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REPORT
1Institut für Neuro- und Sinnesphysiologie, Universität Düsseldorf, Düsseldorf; and 2Lehrstuhl für Zellphysiologie, Ruhr-Universität Bochum, Bochum, Germany
Submitted 14 February 2005; accepted in final form 24 August 2005
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ABSTRACT |
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INTRODUCTION |
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; Finley et al. 1996; Jüngling et al. 2003; Strübing et al. 1995). Moreover, after transplantation to the brain ES cell-derived neurons show organotypic morphology and become synaptically integrated into cortical neuronal networks (Benninger et al. 2003; Wernig et al. 2004). Both major types of synapses, excitatory synapses with AMPA- and N-methyl-D-aspartate (NMDA)-type glutamate receptors and inhibitory GABAergic synapses, have been described in synaptically connected ES cell-derived neurons (Finley et al. 1996; Jüngling et al. 2003).
In addition to the basic properties of synapses, the correct functioning of transplanted ES cell-derived neurons within complex cortical networks requires long-term synaptic plasticity as an essential feature. Long-term synaptic plasticity at excitatory glutamatergic synapses is dependent on action potential activity patterns and is either input-specific (Malenka and Nicoll 1999; Malinow and Malenka 2002) or affects all synapses of a neuron (homeostatic plasticity) (Turrigiano et al. 1998; Turrigiano 1999; Turrigiano and Nelson 2004). Moreover, neurotrophic factors, e.g., brain-derived neurotrophic factor (BDNF), induce long-term synaptic plasticity at cortical glutamatergic synapses (Lessmann et al. 1994; Lessmann 1998; Lu 2003; Lu and Chow 1999). Both, an involvement in the input-specific potentiation of postsynaptic currents evoked by action potentials (Korte et al. 1995; Patterson et al. 1996) and in the cell-wide regulation of spontaneous miniature postsynaptic currents (Collin et al. 2001; McLean Bolton et al. 2000; Paul et al. 2001; Rutherford et al. 1998; Vicario-Abejon et al. 1998) have been described.
Because BDNF readily induces long-term synaptic plasticity, this neurotrophin represents a potentially important co-factor in cell replacement therapy that might boost synaptic integration of transplanted ES cells-derived neurons. However, it has not yet been investigated whether BDNF can induce long-term synaptic changes also in ES cell-derived neurons. To address this, we established a co-culture system consisting of neocortical explants and ES cell-derived neurons and studied glutamatergic synapses electrophysiologically. We focused on BDNF effects on miniature EPSCs because a cell-wide form of plasticity appears more interesting than input-specific plasticity of a few synapses to improve the overall synaptic integration of transplanted cells. Here, we report that ES cell-derived neurons show activity-dependent synaptic scaling of all glutamatergic synapses on a given neuron. Most notably, we demonstrate that BDNF is able to induce a long-term enhancement of miniature EPSCs in ES cell-derived neurons.
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METHODS |
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). In brief, ES cells were cultured on inactivated mouse feeder cells in the presence of LIF. Embryoid bodies (EBs) were formed in hanging drop culture in the presence of retinoic acid. After two further weeks in culture, EBs were dissociated and ES cell-derived neurons were purified by immunoisolation using antibodies against the cell adhesion molecule L1 (Jüngling et al. 2003).
To study the synaptic integration of ES cell-derived neurons into neocortical networks, we used a simple co-culture system consisting of presynaptic explants that innervated dissociated postsynaptic target neurons as described previously (Gottmann et al. 1997; Mohrmann et al. 1999). In brief, mouse neocortical explants were obtained from E18 mouse (C57/black6) fetuses and cultured for 3 days as free-floating explants in Neurobasal medium with addition of B27 supplement, glutamax, and penicillin/streptomycin (all from Invitrogen). Cultivation as free-floating explants led to the formation of "spheres," which was critical to avoid outward migration of neurons from the explants during later cultivation. These explants were added to the purified ES cell-derived neurons that had been seeded at low density on polyornithine-coated culture dishes 1 h previously [day in vitro (DIV) 1]. Cultivation was done at 37°C in 5–6% CO2 atmosphere. The culture medium consisted of a mixture of fresh neurobasal medium (same supplements as in the preceding text) and neurobasal medium that had been conditioned by neocortical explants for 3 days (1:1). At 5 DIV cytosine-
-D-arabinofuranoside (10 µM) was added to inhibit glial proliferation. To exclude outward migration of neurons from the explants, neocortical explants from EGFP-expressing mice (Hadjantonakis et al. 1998) were used. These control experiments were repeated several times (>5). With preculturing the explants freely floating for several days, we never observed EGFP-expressing cells surrounding the explant. Postsynaptic target neurons were always unlabeled and thus represented ES cell-derived neurons.
