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NEUROSCIENCE |
1 Department Neurotransmission and Neuroendocrine Secretion, Institute for Cellular and Integrative Neurosciences (INCI), F-67084 Strasbourg, France
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Abstract |
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(Received 18 July 2006;
accepted after revision 4 September 2006;
first published online 7 September 2006)
Corresponding author F. W. Pfrieger: Dept Neurotransmission INCI, 5, rue Blaise Pascal, F-67084 Strasbourg, France. Email: fw-pfrieger{at}gmx.de
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Introduction |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Methods |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Postnatal Balb/c mice (7 days old; animal facility, Faculty of Medicine, Universite Louis Pasteur, Strasbourg; Elevage Janvier, Le Genest Saint Isle, France) were killed by decapitation according to institutional guidelines. Hippocampi and cerebella were dissected, cut in small pieces and incubated (5% CO2, 37°C) for 60 min in Earle's buffered salt solution (EBSS) (Gibco/Invitrogen, Cergy-Pontoise, France) containing 200 U ml–1 papain (Worthington Biochemical Corporation, Lakewood, NJ, USA), 200 U ml–1 DNAse (Sigma, St-Quentin Fallavier, France), 1.5 mM CaCl2, 1 mM MgSO4 and 0.5 mM EDTA (Sigma). To isolate RGCs, retinae from the same animals were incubated for 45 min at 37°C in D-PBS (Gibco/Invitrogen) containing 160 U ml–1 papain and 200 U ml–1 DNAse. The tissues were then sequentially triturated in D-PBS containing 0.15% trypsin inhibitor (Roche Diagnostics, Meylan, France), 650 U ml–1 DNAse and 1 : 75 rabbit anti-rat macrophage antibody (Sigma) to remove microglial cells. Cells were spun down (800 g for 13 min), resuspended in 1% trypsin inhibitor in D-PBS, spun down again and then resuspended in D-PBS containing 0.02% bovine serum-albumin (fraction V; Sigma). For immunopanning, one (hippocampus, cerebellum) or two (RGCs) subtraction plates (150 mm diameter Petri-dishes; Falcon; BD Biosciences/VWR, Fontenay sous Bois, France) and one selection plate (100 mm diameter Petri-dish) were incubated for > 12 h at 4°C with 10 µg ml–1 secondary antibody in 50 mM Tris-HCl (pH 9.5) (for subtraction: goat anti-rabbit IgG; for selection: hippocampus–cerebellum, goat anti-rat IgG; for RGCs, goat anti-mouse IgG (Jackson Immunoresearch Laboratories/Beckman Coulter, Marseille, France)). After washing for three times with PBS, selection plates were covered with 0.2% bovine serum albumin (fraction V; in D-PBS) and incubated for > 4 h (cerebellum, RGCs) or > 12 h (hippocampus) at room temperature with 0.2 µg ml–1 primary antibody (hippocampus, cerebellum: rat IgG anti-L1 clone 324, Chemicon/Euromedex, Mundolsheim, France; RGCs: mouse IgM anti-Thy1.2, MCA01, Serotec, Cergy Saint-Christophe, France) and then washed with D-PBS. The cell suspensions were filtered through a nylon mesh (Nitex 20 µm, Tetko/Sefar Filtration, Rüschlikon, Switzerland) and incubated on subtraction plates for 30 min. The supernatant was filtered and incubated on the selection plate (hippocampus, cerebellum: 60 min, RGCs: 45 min). Non-adherent cells were thoroughly washed off and bound cells were released by washing with 0.02% BSA (hippocampus, cerebellum) or by trypsination (RGCs: 12 000 U ml–1 in EBSS for 10 min in 5% CO2 at 37°C). Following washing or inactivation of trypsin by 30% fetal calf serum (Gibco/Invitrogen), cells were spun down and resuspended in culture medium.
