Glutamate receptor properties of human ... - Wiley Online Library

JOURNAL OF NEUROCHEMISTRY

| 2009 | 111 | 204–216

doi: 10.1111/j.1471-4159.2009.06315.x

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*Department of Neurology, University of Leipzig, Leipzig, Germany Translational Centre of Regenerative Medicine (TRM), University of Leipzig, Leipzig, Germany àCarl-Ludwig-Institute of Physiology, University of Leipzig, Leipzig, Germany §Institute of Biochemistry, Department of Molecular Biochemistry, University of Leipzig, Leipzig, Germany ¶Paul Flechsig Institute of Brain Research, Department of Neurophysiology, University of Leipzig, Leipzig, Germany

Abstract Human midbrain-derived neural progenitor cells (NPCs) may serve as a continuous source of dopaminergic neurons for the development of novel regenerative therapies in Parkinson’s disease. However, the molecular and functional characteristics of glutamate receptors in human NPCs are largely unknown. Here, we show that differentiated human mesencepahlic NPCs display a distinct pattern of glutamate receptors. In whole-cell patch-clamp recordings, L-glutamate and NMDA elicited currents in 93% of NPCs after 3 weeks of differentiation in vitro. The concentration-response plots of differentiated NPCs yielded an EC50 of 2.2 lM for glutamate and an EC50 of 36 lM for NMDA. Glutamate-induced currents were markedly inhibited by memantine in contrast to 6-cyano-7-nitroquinoxaline-2,3dione (CNQX) suggesting a higher density of functional NMDA than alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA)/kainate receptors. NMDA-evoked currents and calcium signals were blocked by the NR2B-subunit specific antagonist ifenprodil indicating functional expression of NMDA

receptors containing subunits NR1 and NR2B. In calcium imaging experiments, the blockade of voltage-gated calcium channels by verapamil abolished AMPA-induced calcium responses but only partially reduced NMDA-evoked transients suggesting the expression of calcium-impermeable, GluR2containing AMPA receptors. Quantitative real-time PCR showed a predominant expression of subunits NR2A and NR2B (NMDA), GluR2 (AMPA), GluR7 (kainate), and mGluR3 (metabotropic glutamate receptor). Treatment of NPCs with 100 lM NMDA in vitro during proliferation (2 weeks) and differentiation (1 week) increased the amount of tyrosine hydroxylase-immunopositive cells significantly, which was reversed by addition of memantine. These data suggest that NMDA receptors in differentiating human mesencephalic NPCs are important regulators of dopaminergic neurogenesis in vitro. Keywords: dopaminergic neurogenesis, electrophysiology, glutamate receptors, human midbrain-derived neural progenitor cells, NMDA, NR1/NR2B-subunits. J. Neurochem. (2009) 111, 204–216.

L-Glutamate is the major excitatory neurotransmitter in the mammalian CNS acting through ionotropic and metabotropic receptors. Fast synaptic glutamate actions are mediated by different cation-permeable ionotropic receptors which were named after their specific agonists NMDA, alpha-amino-3hydroxy-5-methylisoxazole-4-propionate (AMPA), and kainate. This pharmacological classification is well reflected in the sequence similarity of the 16 subunits (NMDA: NR1, NR2A–D, NR3A–B; AMPA: GluR1–4; kainate: GluR5–7, KA1–2) which may assemble as tetrameric receptors (Hollmann and Heinemann 1994; Dingledine et al. 1999; Kew

Received June 11, 2009; revised manuscript received July 23, 2009; accepted July 24, 2009. Address correspondence and reprint requests to Florian Wegner, Department of Neurology, University of Leipzig, Liebigstr. 20, 04103 Leipzig, Germany. E-mail: fl[email protected] Abbreviations used: AMPA, alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate; ATPA, (RS)-2-Amino-3-(3-hydroxy-5-tert-butylisoxazol-4-yl)propanoic acid; CNQX, 6-cyano-7-nitroquinoxaline-2,3-dione; DAPI, 4¢,6-diamidino-2-phenylindole; DHPG, (S)-3,5-dihydroxyphenylglycine; GFAP, glial fibrillary acidic protein; mGluR, metabotropic glutamate receptor; NPCs, neural progenitor cells; NR, NMDA receptor; TH, tyrosine hydroxylase.

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2009 The Authors Journal Compilation 2009 International Society for Neurochemistry, J. Neurochem. (2009) 111, 204–216