Whole cell patch-clamp recordings from ES cell-derived target neurons at 11–14 DIV were performed at room temperature using an EPC-7 patch-clamp amplifier and a pClamp6 data acquisition system as described previously (Mohrmann et al. 1999). Action potential responses were elicited by depolarizing current injection and were recorded in current-clamp mode. The intracellular solution (in mM) contained 100 K-gluconate, 20 KCl, 0.25 CaCl2, 10 EGTA, and 20 HEPES, pH = 7.3. The extracellular solution (in mM) contained 130 NaCl, 5 KCl, 2 CaCl2, 1 MgCl2, and 20 HEPES, pH = 7.3. Holding potential was –50 mV. For recording AMPA mEPSCs, the extracellular solution contained (in mM) 130 NaCl, 30 KCl, 5 CaCl2, 1 MgCl2, and 20 HEPES, pH = 7.3 with addition of TTX (1 µM) and picrotoxin (100 µM); the intracellular solution (in mM) contained 110 KCl, 0.25 CaCl2, 10 EGTA, and 20 HEPES, pH = 7.3. The holding potential was –60 mV. For recording NMDA receptor-mediated components of mEPSCs, Mg2+ was omitted from the extracellular solution. Miniature EPSC analysis including averaging was performed using AUTESP software as described (Lindlbauer et al. 1998; Mohrmann et al. 1999). Events <5 pA and events with a slow rise time (>2 ms) were excluded from the analysis. mEPSC rise times were determined for each cell after averaging individual mEPSCs. To exclude synaptic connections between ES cell-derived neurons, paired recordings were performed as described previously (Jüngling et al. 2003). Human recombinant BDNF (Tebu-bio) was used for BDNF-treatment. All data are given as means ± SE, and statistical analysis was done using Student's t-test.
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RESULTS |
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). Neocortical explants from mouse fetuses were added to the ES cell-derived neurons and showed massive outgrowth of fibers heavily contacting the surrounding ES cell-derived neurons after a few days in culture. No outward migration (>50 µm) of neurons from the explants was observed. This was confirmed using neocortical explants from EGFP-expressing mice for co-cultures. In these control co-cultures, the fibers arising from the explants were green fluorescent, but the surrounding ES cell-derived target neurons were always completely unlabeled.
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To investigate the formation of functional glutamatergic synapses, we recorded miniature EPSCs (mEPSCs) in the ES cell-derived target neurons using the whole cell patch-clamp technique. At 11–14 DIV, AMPA receptor-mediated mEPSCs were recorded in the presence of TTX (1 µM), picrotoxin (100 µM), and Mg2+ (1 mM) at a holding potential of –60 mV (Fig. 1C). Using an elevated extracellular K+ concentration (30 mM), mEPSCs occurred at a mean frequency of 5.9 ± 0.9 Hz (n = 18) and had a mean amplitude of 13.6 ± 1.3 pA. Their mean rise time was 0.86 ± 0.06 ms (average mEPSCs), and their mean decay time constant was 5.6 ± 0.8 ms. AMPA receptor-mediated mEPSCs were completely blocked by the addition of 6,7-dinitroquinoxalin-2,3-dione (20 µM, n = 6; Fig. 1D). To further study the presence of a NMDA receptor-mediated component, mEPSCs were recorded in Mg2+-free extracellular solution at a holding potential of –60 mV. For analysis, mEPSCs of an individual cell were averaged to obtain a mean mEPSC. Averaged mEPSCs showed a clear NMDA receptor-mediated slow component that was selectively blocked by the addition of D-AP5 (50 µM, n = 5; Fig. 1E). We further compared AMPA mEPSCs from co-cultures containing ES cell-derived neurons as postsynaptic targets with AMPA mEPSCs from control co-cultures consisting of neocortical explants and dissociated neocortical target neurons. The mean frequency (10.3 ± 1.5 Hz, n = 21) and the mean amplitude (12.7 ± 0.6 pA) of AMPA mEPSCs in neocortical neurons were not significantly different (Fig. 1F). Thus glutamatergic mEPSCs in ES cell-derived neurons had properties very similar to those of glutamatergic mEPSCs in primary cultured neocortical neurons in the same type of co-culture system (Mohrmann et al. 1999). Functional glutamatergic synapses between ES cell-derived neurons or glutamatergic autapses were observed only very rarely, thus demonstrating that synaptic input to ES cell-derived neurons in our co-culture system is strongly dominated by the explant fibers. In whole cell recordings from pairs of ES cell-derived neurons action potential-evoked AMPA EPSCs were detectable in only 1 of 14 potential connections tested. Similarly, autaptic AMPA EPSCs were detected in only 2 of 14 cells studied. In both cases, the amplitudes of evoked AMPA EPSCs were very small (<5 pA) in the range of AMPA mEPSCs, suggesting the presence of only a single release site.