After determination of cell counts (haemocytometer using tryptan blue staining, Sigma), cells were plated at indicated densities. For electrophysiological recordings and immunocytochemical staining, neurons were plated at 600 cells mm–2 in a small circle (10 mm diameter) centred on tissue culture plates (35 mm diameter; Falcon, BD Biosciences) coated with 5 µg ml–1 poly D-lysine (MW 
40 kDa; Sigma) with (hippocampus) or without (cerebellum, RGCs) 10 µg ml–1 laminin (Sigma). For viability (3 h) and survival assays at 3 days in vitro (DIV), cells were plated on 96-well plates at 5000 cells per well in indicated culture media. For the proliferation assay, cerebellum cells were plated at 1000 cells mm–2 in 24-well culture plates (Falcon, BD Bioscience). Cells were cultured in Neurobasal medium (Gibco/Invitrogen) supplemented with (all from Sigma, except where indicated) pyruvate (1 mM), glutamine (2 mM; Gibco/Invitrogen), N-acetyl-L-cysteine (60 µg ml–1), putrescine (16 µg ml–1), selenite (40 ng ml–1), bovine serum albumin (100 µg ml–1; fraction V, crystalline grade), streptomycin (100 µg ml–1), penicillin (100 U ml–1), triiodothyronine (40 ng ml–1), holotransferrin (100 µg ml–1), insulin (5 µg ml–1) and progesterone (62 ng ml–1). This medium is referred to as minimally supplemented medium (MSM). To support neuronal survival, this medium was further supplemented with B27 (1 : 50, Gibco/Invitrogen), brain-derived neurotrophic factor (BDNF; 25 ng ml–1; PeproTech, London, UK), ciliary neurotrophic factor (CNTF; 10 ng ml–1; PeproTech) and forskolin (10 µM; Sigma). This medium is referred to as fully supplemented medium (FSM). For co-cultures, neurons were plated with glial cells from the respective region except for RGCs, which were cultured with cortical glia (Mauch et al. 2001). For treatment with soluble glial factors, GCM was obtained from primary cultures of mouse hippocampal, cerebellar and cortical glial cells according to a standard protocol (Pfrieger & Barres, 1997). Briefly, papain-digested and triturated tissue from the different brain regions of 7-day-old mice was cultured in PDL-coated tissue culture flasks (25 cm2, TPP/VWR) in a medium containing (all Gibco/Invitrogen) DMEM, heat-inactivated fetal calf serum (10%), penicillin (100 units ml–1), streptomycin (100 µg ml–1), glutamine (2 mM) and sodium pyruvate (1 mM). After 1 week, culture flasks were washed with PBS, and glial cells were cultured in MSM. Three times a week, half of the GCM was harvested and replaced by fresh MSM. GCM was spun down (5 min at 3000 g) to remove cellular debris and added to 1-day-old cultures (1.7 ml GCM to 2 ml culture medium). For some experiments, cholesterol (5 µg ml–1 from a 1000-fold ethanolic stock solution; Sigma) was added to 1-day-old cultures of RGCs. To some hippocampal cultures, recombinant murine tumour necrosis factor alpha (100 ng ml–1; R & D Systems, Lille, France) was added at 4 DIV for 48 h.
Survival and proliferation assay
The neuronal survival rate was determined by the live/dead assay (Molecular Probes/Invitrogen) according to the manufacturer's instructions. Calcein, ethidium and bisbenzimide fluorescence were viewed on an inverted microscope (Axiovert 135TV; Zeiss, Göttingen, Germany) using suitable filter sets (Omega Optical/PhotoMed GmbH, Seefeld, Germany) and digitized by an air-cooled monochrome CCD camera (Sensicam, PCO Computer Optics, Kehlheim, Germany). The percentage of living cells was analysed by a custom-written routine (Labview; National Instruments, Le Blanc-Mesnil, France). For the proliferation assay, cells were incubated 3 h (n = 2) and 21 h (n = 2) after plating for 6 h in culture medium with 5-bromo-2-deoxyuridine (BrdU; 10 µM; Sigma), fixed, immunostained (mouse anti-BrdU; clone BU33; 1 : 1000; Sigma) and viewed on the same set-up as described above.
