and is mimicked by the A1AR agonist 2-chloro-N6-cyclopentyladenosine (CCPA), while ADP effect, that is completely blocked by the P2x, P2y receptors noncompetitive antagonist suramine, is restored in the presence of the selective P2x purinoceptors agonist β,γ-methylene- -ATP. These findings demonstrate that adenosine and ADP markedly inhibit the program of cell death in cerebellar granule cells and suggest that such an action is mediated via interaction with, respectively, A1 and P2x receptors.     

Depolarization-dependent survival of cultured mouse cerebellar granule neurons is strain-restrained

Cerebellar granule neurons (CGNs) isolated from the rat are often used as a model system for the analysis of activity-dependent survival of neurons. These cells do not survive in culture without addition of a depolarizing agent (KCl or glutamate) to the medium. However, it has been reported that mouse CGN behave differently. Here we found that the requirement for depolarization for the survival of the mouse CGN was strain-dependent, which may be important for future analyses using transgenic animals. CGNs from the Balb/C mouse could survive without KCl addition, whereas CGNs from the C57Bl/6 mouse could not (similar to the rat CGN). The survival-promoting activity of the Balb/C mouse CGNs was transferable by coculturing. However, the medium of the Balb/C mouse CGN culture was ineffective, suggesting that the neurotrophic substance that is supposed to be released is labile. Although we are yet to specify the substance, it might be independent of neurotrophins. It is necessary to select the strain of mouse in the production of transgenic animals for the analyses of activity-dependent neuronal survival.     

Tamoxifen对星形胶质细胞增殖的作用

目的观察体积调节性阴离子通道阻滞剂Tamoxifen对体外培养星形胶质细胞增殖的的影响。方法采用原代培养的大鼠大脑皮层星形胶质细胞,实验分为对照组(正常培养基培养)与Tamoxifen干预组;在不同时间点(0h、12h、24h、48h),应用流式细胞技术以及免疫细胞荧光双标法(Brdu/DAPI)检测各实验组星形胶质细胞增殖及细胞周期进展的情况。结果与对照组相比,Tamoxifen干预组在12h、24h时星形胶质细胞增殖率较正常对照组降低(P0.05);处于S期细胞的百分率相对正常对照组明显下降,处于G0-G1期细胞的百分率较正常组增高(P0.01)。结论体积调节性阴离子通道阻滞剂Tamoxifen可以显著抑制体外培养星形胶质细胞增殖及细胞周期进展,提示VRAC参与了体外培养星形胶质细胞的增殖。     

Transport of 5-formyltetrahydrofolate into primary cultured cerebellar granule cells

Transport of 5-formyltetrahydrofolate (5-FTHF) into primary cultured cerebellar granule cells (CGC) was studied. Uptake of 5-FTHF into CGC was saturable with K(m)=2.86 microM and V(max)=40.8 pmol/mg protein/45 min in pH 7.4 medium. Uptake of 5-FTHF in the astrocytes has a similar style in the time curve. Uptake of 5-FTHF is characterized by countertransport because adding unlabeled 5-FTHF in the medium resulted in the efflux of labeled 5-FTHF. Uptake of 5-FTHF was inhibited by the structural analogs 5-methyltetrahydrofolate, methotrexate and folic acid (K(i)=6.64, 7.69, and 19.38 microM, respectively). Uptake was significantly decreased by high concentrations of sodium azide and sodium arsenate but not by sodium cyanide. Uptake was also inhibited by p-chloromercuriphenylsulfonate and by the anions probenecid and 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid. Acute exposure of the cells to ethanol (100 mM) did not affect the uptake. It is concluded that CGC have a carrier-mediated system for the uptake of 5-FTHF and other folates.