We next wanted to demonstrate that glutamatergic synapses in ES cell-derived neurons show synaptic scaling, a homeostatic form of activity-dependent long-term plasticity (Turrigiano et al. 1998). Synaptic scaling has been described in primary cultured neocortical neurons as a selective increase in the amplitude of AMPA mEPSCs after activity blockade by TTX, which is caused by a postsynaptic change in AMPA receptors. We added TTX (1 µM) to our co-cultures at 8 DIV and recorded AMPA mEPSCs at 11–14 DIV as described in the preceding text. Intriguingly, we observed a significant (P < 0.01) increase in the mean amplitude of AMPA mEPSCs from 10.8 ± 0.9 pA (n = 16) in parallel control cultures to 14.5 ± 0.9 pA (n = 22) in TTX-treated cultures, whereas the mean frequency (4.9 ± 0.8 and 4.1 ± 0.4 Hz) was unchanged (Fig. 2). No significant change in the mean rise time of average AMPA mEPSCs was observed (control: 0.78 ± 0.05 ms; TTX-treated: 0.8 ± 0.05 ms; Fig. 2E) supporting a postsynaptic mechanism. These results clearly indicate that ES cell-derived neurons integrated in neocortical networks are capable of cell-wide synaptic long-term plasticity involving postsynaptic AMPA receptor regulation.
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; Vicario-Abejon et al. 1998).
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DISCUSSION |
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; Strübing et al. 1995). In addition, cultured ES cell-derived neurons form both excitatory, glutamatergic synapses with postsynaptic AMPA and NMDA receptors and inhibitory, GABAergic synapses (Finley et al. 1996; Strübing et al. 1995). On transplantation of ES cell-derived neural precursor cells in slice cultures and in vivo, the ES cell-derived neurons exhibited functional properties similar to cortical neurons, i.e., action potential discharge and glutamatergic and GABAergic synaptic input (Benninger et al. 2003; Wernig et al. 2004). However, in addition to these basic properties, long-term synaptic plasticity is required for the proper functioning of central synapses within neuronal networks. Although the preceding studies have already well established a basic functional integration, the demonstration that ES cell-derived neurons are capable of long-term synaptic plasticity is still lacking. In this paper, we describe a simple model system that allows to study the functional integration of ES cell-derived neurons under well-defined in vitro conditions. Our co-cultures consisted of mouse neocortical explants that innervated spatially separated mouse ES cell-derived neurons. This system allows for an efficient synaptic integration of ES cell-derived neurons in a neocortical network while preserving spatial separation. In particular, chronic application of pharmacological substances and neurotrophic factors at well-defined concentrations is easily possible in our co-culture system.
Long-term synaptic plasticity of glutamatergic synapses is an essential feature of cortical neuronal networks and is thought to underlie developmental maturation of connectivity and memory formation. In addition to classical input-specific long-term potentiation/depression, a cell-wide, homeostatic form of plasticity, synaptic scaling, has been described (Turrigiano 1999; Turrigiano and Nelson 2004; Turrigiano et al. 1998). In neocortical neurons, chronic blockade of activity increased the amplitudes of AMPA receptor-mediated miniature EPSCs, while enhancing activity decreased miniature EPSC amplitudes. Both changes are caused by alterations in the number and postsynaptic accumulation of postsynaptic glutamate receptors (Desai et al. 2002; Turrigiano et al. 1998; Watt et al. 2000; Wierenga et al. 2005). Homeostatic plasticity, in particular the upscaling of mEPSCs, represents a cellular mechanism that could enhance the functional synaptic integration of ES cell-derived neurons into cortical networks. Here we demonstrated that a very similar scaling up of the amplitudes of AMPA receptor-mediated mEPSCs also occurs in neocortical explant-ES cell-derived neuron co-cultures during chronic activity blockade. This finding strongly suggests that glutamatergic synapses in ES cell-derived neurons are capable of homeostatic long-term plasticity.