Glutamate receptor properties in human neural progenitors | 205

and Kemp 2005; Lujan et al. 2005). The G-protein-coupled metabotropic glutamate receptor family contains at least eight subtypes (mGluR1–8) divided into three groups based on their sequence homology, pharmacological profile, and second messenger coupling (group I: mGluR1 and 5 activate phospholipase C via Gq/G11; both group II: mGluR2–3 as well as group III: mGluR4 and 6–8 inhibit adenylate cyclase via Gi/Go). Generally mGluR members exert a more modulatory role, regulating neuronal excitability, synaptic transmission and plasticity (Conn and Pin 1997; ManahanVaughan et al. 1998; Kew and Kemp 2005; Lujan et al. 2005; Root et al. 2008). The functional NMDA receptor subunit NR1 is present throughout the brain during pre- and post-natal development, while the modulatory subunits (NR2A–D) are differentially expressed. The NR2B-subunit is detected in the entire brain at prenatal stages and shows a restricted expression to the forebrain post-natally in contrast to NR2A that is ubiquitously increased over time (Watanabe et al. 1993; Takai et al. 2003; Lujan et al. 2005). This developmental switch in the NMDA receptor subunit composition has been suggested to promote rapid and stable growth of immature synapses (Kubota and Kitajima 2008). Evidence is emerging that NMDA receptors may regulate proliferation, migration, and neuronal differentiation in the developing and adult CNS (Simon et al. 1992; Rossi and Slater 1993; Gould et al. 1994; Cameron et al. 1995; Luk et al. 2003; Lujan et al. 2005; Bursztajn et al. 2007; Joo et al. 2007; Nacher et al. 2007; Grubb et al. 2008; Wang and Kriegstein 2008) as well as the expansion, survival, and neurogenesis of neural progenitor cells (NPCs) in vitro (Luk et al. 2003; Suzuki et al. 2004, 2006; Mochizuki et al. 2007; Georgiev et al. 2008; Hu et al. 2008; Klimaviciusa et al. 2008). However, analyses of glutamate receptor function in human NPCs are rare particularly regarding the role of NMDA receptors that have been shown to mediate the mitotic and neurogenic effects of the neurosteroid dehydroepiandrosterone and glutamate in vitro (Suzuki et al. 2004, 2006). The propagation and differentiation of NPCs enables to study human neurogenesis in vitro (Suzuki et al. 2004, 2006; Milosevic et al. 2006, 2007; Wegner et al. 2008b; Schaarschmidt et al. 2009) and may deliver a tissue source for drug screening and cell therapy to treat neurodegenerative diseases (Schwarz et al. 2006a,b; Redmond et al. 2007; Hermann and Storch 2008). Long-term expanded human mesencephalic NPCs maintain their proliferative capacity and continue to give rise to neurons that express tyrosine hydroxylase and also release dopamine (Storch et al. 2001). The present study provides detailed analyses of human midbrain-derived NPCs in respect to glutamate receptor expression and function. We also investigated whether Lglutamate or NMDA is able to influence dopaminergic neurogenesis in vitro.

Materials and methods Cell culture Human neural progenitor cells were derived from CNS tissue of aborted human fetuses (gestational week 10–18) with mother’s consent. All experiments were approved by the Ethics Committee of the University of Leipzig, Germany and in accordance with all state and federal guidelines. The human fetal tissue was washed with sterile Hank’s buffered salt solution, dissected into mesencephalic and non-mesencephalic primary tissue samples. The tissue samples were mechanically separated into small pieces, incubated in 0.1 mg/ mL papain solution (Roche, Mannheim, Germany), supplemented with 10 lg/mL DNase (Roche) for 30 min at 37C, then washed three times with Hank’s buffered salt solution followed by an incubation with 50 lg/mL anti-pain solution (Roche) for 30 min at 37C. After three further washing steps the samples were homogenized by gentle trituration using fire-polished pasteur pipettes. The quality of the tissue was assessed as described previously (Milosevic et al. 2006, 2007). Propagation of human mesencephalic neural progenitor cells (NPCs) was performed in a monolayer by plating onto poly-Lornithine (Sigma, Taufkirchen, Germany) and fibronectin (Millipore, Billerica, MA, USA) coated culture dishes at a density of 30 000 cells/cm2. For expansion of NPCs, a xeno-free medium (Dulbecco’s modified Eagle’s medium/Ham’s F12) supplemented with epidermal growth factor and basic fibroblast growth factor (20 ng/mL each; both from PromoCell, Heidelberg, Germany), 2% B27 (Invitrogen, Karlsruhe, Germany), and 1% penicillin/streptomycin. Cells could be expanded for prolonged periods (> 10 passages) in reduced atmospheric oxygen (3%) as described previously (Storch et al. 2001; Milosevic et al. 2005). For differentiation NPCs were plated on poly-L-lysine coated 35 mm dishes (Sigma) at a density of 1 · 105/cm2. Neuronal differentiation was induced by replacement of the expansion medium by a mitogen-free medium consisting of Neurobasal (Invitrogen) supplemented with 2% B27 (Invitrogen), 1% Glutamax, interleukin-1b (100 pg/mL; Sigma), 5 lM forskolin (Sigma), and 0.1% gentamicin. The differentiation medium was replaced twice a week during the whole incubation protocol. Note, the expansion medium contained 50 lM L-glutamate, while the standard differentiation medium was free of glutamate. Electrophysiology Whole-cell patch-clamp recordings of ligand- and voltage-gated ion channels were performed on human midbrain-derived NPCs that had been differentiated for 3 weeks in vitro. Experiments were carried out in the voltage- or current-clamp mode at 20–22C using an EPC-9 amplifier and PulseFit software (HEKA, Lambrecht, Germany) as described previously (Wegner et al. 2008b). The external bath solution contained (in mM): 162 NaCl, 1.2 CaCl2, 2.4 KCl, 0.01 glycine, 11 glucose, 10 HEPES (pH 7.3; 320 mOsm). Micropipettes were formed from thin-walled borosilicate glass (BioMedical Instruments, Zo¨llnitz, Germany) with a Flaming Brown electrode puller P-97 (Sutter Instrument Co., Novato, CA, USA) and a Micro Forge (Narishige, Tokyo, Japan). Electrodes had resistances of 3–5 MW when filled with the internal solution containing (in mM): 95 CsCl, 6 MgCl2, 1 CaCl2, 10 HEPES, and 11 EGTA (pH 7.2; adjusted to 300 mOsm with glucose).