Electrophysiological recordings
Recordings of spontaneous postsynaptic currents were performed and analysed as described earlier (Nagler et al. 2001; Goritz et al. 2005). The intracellular recording solution contained (mM, all Sigma): 100 potassium gluconate, 10 KCl, 10 EGTA and 10 Hepes adjusted to pH 7.3 with KOH. The extracellular solution contained (mM, all Sigma): 120 NaCl, 3 CaCl2, 2 MgCl2, 5 KCl and 10 Hepes adjusted to pH 7.4 with NaOH. Analysis of postsynaptic currents was performed automatically by custom-written Labview routines. The frequency of excitatory postsynaptic currents (EPSCs) was determined from inward currents recorded at –70 mV holding potential. EPSCs could be distinguished from inhibitory postsynaptic currents (IPSCs), which were also inwardly directed at –70 mV due to their faster time course (Fig. 2): half-widths of EPSCs ranged from 0.8 to 6 ms whereas those from IPSCs ranged between 6 and 40 ms. The frequency of IPSCs was determined from outward currents recorded at –30 mV. All neurons tested were electrically excitable as indicated by the presence of voltage-activated sodium currents in response to depolarizing voltage steps to 0 mV holding potential. Miniature EPSCs (mEPSCs) and IPSCs (mIPSCs), which are due to action potential-independent transmitter release, were recorded in the presence of tetrodotoxin (1 µM; Sigma).
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Immunocytochemical staining was carried out using standard procedures (Nagler et al. 2001). To determine the purity of neuronal preparations, cells were stained 24 h after plating with combinations of cell-type specific antibodies: for oligodendrocytes, mouse anti 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase; 1 : 500; Sigma) and mouse anti-O4 (oligodendrocyte lineage; 1 : 20; Roche Diagnostics); for astrocytes, rabbit anti-S100ß (astrocytes; 1 : 1000; Swant, Bellinzona, Switzerland) and rabbit anti-glial fibrillary acid protein (GFAP; astrocytes; 1 : 2000; Dako Cytomation, Trappes, France); for fibroblasts, rabbit anti-fibronectin (fibroblasts) (1 : 500; Sigma). Microglial cells were detected by Griffonia (bandeiraea) simplicifolia (GS) lectin I isolectin B4-FITC (1 : 100; Vector Laboratories/Alexis, Grunberg, Germany), which was applied together with secondary antibodies. GABAergic neurons were stained with a rabbit anti-glutamate decarboxylase isoform 65 (GAD65; 1 : 1000; Chemicon/Euromedex). Control staining ensured that the antibodies detected the respective cell type. To determine the total number of cells in a field of view, the non-selective DNA marker H33342/bis-benzimide (20 µg ml–1; Sigma) was added together with secondary antibodies. The percentage of specific cell types was determined by counting the total number of fluorescent nuclei and then the number of antibody-labelled cells. For synapse staining, neurons were labelled by mouse anti-synapsin I (clone 46.1; 1 : 200; Synaptic Systems, Göttingen, Germany), rabbit anti-PSD95 (1 : 50, Synaptic Systems) and rabbit anti-GABAA receptor (1 : 400; Upstate Biotechnology/Euromedex). For fluorescence labelling, Alexa 488-, Alexa 555- (Molecular Probes/Invitrogen), Cy2- and Cy3-conjugated (Jackson Immunoresearch Laboratories) goat anti-mouse or goat anti-rabbit IgG antibodies were used.
Data representation and statistical analysis
Each data set was obtained from at least three independent preparations. Statistical analysis was performed using STATISTICA 7.1 (StatSoft Inc., Maison-Alfort, France). Graphs were created by SigmaPlot 9.01 (Systat Software GmbH, Erkrath, Germany). Data samples were tested for normality (Shapiro-Wilk's test) to select appropriate statistical tests and graphical representation. Non-normally distributed values were represented by box plots (horizontal line, median; box limits, 1st and 3rd quartile; whiskers, 10th and 90th percentile) or cumulative relative frequency plots and were tested for statistically significant changes by appropriate tests (two independent samples: Mann-Whitney U test; multiple independent samples: Kruskal-Wallis ANOVA by ranks test followed by post hoc comparison of mean ranks; two dependent samples: Wilcoxon signed rank test). Changes in percentages of cells were analysed using Pearson's 
2 test. Parameters with large sample sizes (n > 500) were analysed by one-way ANOVA followed by Dunnett's post hoc test to detect changes compared with control condition. To adjust for unequal sample size, random subsamples were analysed as indicated. Levels of significance are indicated by asterisks (*P < 0.05; **P < 0.01; ***P < 0.001). Mean values are indicated together with (±) standard deviation (S.D.).