The neurotrophin BDNF has been demonstrated to control several types of input-specific long-term synaptic plasticity at central glutamatergic synapses (for review, see Lessmann 1998; Lu 2003; Lu and Chow 1999). Both pre- and postsynaptic mechanisms for persistent increases in synaptic strength have been shown to depend on BDNF (e.g., Itami et al. 2003; Zakharenko et al. 2003). Moreover, BDNF is also well known to affect the cell-wide homeostatic regulation of miniature synaptic currents. Chronic BDNF application has been shown to enhance both the frequency (Collin et al. 2001; Paul et al. 2001; Vicario-Abejon et al. 1998) and the amplitude of AMPA mEPSCs (McLean Bolton et al. 2000) in primary cultured hippocampal neurons. In addition, effects of BDNF on AMPA mEPSC amplitudes have been shown to be strongly dependent on the neuronal cell type investigated with opposing effects occurring in neocortical pyramidal cells as compared with GABAergic interneurons (Leslie et al. 2001; Rutherford et al. 1998).
In our co-culture system, we observed a strong BDNF-induced increase in the frequency of AMPA mEPSCs in ES cell-derived neurons similar to the effects of BDNF in immature hippocampal neurons (Collin et al. 2001; Vicario-Abejon et al. 1998). We found in addition an increase in mEPSC amplitudes suggesting that chronic BDNF application affects both presynaptic release properties (mEPSC frequency) and postsynaptic AMPA receptors. Although postsynaptic mechanisms acting on AMPA receptors appear more likely, changes in mEPSC amplitudes can in principle be explained also by presynaptic mechanisms such as an increased transmitter content of synaptic vesicles. Because presynaptic mechanisms would lead to an increased glutamate concentration in the synaptic cleft, a change in the rise time of mEPSCs would be expected. However, we did not observe any significant change in AMPA mEPSC rise times in our experiments.
In contrast, in mature neocortical pyramidal neurons a BDNF-dependent downregulation of the amplitude of AMPA mEPSCs has been described (Rutherford et al. 1998). Taken together, these results suggest that BDNF might affect mEPSCs differently in different types of cells: in immature (precursor) cells, BDNF appears to enhance frequency and amplitude, whereas in mature neurons, BDNF limits quantal size. To this end, our results in ES cell-derived neurons indicate that ES cell-derived neurons are capable of BDNF-dependent long-term synaptic plasticity. The effects of BDNF in our ES cell-derived neurons more closely resembled BDNF effects in immature hippocampal neurons as compared with those in mature neocortical neurons. Whether this is appropriate for synaptic integration and plasticity in neocortical circuits needs further investigation in organotypic systems at different stages of differentiation. Nevertheless BDNF appears to represent a promising cofactor in cell replacement therapies of neurodegerative diseases that has the potential to enhance the functional synaptic integration of stem cell-derived neurons into cortical neuronal networks.
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GRANTS |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Address for reprint requests and other correspondence: K. Gottmann, Institut für Neuro- und Sinnesphysiologie, Heinrich-Heine-Universität Düsseldorf, Universitätsstr. 1, D- 40225 Düsseldorf, Germany (E-mail: Kurt.Gottmann{at}uni-duesseldorf.de)
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ABSTRACT
INTRODUCTION
) and from TTX-treated cultures (
). *, significant difference. n is indicated above bars. D: plot of AMPA mEPSC frequency vs. mean AMPA mEPSC amplitude for each individual cell analyzed from control co-cultures (
) and from TTX-treated co-cultures (
). Note the increased amplitudes in TTX-treated co-cultures. E: time course of AMPA mEPSCs was not affected by TTX treatment. Mean mEPSCs from a control cell and a TTX-treated cell obtained by averaging individual mEPSCs are superimposed.