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All solvents and chemicals were purchased from Sigma. The stock solutions were prepared in dimethylsulfoxide or external recording solution as appropriate (1–100 mM). The drugs were dissolved in external solution containing dimethylsulfoxide at a maximal final concentration of 0.1%. All drugs were applied rapidly via gravity using a modified SF-77B perfusion fast-step system (Warner Instruments Inc., Hamden, CT, USA) as described previously (Wegner et al. 2007). For glutamate and NMDA concentration-response curves, nine increasing concentrations were applied for 2 s on NPCs every 30 s. The glutamate receptor currents recorded in pharmacological experiments using 2 s co-applications of antagonists or modulators were measured 1 s after activation to allow appropriate receptor binding. After application of antagonists, 1-min intervals were allowed for wash out. Whole cell currents were low-pass filtered at 1–10 kHz, digitized at 5–50 kHz, and analyzed with PulseFit (HEKA) and GraphPad Prism version 4.00 for Windows (GraphPad Software, San Diego, CA, USA). Glutamate- and NMDA-evoked peak currents of investigated cells were normalized and fitted to a sigmoidal function using a four parameter logistic equation with a variable slope to obtain nonlinear regression concentration-response plots as described previously (Wegner et al. 2008a). The equation used to fit the concentration-response relationship was I¼

Imax 1 þ 10ðLogEC50 Logdrug Þ Hill slope

where I was the peak current at a given concentration. Numerical data of all experiments were expressed as means ± SEM, statistical significance using Student’s t-test (two tailed, unpaired) and F test to compare the best-fit values of Log EC50 was taken as p < 0.05. Quantitative real-time PCR After 3 weeks of differentiation in vitro, human mesencephalic NPCs (three cell lines) were harvested and total RNA isolated using Trizol reagent (Invitrogen). Reverse transcription of 800 ng total RNA per reaction was carried out using oligo-dT primer and Superscript II reverse transcriptase (Invitrogen). Oligonucleotide primers for the human glutamate receptor subunits (Tables S1–S3) were designed to flank intron sequences, if feasible, using Primer 3 software (http://frodo.wi.mit.edu). Quantitative real-time PCR was performed with cDNA from 30 ng total RNA, 0.6 lM forward and reverse primers, PlatinumSYBR Green qPCR Supermix (Invitrogen), and 100 nM 6carboxy-X-rhodamine (ROX) using the following protocol in an MX 3000P instrument (Stratagene, La Jolla, CA, USA): 2 min 50C, 2 min 95C and 50 cycles of 15 s 95C, 30 s 60C. To confirm a single amplicon a product melting curve was recorded. The correct amplicon size was asserted by agarose gel electrophoresis using low molecular weight DNA ladder (New England Biolabs, Ipswich, MA, USA). Threshold cycle (Ct) values were placed within the exponential phase of the PCR as described previously (Engemaier et al. 2006). Ct values of three independent experiments, each performed in duplicate, were normalized to b2-microglobulin (Ct)Ct b2-microglobulin = DCt). DCt values were used to calculate the relative subunit expression levels and are given as means ± SEM (Tables S1–S3). The expression of glutamate receptor subunits was statistically evaluated by subjecting DCt values to a one-way

and Newman–Keuls post-test for multiple comparisons taking statistical significance as p < 0.05.

ANOVA

Calcium imaging Measurements of the intracellular Ca2+ concentration [Ca2+]i in differentiating human mesencephalic NPCs were carried out using the fluorescent indicator fura-2 in combination with a monochromator-based imaging system (T.I.L.L. Photonics, Gra¨felfing, Germany) attached to an inverted microscope (BX51WI, Olympus, Hamburg, Germany). Emitted fluorescence was collected by a charge-coupled device camera. NPCs were differentiated on poly-Llysine coated 15 mm coverslips in 12-well microplates. Cells were loaded with 5 lM fura-2-AM (Invitrogen) supplemented with 0.01% Pluronic F127 for 30 min at 20–22C in a standard bath solution containing (in mM): 140 NaCl, 5 KCl, 2 CaCl2, 10 glucose and 10 HEPES, adjusted to pH 7.4 with NaOH. For measurements of [Ca2+]i, fura-2 fluorescence was excited at 340 and 380 nm. Fluorescence emission from single cells was acquired in intervals of 2 s. After correction for the individual background fluorescence, the fluorescence ratio R = F340/F380 was calculated. Calibration of [Ca2+]i was performed as described previously (Wegner et al. 2008b). Drug treatment of NPCs To investigate the influence of L-glutamate, NMDA, and NMDA + memantine on NPCs in vitro, additional separate experiments were performed in which at least three different cell lines were treated with two distinct concentrations (10 or 100 lM) during proliferation (2 weeks) and differentiation (1 week). The drugs were renewed with every media change. The stock solution was prepared by dissolving drugs (Sigma) in distilled water at a concentration of 100 mM. The determination of cell number and protein content of treated and untreated NPCs was performed as described previously (Milosevic et al. 2006). Additionally, we treated some NPC cultures with glutamate (50 lM), NMDA (100 lM), or NMDA + memantine (each 100 lM) during the first week of differentiation in vitro. We chose these drug concentrations because evaluation of cell viability had shown no significant reduction. After two more weeks of differentiation without drug treatment, the voltage-gated sodium and potassium currents of NPCs were recorded to investigate their functional neuronal differentiation. Immunocytochemistry For immunocytochemical stainings of the NMDA receptor subunits NR1 and NR2B in NPCs after 3 weeks differentiation (Fig. 1) we used the same antibodies and protocol as described previously (Garcia de Arriba et al. 2006). For stainings of NPCs after drug treatment, the following protocol was applied: After fixation of differentiated cells with 4% paraformaldehyde, unspecific binding was blocked in phosphatebuffered saline supplemented with 0.3% Triton X-100 and 2% bovine serum albumin or 10% fetal calf serum. Then, cultures were incubated for 2 h at 20–22C with the following primary antibodies: rabbit polyclonal anti-tyrosine hydroxylase (TH; Santa Cruz, Heidelberg, Germany) diluted 1 : 500, mouse monoclonal anti-btubulin III (1 : 500, Sigma) or rat monoclonal anti-glial fibrillary acidic protein (GFAP; 1 : 500, Zymed, San Francisco, CA, USA). After washing, the cells were incubated for 1 h at 20–22C with