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Results |
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To test whether neurons of the mouse CNS depend on glia to form synapses, we developed protocols to immunoisolate hippocampal and cerebellar neurons from postnatal mice using an antibody against the cell adhesion molecule L1 (Rathjen & Schachner, 1984). We also purified RGCs from postnatal mice using a modified version of the protocol for rat RGCs (Barres et al. 1988). The yield of cells per animal that could be harvested from the selection plates varied with the brain region (Table 1). Immunopanning of mouse RGCs yielded about a third of cells (Table 1) compared with their isolation from postnatal rats (71 000 ± 14 000; n = 75). Regardless of the brain region, the initial viability of cells, determined 3 h after plating by a microfluorometric assay, averaged at 75% (Table 1). To determine the purity of the neuronal preparations, we performed immunocytochemical staining with combinations of cell-type-specific markers. Fractions of GFAP/S100ß-positive astrocytes, of O4/CNPase-positive oligodendrocytes, of fibronectin-positive fibroblasts and of GS lectin I-positive microglia were very low (Table 1) indicating that the procedures isolated > 99% pure populations of neurons from these regions. As expected from previous studies on rat (Barres et al. 1988) and chick (Butowt et al. 2000; Annies & Kroger, 2002), our preparation of mouse RGCs immunoisolated by a Thy1-specific antibody also contained very few non-neuronal cells (Table 1).
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Synapse development in CNS neurons in the absence of glia
Next, we used our new culture preparations to test whether hippocampal and cerebellar neurons establish functional synaptic connections in the absence of glial cells. Neurons from each region were cultured for 1 week at similar densities under defined, glia-free conditions. The presence of functional synapses was determined by whole-cell patch-clamp recordings of excitatory and inhibitory synaptic activity from randomly selected cells (Figs 2 and 3). The large majority of hippocampal (Figs 2 and 7A) and of cerebellar (Figs 3 and 7B) neurons showed excitatory synaptic activity, when cultured for 1 week without glia. In both cultures, spontaneous EPSCs (sEPSCs) reached frequencies of 1000 events min–1 and higher (Figs 2B and 3B). Spontaneous inhibitory synaptic activity was observed in more than half of hippocampal neurons (Figs 2 and 9A) with spontaneous IPSCs (sIPSCs) occurring at frequencies up to 250 events min–1 (Figs 2B and 9A). The fraction of cerebellar neurons showing spontaneous IPSCs (sIPSCs) and their frequencies (Figs 3B and 9B) were lower than in hippocampal neurons (Figs 2B and 9A) reflecting the smaller percentage of GABAergic neurons in the cerebellar culture preparation.
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To study, whether glial signals affected synapse development, immunoisolated neurons were co-cultured with glial cells or treated> with GCM, which contains secreted factors. We observed different glial effects depending on the brain region and the type of synapse.
In randomly selected hippocampal neurons, co-culture with glial cells induced a slight but significant enhancement in the fraction of cells showing sEPSCs, but glial signals did not change the frequency of sEPSCs (Fig. 7A). Furthermore, co-culture with glia enhanced the size of sEPSCs in a large fraction of neurons (Fig. 8A). Inhibitory activity was not affected by the presence of glia (Fig. 9A). In pyramidal cells, glial cells also enhanced strongly the size of sEPSCs (Fig. 8A), but left their incidence and frequency unaffected (Fig. 7A). Moreover, they raised significantly the incidence of sIPSCs and their size (Fig. 9A). In cerebellar neurons, co-culture with glia increased the size of sEPSCs (Fig. 8B) and raised the incidence of inhibitory activity (Fig. 9B), but left all other parameters unaffected. Notably, changes observed in neuron-glia co-cultures were only mimicked in part by GCM.
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2 test) and increased the frequency of sEPSCs significantly compared with control cultures (P < 0.05; n
= 25; Mann-Whitney U test). However, cholesterol did not mimick the glia-induced increase in the size of sEPSCs compared with glia-free cultures (P
= 0.58; n
= 25; Mann-Whitney U test). Effects of glial cells on miniature postsynaptic currents
The observed glia-induced changes in synaptic activity in the different neuronal cell types could have been due to enhanced neuronal excitability and thus a higher level of action potential-driven activity. To address this, we tested how glial cells affected miniature postsynaptic currents, which are due to action potential-independent quantal transmitter release.