Fig. 1 Immunocytochemistry of human mesencephalic neural progenitor cells (NPCs) after 3 weeks differentiation in vitro. Confocal laser-scans of NPCs immunoreactive for b-tubulin III (Tuj1; a, e) and tyrosine hydroxilase (TH; i) as well as the NMDA receptor subunits NR1 (b, j) and NR2B (f); nuclei were counter-stained with DAPI (c, g, k). Merged pictures illustrate that b-tubulin III-positive NPCs express NR1 and NR2B in contrast to the b-tubulin III-negative cells (d, h). A representative TH-immunoreactive NPC indicates expression of the subunit NR1 in dopaminergic cells (l).

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fluorescent secondary antibodies conjugated to Alexa Fluor 488 or Alexa Fluor 594 (1 : 500, Invitrogen), respectively. Nuclei were stained with 4¢,6-diamidino-2-phenylindole (DAPI, 0.5 mg/mL; Calbiochem, San Diego, CA, USA) for 30 min at 20–22C. Immunostainings were visualised by fluorescence microscopy (Axiovert 200, Zeiss, Jena, Germany). Digital images were acquired with an AxioCam MRc camera using the image-analysis software AxioVision 4 (Zeiss) or by a confocal laser-scanning microscopy (Zeiss LSM 510 Meta). The number of cells immunoreactive for TH, GFAP, and b-tubulin III was determined by counting the number of positive cells in relation to the number of DAPI stained nuclei. A total of approximately 800–1000 cells were counted within three randomly selected fields per well. Additionally, all THimmunopositive NPCs were counted per well.

Results Functional properties of ionotropic glutamate receptors in differentiated NPCs We investigated human midbrain-derived neural progenitor cells (NPCs) that had been differentiated for 3 weeks in vitro under standard conditions. Immunocytochemical stainings showed that b-tubulin III-positive NPCs express the NMDA receptor subunits NR1 and NR2B in contrast to b-tubulin III-

negative cells (Fig. 1a–h). All investigated TH-immunoreactive NPCs also expressed the NR1-subunit (Fig. 1i–l). The expression of NR1 is a prerequisite for the assembly of functional NMDA receptors (Kew and Kemp 2005). Whole-cell recordings of differentiated NPCs were performed in the voltage-clamp mode to measure ionotropic glutamate receptor currents. A current response (> 5 pA) during rapid applications of L-glutamate or NMDA was detected in 93% of cells (n = 124/134). The mean maximal sodium current (98 pA, n = 120), the membrane capacity (9 pF, n = 124), and input resistance (632 MW, n = 118) of these NPCs were not significantly different compared to cells without detectable glutamate-/NMDA-induced current. Rapid applications of increasing glutamate concentrations (100 nM to 1 mM) on differentiated NPCs elicited inward currents in a concentration-dependent manner (Fig. 2a). The glutamate concentration-response plot (Fig. 2b) indicated an EC50 of 2.2 lM (95% confidence interval of log EC50 value )5.755 to )5.566, slope 0.9, n = 20). Mean peak currents induced by L-glutamate were 231 pA (n = 84). Currents evoked by 100 lM glutamate (EC90) could be antagonized by co-application of memantine (Fig. 2c) while only a slight inhibition was caused by 6-cyano-7-nitroquinoxaline-2,3-



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dione (CNQX; Fig. 2d). NMDA- and (±)-AMPA-induced currents were blocked by memantine or CNQX, respectively (Fig. 2c and d). Currents through AMPA and kainate receptors induced by the specific agonists S-AMPA and (RS)-2-Amino-3-(3-hydroxy-5-tert-butylisoxazol-4-yl)propanoic acid (ATPA), respectively (Kew and Kemp 2005), were markedly smaller than NMDA-evoked responses (Fig. 2e). In summary, the glutamate receptor pharmacology of NPCs suggests a predominance of NMDA receptor over AMPA/ kainate receptor currents (Fig. 2e and f). Next, we analyzed the NMDA receptor pharmacology of NPCs in detail. Whole-cell recordings of inward currents during rapid applications of increasing NMDA concentrations (300 nM to 3 mM) showed a concentration-dependency (Fig. 3a). Mean peak currents evoked by NMDA were 169 pA (n = 43). The concentration-response curve for NMDA (Fig. 3b; EC50 = 36 lM, 95% confidence interval

Fig. 2 Functional analysis of glutamate receptors in human mesencephalic NPCs differentiated for 3 weeks in vitro. (a) Whole cell recordings of inward currents elicited by rapid application of increasing glutamate concentrations (100 nM–1 mM). (b) The glutamate concentration-response curve suggests an EC50 of 2.2 lM (n = 20). (c) Whole cell currents evoked by glutamate and NMDA were markedly inhibited by coapplication of the non-competitive NMDA receptor antagonist memantine. (d) Coapplications of the competitive AMPA/kainate receptor antagonist CNQX reduced a glutamate-induced current only slightly, while the small current evoked by the AMPA/kainate receptor agonist (±)-AMPA was almost completely blocked. (e) Bar graph illustrates glutamate receptor peak currents in pA determined by application of the specific agonists NMDA, S-AMPA, and ATPA indicating a predominance of NMDA over AMPA/kainate receptor peak currents (n = 6–28, data are given as means ± SEM). (f) The inhibition of glutamate receptor currents by co-application of the antagonists memantine and CNQX is shown in % residual current. Note, current amplitudes were measured 1 s after activation (n = 7–16, data are given as means ± SEM).

of log EC50 value )4.532 to )4.364, slope = 0.8, n = 16) indicates a significantly different EC50 value than for glutamate (p < 0.0001, F test). Similar high EC50 values were calculated for NMDA concentration-response plots of rat primary mesencephalic neurons at different time points of embryogenesis [21 lM at E8 (n = 15) and 26 lM at E14 (n = 16), data not shown]. In some differentiated human NPCs displaying large voltage-gated sodium currents, rapid applications of 3 mM NMDA evoked depolarising inward currents that elicited single action potentials (Fig. 3c). To distinguish the particular NMDA receptor subunit NR2 coexpressed in a functional assembly with NR1, we investigated the pharmacology of different subtype-specific antagonists and modulators at NPCs (Fig. 3d). NMDA-induced currents were not reduced by a low concentration of Zn2+ (100 nM) which only inhibits receptors containing the NR2A-subunit (Rachline et al. 2005). In contrast, a high Zn2+ concentration



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Fig. 3 NMDA receptor pharmacology of human midbrain-derived NPCs after 3 weeks differentiation. (a) Whole-cell recordings of inward currents evoked by application of increasing NMDA-concentrations (300 nM–3 mM). (b) The concentration-response curve for NMDA suggests an EC50 of 36 lM, which is significantly different from the glutamate EC50 (p < 0.0001, F test). (c) Recordings show large voltage-gated sodium currents (left, 1238 pA at )20 mV) elicited by stepwise depolarisation in increments of 10 mV from a holding potential of )70 mV. In the same cell rapid application of 3 mM NMDA evoked an inward current (middle, 228 pA) and induced an action potential (right, peak at )10 mV) measured in whole-cell current clamp mode. (d) NMDA-induced currents are not decreased by a low

concentration of Zn2+ (100 nM) which is most sensitive at receptors containing the NR2A-subunit. In contrast the co-application of a high Zn2+ concentration (100 lM), known to reduce NMDA-evoked currents at all NR2-containing receptors, and the NR2B-specific antagonist ifenprodil (10 and 100 lM) inhibited a NMDA EC70 in all NPCs. The neurosteroid pregnenolone sulphate potentiated NMDA-evoked currents in all cells as described for receptors with NR2A or NR2Bsubunits. (e) NMDA-receptor pharmacology of NPCs suggesting expression of NR2B-subunits is summarized in the bar graph. Note, current amplitudes were measured 1 s after activation (n = 13–15; data are given as means ± SEM).

(100 lM), known to decrease NMDA-evoked currents at all NR2-containing receptors (Rachline et al. 2005), and the NR2B-specific antagonist ifenprodil (10 and 100 lM; Williams 1993) blocked a NMDA EC70 (100 lM) in all NPCs. The neurosteroid pregnenolone sulphate potentiated NMDAevoked currents in all cells as described for receptors containing NR2A- or NR2B-subunits (Malayev et al. 2002).

In summary, the NMDA receptor pharmacology of NPCs indicates expression of NR2B-subunits (Fig. 3e). Measurements of the intracellular Ca2+ concentration We determined the effect of different glutamate receptor agonists and antagonists on Ca2+ signaling in differentiated NPCs. Bath application of the specific group I metabotropic



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Fig. 4 Glutamate receptor-induced Ca2+ signaling in fura-2 loaded NPCs. (a) Transient [Ca2+]i changes induced by bath application of 100 lM DHPG, 100 lM ATPA, 100 lM S-AMPA, 100 lM NMDA/ 10 lM glycine, and 50 mM KCl. The trace is representative of recordings from 54 cells in four experiments. (b) Summary of [Ca2+]i response amplitudes (means ± SEM) and fractions of cells responding to different stimuli was obtained from experiments as shown in (a). (c–d) The [Ca2+]i increases evoked by 100 lM NMDA/10 lM glycine

were blocked by 100 lM memantine (MEM) and 100 lM of the NR2Bspecific inhibitor ifenprodil (IFP). (e–f) S-AMPA- and ATPA-induced [Ca2+]i increases were blocked by 100 lM CNQX. (g) Verapamil (VER, 100 lM), a broad-spectrum blocker of voltage-gated [Ca2+]i channels, differentially suppressed [Ca2+]i increases evoked by bath application of 50 mM KCl, 100 lM S-AMPA, and 100 lM NMDA/10 lM glycine. (h) Summary of blocked [Ca2+]i increases (n = 18–27; means ± SEM) obtained from experiments as shown in (c–g).