In hippocampal neurons including pyramidal cells, glial cells affected mEPSCs in a similar manner as spontaneous excitatory events: compared with glia-free cultures, co-culture with glia increased the fraction of neurons showing mEPSCs (Fig. 7A) and their size (Fig. 8A). The glia-induced changes in the incidence and size of sIPSCs of pyramidal cells were not observed in mIPSCs (Fig. 9A). This indicated that glial cells affected the development or function of excitatory, but not of inhibitory synaptic connections in hippocampal neurons. Recent studies showed that glia-derived tumour necrosis factor alpha enhances the size of mEPSCs in hippocampal cultures (Beattie et al. 2002; Stellwagen & Malenka, 2006). However, this component did not mimick the glia-induced increase in mEPSC size in hippocampal neurons (n = 15 cells, 3 preparations). In cerebellar cultures, glial signals decreased the size of mEPSCs (Glia: n = 38; GCM: n = 39) compared with glia-free cultures (n = 66; Fig. 8B) indicating that the enhanced size of sEPSCs in these neurons was due to a change in action potential-dependent synaptic activity. In RGCs, the glia-induced increase in the incidence, frequency and size of sEPSCs was also observed for mEPSCs (Figs 7C and 8C), thus confirming previous observations that glial cells promote the development of synapses in these neurons.
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Discussion |
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Hippocampal and cerebellar neurons, but not RGCs show robust synapse formation in the absence of glia
A main finding of our study is that CNS neurons differ in their ability to form synapses without glia. Within 1 week in culture, most hippocampal and cerebellar neurons formed functional excitatory and inhibitory synaptic contacts in the absence of glia as indicated by robust spontaneous synaptic activity and by the presence of immunocytochemically stained synapses. The frequencies of EPSCs and IPSCs in glia-free cultures were similar or even higher than those reported previously in conventional hippocampal (Mennerick et al. 1995; Bouron & Reuter, 1997; Boehm, 1999) and cerebellar (Virginio et al. 1995; Losi et al. 2002; Fiszman et al. 2005) cultures derived from postnatal rats or mice. This indicated that under the present culture conditions, purified neurons reach the same level of connectivity as those growing in glia-containing primary cultures. This was in contrast to cultured RGCs, which formed only very few connections and showed a very low level of activity in the absence of glia thus confirming previous studies (Pfrieger & Barres, 1997; Nagler et al. 2001; Ullian et al. 2001).
Our isolation procedure and culture conditions may have selected for those neurons that form synapses autonomously. However, hippocampal and cerebellar cultures contained inhibitory neurons as well as principal neurons like pyramidal and Purkinje cells at ratios that can be expected from the situation in vivo. Thus, our preparation preserved the diversity of neuronal cell types rather than selecting for synaptogenesis-competent cells. It may also be argued that synaptic connections in our cultures were ‘non-natural’ and that their formation proceeds independently from glia. This argument does not apply for hippocampal cultures, where pyramidal cells, which represent principal synaptic targets, received synaptic input from excitatory and inhibitory neurons. In cerebellar cultures, which consisted mainly of granule cells, excitatory synaptic activity originated from somewhat non-natural synapses among granule cells, as these are the only cerebellar cell type that can form excitatory glutamatergic connections. However, the same argument applies for cultures of immunoisolated RGCs (Nagler et al. 2001; Ullian et al. 2001) and motoneurons (Ullian et al. 2004b), which lack natural partner neurons. In contrast to cerebellar granule cells, however, these neurons required glia to form non-natural synaptic connections. This corroborates our statement that neurons differ in their ability to form synapses autonomously. Due to the rare occurrence (< 0.5%) of Purkinje cells in our cultures it was not possible to determine whether they receive natural input from granule cells.