glutamate receptor (mGluR1 and 5) agonist (S)-3,5-dihydroxyphenylglycine (DHPG) and of specific ionotropic glutamate receptor agonists induced [Ca2+]i changes in single NPCs (Fig. 4a). The [Ca2+]i response amplitudes evoked by NMDA/glycine and S-AMPA exceeded those induced by ATPA, DHPG and 50 mM KCl (Fig. 4b). The percentage of NPCs responding to the different stimuli were 96–98% with the exception of 44% DHPG-responsive cells (Fig. 4b). Because glycine is a co-agonist at NMDA-receptors composed of NR1 and NR2A–D but induces activity of NR1/ NR3A or -3B receptors in the absence of NMDA (Chatterton et al. 2002), we applied 10 lM glycine alone on NPCs. In 61% of cells an increase in [Ca2+]i could be observed (data

not shown), suggesting the presence of excitatory glycine receptors of the NMDA receptor family. Similarly to the current responses, NMDA-induced [Ca2+]i increases were completely suppressed by memantine (by 97%; Fig. 4c and h). Ifenprodil was slightly less effective than memantine (by 89%; Fig. 4d). Responses evoked by S-AMPA and ATPA were blocked in the presence of CNQX by 67% and 86%, respectively (Fig. 4e and f). In order to revise the Ca2+ permeability of different glutamate receptor subtypes we tested the effect of the unspecific inhibitor of voltage-gated Ca2+ channels, verapamil, on Ca2+ signaling in NPCs. Verapamil suppressed [Ca2+]i increases evoked by bath application of 50 mM KCl and S-AMPA for about 80% and



76%, respectively (Fig. 4g and h), suggesting the predominant expression of Ca2+-impermeable AMPA receptors. Responses to NMDA/glycine were only inhibited by about 31% (Fig. 4g and h). To gain insight into the changes of ionotropic glutamate receptor expression during differentiation we performed similar Ca2+ imaging experiments in proliferating NPCs. In contrast to the above results, proliferating cells showed no [Ca2+]i increases at all when NMDA/glycine (100 lM/ 10 lM) or 50 mM KCl were applied (n = 125; data not shown). However, S-AMPA and ATPA (100 lM each) induced [Ca2+]i increases in 94% and 85% of tested cells, respectively (n = 125; data not shown). Quantification of glutamate receptor subunits in differentiated NPCs For quantitative expression analysis of glutamate receptor subunits, human midbrain-derived NPC lines (n = 3) were investigated by real-time PCR after 3 weeks of differentiation in vitro. Significant differences between DCt values are indicated for the most frequent subunit of a particular receptor subtype (Fig. 5). The predominant expression of NR2A and NR2B was not significantly different from the other NMDA receptor (a)

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Fig. 5 Real time PCR analysis of glutamate receptor subunits expressed by human mesencephalic NPCs after 3 weeks differentiation. RNA from three NPC lines was subjected to reverse transcriptase reaction. Quantitative real-time PCR was performed for each transcript and control (b2-microglobulin). Threshold cycle (Ct) values were normalized to the Ct values of the control and are given as log2)DCt (DCt = Ct)Ct b2-microglobulin). Data are presented as means ± SEM of three independent experiments per subunit, each performed in duplicate. Significant differences between DCt values

subunits (Fig. 5a). Surprisingly, NR1 was expressed to a rather low extent although it is obligatory to form a functional NMDA receptor. The most frequent glutamate receptor subunit GluR2 plays important roles in determining the Ca2+-sensitivity of AMPA receptor subtypes and in longterm synaptic plasticity (Isaac et al. 2007). GluR2 was expressed to a significantly higher extent (Fig. 5b, *p < 0.05, ANOVA) than all other AMPA receptor subunits and GluR7 was found more frequently than other kainate receptor subunits (Fig. 5c, *p < 0.01, ANOVA). Among metabotropic glutamate receptor subunits mGluR3 was prominently expressed (Fig. 5d, ***p < 0.001, ANOVA). Drug treatment of NPCs Drug treatment of human mesencephalic NPCs with Lglutamate (10 or 100 lM) during expansion (2 weeks) did not reveal significant changes compared to untreated controls in respect to the number of living cells and protein content. Also, the immunocytochemical analysis of drugtreated NPCs after differentiation (1 week) using the markers GFAP and b-tubulin III was not different from controls, whereas the total number of TH-immunopositive cells per well was significantly reduced to 54.4% and 26.8% of untreated controls by L-glutamate (10 lM and (b)

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are indicated for the most frequent subunit of a particular receptor subtype (*p < 0.05, **p < 0.01, ***p < 0.001, ANOVA and Newman– Keuls post-test). (a) Bar graph shows predominant expression of NMDA receptor subunits NR2A and NR2B. (b) GluR2 expression was significantly more pronounced than the expression of all other AMPA receptor subunits. (c) GluR7 was expressed to a significantly higher extent than the other kainate receptor subunits. (d) All metabotropic glutamate receptor subunits were expressed significantly lower than mGluR3.



100 lM, respectively; n = 3, p < 0.001, ANOVA, data not shown) showing a similar effect as GABA (Wegner et al. 2008b). Using the same protocol, the treatment of NPCs with NMDA (10 or 100 lM) did not result in significant differences from controls in cell number, proliferation rates, protein content, GFAP- and MAP2-immunoreative NPCs. However, the number of b-tubulin III/DAPI-positive cells was significantly reduced from 34.9% in controls to 25.3% by 100 lM NMDA (n = 8, p < 0.05, ANOVA; data not shown). Surprisingly, treatment of NPCs with 100 lM NMDA revealed a significant increase in the percentage of TH/DAPI-positive cells and in the total number of TH(a)

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immunoreactive cells per well (Fig. 6; n = 3–8, p < 0.05, ANOVA), while the addition of 10 lM NMDA had no effect. Adding the NMDA receptor antagonist memantine (100 lM) during the treatment with 100 lM NMDA, resulted in a marked reduction of TH-immunopositive NPCs compared to treatment with NMDA alone (Fig. 6i and j; n = 3, p < 0.05, ANOVA). Drug treatment of NPCs with glutamate (50 lM), NMDA (100 lM), or NMDA + memantine (each 100 lM) during the first week of differentiation in vitro did not induce a significant difference in the peak currents of voltage-gated sodium and potassium channels recorded in cells after differentiation for 3 weeks (data not shown).

Fig. 6 Immunocytochemistry of human midbrain-derived NPCs after treatment with NMDA during 2 weeks of proliferation and 1 week of differentiation in vitro (a–d: control, e–h: +100 lM NMDA). Confocal laserscans of NPCs immunoreactive for b-tubulin III (a, e) and TH (b, f); nuclei were stained with DAPI (c, g). Merged pictures illustrate that the number of NPCs expressing TH (d, untreated control) can be markedly increased by addition of 100 lM NMDA (h). The TH-immunopositive NPCs in untreated controls and treated cultures (+10 lM NMDA, +100 lM NMDA, +NMDA/ memantine each 10 lM, +NMDA/memantine each 100 lM) were quantified in relation to DAPI-stained cells (i) and normalized to the total number of TH-positve cells per well under control conditions (j). Treatment of mesencephalic NPCs with 100 lM NMDA significantly enhanced dopaminergic neurogenesis while the addition of 100 lM memantine blocked this effect (i–j, n = 3–8; data are given as means ± SEM; *p < 0.05, **p < 0.01, ANOVA and Newman–Keuls posttest).



Discussion In this study, electrophysiological recordings and calcium imaging data demonstrated the expression of functional glutamate receptors in differentiated human midbrain-derived NPCs. Previous approaches to characterize human fetal neural progenitors revealed intracellular Ca2+ responses to glutamate with a lower magnitude and frequency (12%) after 2–4 weeks of differentiation (Piper et al. 2000, 2001) suggesting an immature state of glutamate receptor expression and function. Recently, we showed that depolarizing followed by hyperpolarizing culture conditions enable developing human NPCs to adopt more mature functional qualities in respect to voltage-gated sodium and potassium channels, however, AMPA-induced Ca2+ signals decreased significantly (Schaarschmidt et al. 2009). Thus, in vitro conditions promoting the functional maturation of voltagegated ion channels in NPCs may not equally stimulate the development of ionotropic glutamate receptors. In electrophysiological recordings of neural stem-like cells derived from human umbilical cord blood and differentiated for up to 4 weeks, NMDA failed to induce a current, while kainic acid evoked non-desensitizing inward currents in few cells suggesting expression of ionotropic non-NMDA glutamate receptors (Sun et al. 2005; Buzanska et al. 2006). In contrast, human fetal progenitor cells grown on rat glial feeder layers gave rise to neurons after 22–70 days in vitro showing mature ionotropic glutamate receptor properties qualitatively similar to rat cortical neurons (ChalmersRedman et al. 1997). These studies indicate that the expression of functional ionotropic glutamate receptors in human neural progenitors is likely to be dependent on the cell line, differentiation protocol, and time of in vitro maturation. We observed an EC50 value for the glutamate concentration-response plot of human NPCs (2.2 lM) close to the value of neural stem cells from the adult mouse tegmentum (8.2 lM; Hermann et al. 2006). Also, similar high EC50 values were calculated for NMDA concentration-response plots of rat primary mesencephalic neurons at different time points of embryogenesis (21–26 lM; F. Wegner, unpublished observation) as for human NPCs (36 lM). These EC50 values may reflect rather low species differences in terms of glutamate and NMDA receptor pharmacology of midbrainderived neural cells. In human mesencephalic NPCs, specific agonists at AMPA (S-AMPA) and kainate (ATPA) receptors induced relatively small current amplitudes and glutamate-evoked currents were only slightly reduced by CNQX. By comparison, NMDA-induced currents were of higher amplitude and glutamate-evoked currents were blocked by memantine for the predominant part. Differential increases in [Ca2+]i caused by these agonists also suggest a graded expression of ionotropic glutamate receptors with a predominance of

the NMDA type which plays an important role for the regulation of proliferation and differentiation in NPCs (Luk et al. 2003; Suzuki et al. 2004, 2006; Mochizuki et al. 2007; Georgiev et al. 2008; Hu et al. 2008; Klimaviciusa et al. 2008). The NMDA receptor pharmacology of NPCs can be characterized by the differential sensitivity to Zn2+, the neurosteroid pregnenolone sulphate, and the NR2B-subunit specific antagonist ifenprodil (Williams 1993; Perin-Dureau et al. 2002). While pregnenolone sulphate potentiated NMDA-induced currents in all NPCs as described for receptors containing NR2A- or NR2B-subunits (Malayev et al. 2002), a low concentration of Zn2+ (100 nM), which is most sensitive at receptors with NR2A-subunits (Rachline et al. 2005), did not inhibit NMDA-evoked currents contrasting the block by 10 and 100 lM ifenprodil. Although a high ifenprodil concentration (100 lM) also shows inhibitory effects on NR2A-containing receptors (IC50 = 146 lM, Williams 1993), the nearly complete suppression of NMDAevoked currents by 10 and 100 lM ifenprodil indicates NR2B-subunit expression. Furthermore, ifenprodil suppressed NMDA-induced Ca2+ responses almost as effective as memantine. Although quantitative PCR showed that both NR2A- and NR2B-subunits were expressed similarly pronounced, the immunocytochemical and functional analyses suggest a predominant role for NMDA receptors containing NR1- and NR2B-subunits in NPCs. Like NMDA receptors, the native AMPA receptors are probably heteromeric in composition. The AMPA receptor GluR2-subunit dictates critical biophysical properties including the Ca2+-permeability of the receptor and strongly influences receptor assembly and trafficking as well as long-term synaptic plasticity. AMPA receptors devoid of GluR2 are Ca2+-permeable and show marked inward rectification (Kew and Kemp 2005; Isaac et al. 2007). In human mesencephalic NPCs, co-application of verapamil with glutamate receptor agonists or KCl revealed that the AMPA-induced [Ca2+]i increases were primarily because of depolarisation-induced activation of voltage-gated calcium channels. By contrast, the weak effect of verapamil on NMDA-evoked Ca2+ signals argue for the Ca2+-permeability of this ionotropic receptor. These results correspond well to the quantitative PCR showing a most pronounced expression of GluR2 and indicate the predominance of a Ca2+-impermeable, GluR2-subunit containing AMPA receptor in NPCs. Unlike the kainate receptor subunits KA1–2, which are barely expressed in human midbrain-derived NPCs, the subunits GluR5–7 can form functional homomeric kainate receptors or combine with KA1–2 to form heteromeric receptors (Alt et al. 2004). A previous study of recombinant homomeric receptors concluded that the kainate receptor subunit GluR7 exhibits a low affinity for glutamate and kainate relative to GluR5 and GluR6 (Schiffer et al. 1997). The significantly higher expression of GluR7 in NPC-PCR