Glial signals promote formation of synapses by RGCs, but not by hippocampal or cerebellar neurons
Glial signals did not enhance the frequency of excitatory or inhibitory synaptic events in hippocampal and cerebellar cultures indicating that they did not promote the formation of new functional connections by these neurons. This is different from previous reports that glial signals promote synaptic activity and synapse formation in cultures of hippocampal neurons (Yang et al. 2003; Hama et al. 2004; Elmariah et al. 2005). The divergence may be explained by differences in culture conditions and in the age of animals from which neurons were obtained. The cited studies prepared neurons from embryonic animals. At this stage, only few glial cells have been generated and so the culture preparations can be regarded as glia-free. It is possible that at the embryonic stage, neurons require glial signals for synaptogenesis, whereas postnatally they become autonomous. We note that the glia-induced increase in a small fraction of hippocampal neurons (but not pyramidal cells) that showed excitatory synaptic events may reflect an induction of synaptic input and thus synapse formation in a subset of hippocampal neurons.
In contrast to hippocampal and cerebellar neurons, glial signals strongly promoted synapse formation in RGCs as indicated by the large increase in the incidence of synaptic activity and in the frequency of spontaneous and miniature synaptic events. This confirmed previous reports that the glia-induced enhancement of synaptic activity in these neurons is due to enhanced synaptogenesis (Nagler et al. 2001; Ullian et al. 2001). The lack of effect by mouse GCM, which differs from the situation in the rat (Pfrieger & Barres, 1997; Nagler et al. 2001), was possibly due to a lower cholesterol concentration in mouse GCM, since addition of cholesterol mimicked the effects of co-culture on the frequency of sEPSCs at least in part.
Glial cells enhance the size of miniature synaptic currents in hippocampal neurons and RGCs
Glial cells enhanced the size of spontaneous excitatory postsynaptic currents in all three culture preparations. In hippocampal neurons and RGCs, this was due to an increase in the size of action potential-independent miniature synaptic currents. In cerebellar cultures, which consist mainly of granule cells, the effect was action potential-dependent and may have been caused by an increased excitability or by an increased efficacy of evoked transmitter release. Spontaneous inhibitory synaptic currents were only affected by glia in pyramidal cells, where co-culture led to a higher incidence and larger size of sIPSCs. This effect, which did not occur in miniature inhibitory synaptic currents and was therefore action potential-dependent, may have been indirect via elevated excitatory synaptic activity. This may have excited GABAergic interneurons providing synaptic input to pyramidal cells. Alternatively, glial cells, namely astrocytes, may have directly activated interneurons as reported recently (Liu et al. 2004). Apart from this effect in pyramidal cells, glial cells increased only the size of excitatory postsynaptic currents. Such a selective effect on glutamatergic rather than GABAergic synapses is in line with the fact that inhibitory synapses develop before glutamatergic synapses (Ben-Ari, 2002), and thus may not require glial signals.
To increase the size of mEPSCs in hippocampal neurons and RGCs, glial signals may have enhanced postsynaptically the efficacy of glutamate receptors or presynaptically the glutamate concentration in synaptic vesicles. These changes may have been due to enhanced maturation of excitatory synaptic connections, for example by increased clustering of postsynaptic glutamate receptors, or due to enhanced function of normally developed synapses, for example by glial support of the glutamate–glutamine cycle (Hertz & Zielke, 2004). Except for hippocampal pyramidal cells, the glia-induced increase in miniature size was only seen in neuron–glia co-cultures and was not mimicked by soluble glial factors contained in GCM. This is in line with our previous observation on immunoisolated rat RGCs that co-culture with glia enhanced the size of mEPSCs more strongly than GCM (Nagler et al. 2001). Astrocyte-derived tumour necrosis factor alpha, which has been shown to enhance the surface expression of glutamate receptors (Beattie et al. 2002; Stellwagen & Malenka, 2006), did not mimick the glia-induced increase in miniature size in our cultures.
Taken together, our results indicate that in the mammalian CNS the way that glial signals promote the formation and efficacy of excitatory synapses varies with the neuronal cell type. This underlines the importance of comparative studies on different neurons. Our new culture preparations may serve as first experimental models to study the relevance of glial signals on postnatal neuronal development.
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  M. A. Johnson, J. P. Weick, R. A. Pearce, and S.-C. Zhang Functional Neural Development from Human Embryonic Stem Cells: Accelerated Synaptic Activity via Astrocyte Coculture J. Neurosci., March 21, 2007; 27(12): 3069 - 3077. [Abstract] [Full Text] [PDF]
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nbsp;2000) and cerebellar neurons (n