may therefore hint to kainate receptors with low glutamate affinity. Besides the presence of kainate receptors in nearly all cells, the specific group I metabotropic glutamate receptor (mGluR1 and 5) agonist DHPG induced Ca2+ signals in a fraction of NPCs. Group 1 metabotropic glutamate receptors play a critical role in both NMDA receptor and voltage-gated calcium channel-dependent long-term potentiation (Manahan-Vaughan et al. 1998). However, quantitative PCR of NPCs showed a predominant expression of mGluR3 (group 2) that was found to inhibit adenylate cyclase via Gi/Go in glial cells and synapses with a predominantly pre-synaptic distribution, consistent with its function as an inhibitory autoand heteroceptor suggesting particular roles for mGluR3 in long-term depression, glial function, and neuroprotection (Tamaru et al. 2001; Kew and Kemp 2005; Lujan et al. 2005; Harrison et al. 2008). Glutamate (10 lM) can significantly increase the neurosphere size, proliferation rates, and neurogenesis of human fetal cortical NPCs (Suzuki et al. 2006), which were shown to express the NMDA receptor subunits NR1 and NR2B by reverse transcription-PCR (Suzuki et al. 2004). The enhanced proliferation in vitro could be blocked by the NMDA receptor antagonist MK-801 (10 lM) but not by AMPA/ kainate or metabotropic glutamate receptor antagonists (Suzuki et al. 2006). To investigate the influence of Lglutamate and NMDA on human mesencephalic NPCs regarding dopaminergic neurogenesis, we treated at least three different cell lines with two drug concentrations (10 or 100 lM) during proliferation and differentiation. In proliferating NPCs, NMDA was unable to induce Ca2+ signals and did not affect the rate of expansion, suggesting the absence of functional NMDA receptors under the appropriate culture conditions. Because we frequently observed Ca2+ responses upon stimulation with S-AMPA and ATPA but not with DHPG, we suppose that AMPA and kainate receptors are the dominant glutamate receptors in proliferating NPCs. During differentiation, however, the addition of 100 lM NMDA but not of 10 lM NMDA and glutamate (10 or 100 lM) significantly increased the percentage of TH/DAPI-stained cells as well as the total number of TH-positive NPCs. The NMDA-evoked increase of TH-positive NPCs could be blocked by memantine indicating the enhancement of dopaminergic neurogenesis through NMDA receptor activity. Although NMDA is of lower potency than glutamate and not quite a full agonist at NMDA receptors, it is not a substrate for glutamate degradation or uptake and acts therefore more effectively as NMDA receptor agonist (Kew and Kemp 2005). In contrast to NMDA actions, the continuous stimulation of AMPA, kainate, and metabotropic glutamate receptors may lead to a reduced dopaminergic neurogenesis of NPCs. In conclusion, the analyses of differentiated NPCs derived from human fetal midbrain tissue show the functional expression of ionotropic and metabotropic glutamate recep-

tors indicating a predominant role for NMDA receptors containing NR1- and NR2B-subunits. Treatment of NPCs with 100 lM NMDA in vitro during proliferation and differentiation significantly enhanced dopaminergic neurogenesis. This study suggests that human mesencephalic NPCs acquire essential glutamate receptor properties during neuronal maturation in vitro.

Acknowledgements The work presented in this paper was made possible in part by funding from the German Federal Ministry of Education and Research (BMBF, PtJ-Bio, 0313909), the IZKF-Leipzig (TP C27), and the DFG (Schw 704/5-1). The authors wish to thank Mrs. Annett Brandt and Mrs. Ute Roemuss for excellent technical assistance as well as Dr. Jens Grosche (IZKF, University of Leipzig, Germany) and Dr. Claudia Heine (TRM, University of Leipzig, Germany) for support in confocal laser-scanning microscopy.

Supporting Information Additional Supporting Information may be found in the online version of this article: Table S1. Quantitative real-time PCR analysis of NMDA receptor subunit expression in human mesencephalic NPCs after 3 weeks differentiation in vitro. Table S2. Quantitative real-time PCR analysis of AMPA/kainate receptor subunit expression in human mesencephalic NPCs after 3 weeks differentiation in vitro. Table S3. Quantitative real-time PCR analysis of metabotropic glutamate receptor subunit expression in human mesencephalic NPCs after 3 weeks differentiation in vitro. As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer-reviewed and may be re-organized for online delivery, but are not copy-edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.

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