CHAPTER 2: CELLULAR PHENOTYPES OF HUMAN MODEL NEURONS (NT2) AFTER
5. Concluding remarks
Our rapid differentiation protocol involving the use of floating aggregates, instead of adherent culture, generates 95% pure, astrocyte-free neurons that display a variety of neurotransmitter phenotypes. A large portion of these neurons is cholinergic and exhibits motor neuronal characteristics. The rapidity of the differentiation protocol should enable fast high-throughput screening of chemical compounds, e.g., for the neuroprotection of motor neurons. Moreover, our method should improve the suitability of the NT2 system as a model for the differentiation of human nerve cells and developmental neurotoxicity (Hill et al. 2008; Radio and Mundy 2008).
Acknowledgements
We are grateful to Jan de Vente, Maastricht University for the gift of the cGMP antiserum, to O. P.
Ottersen, Department of Anatomy, University of Oslo for the gift of the glutamate antiserum, to Alexandra Kotsiari, Daniela Ragancokova, and Martin Stangel, Hannover Medical School for the gift of primary rat astrocytes, to the Department of Physiology, University of Veterinary Medicine, Hannover for the gift of mouse CNS tissue, and to Sabine Knipp for helpful discussions.
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Chapter 3: Synaptic currents and transmitter responses in human NT2 neurons differentiated in aggregate culture
Grzegorz Podrygajloa,b, Yunping Songb,c , Friedrich Schlesingerc, Klaus Krampflb,c, Gerd Bickera,c
a Div. of Cell Biology, Institute of Physiology, University of Veterinary Medicine Hannover, Hannover, Germany
b Center for Systems Neuroscience (ZSN) Hannover, Germany
c Department of Neurology and Clinical Neurophysiology, Hannover Medical School, Hannover, Germany
Corresponding author:
Gerd Bicker; Div. of Cell Biology, Institute of Physiology, University of Veterinary Medicine Hannover, Bischofsholer Damm 15, Haus 102, D-30173 Hannover, Germany;
email: gerd.bicker@tiho-hannover.de
Submitted to: Neuroscience Letters
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1. A
BSTRACTPostmitotic neurons were generated from the human NT2 teratocarcinoma cell line in a novel cell aggregate differentiation procedure. The NT2 model neurons express punctate immunoreactivity for synapsin and for cell markers related to GABAergic and glutamatergic neurotransmission. Using the outside-out patch-clamp configuration, we characterized the kinetics of currents elicited by a rapid application of the amino acid neurotransmitters. Moreover, we detected spontaneous postsynaptic currents in glia free cell cultures that may result from the firing activity of glutamatergic and GABAergic NT2 neurons. These cultured spontaneously active networks might serve as a useful tool to analyze factors that modulate the formation and efficacy of synapses bet ween human neurons.
Key words: human NT2 neurons, GABA, glutamate, patch clamp, spontaneous activity
Neurons derived from the human teratocarcinoma cell line Ntera 2 (NT2) by retinoic acid treatment have been used in a variety of biomedical investigations including cell culture studies, cell grafts into the nervous system of experimental animals, and successful transplantations into human stroke patients (Pleasure et al. 1992, Saporta et al. 2002, Nelson et al. 2002). Gene profiling studies of retinoic acid-treated teratocarcinoma cells indicate the similarity of NT2 neural differentiation to vertebrate neurogenesis (Przyborski et al. 2000, Houldworth et al 2001, Przyborski et al 2003). Cultures of NT2 neurons may be useful to investigate mechanisms of synaptogenesis among human nerve cells.
However, rather few studies have so far been dedicated to the question of synaptic connectivity between NT2 neurons. Hartley et al. (1999) demonstrated the formation of functional synapses between NT2 neurons grown on rat astrocytes using the patch-clamping technique. The presence of synaptic profiles has been detected in electron-microscopic sections (Guillemain et al. 2000). An increased expression of synapsins during neural differentiation (Leypoldt el al. 2002) and immunocytochemica l stainings against various neurotransmitter related markers (Guillemain et al. 2000) add supporting lines of evidence for synaptic neurotransmission. NT2 neurons cultured in the absence of glia cells on multi-electrode arrays showed spontaneous action potentials and signal propagation with a speed typical for non-myelinated neurons (Görtz et al., 2004). However, NT2 neurons did not show the synchronized firing activity that is typical for networks of embryonic rodent cortical neurons, a finding that was attributed to insufficient synaptic coupling under these culture conditions.
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Previously we have demonstrated a novel cell aggregate method for differentiation of NT2 neurons from NT2 precursor cells what resulted in generation of postmito tic neurons in a less than one month (Paquet-Durand et al. 2003, Paquet-Durand and Bicker 2007). These neurons elaborate extensive neurite connections in culture which show punctate stainings for the synaptic markers synapsin and synaptotagmin (Podrygajlo et al. 2009). The present work addresses several issues: using whole-cell patch clamp techniques we investigated whether spontaneous postsynaptic currents are present in networks of NT2 neurons differentiated by our cell aggregate method. Then we use the outside out configuration together with an ultrafast solution exchange system for the application of l-glutamate and GABA to determine the time constants for neurotransmitter-induced currents.
Electrophysiological experiments together with immunocytochemical staining provided evidence for the presence of glutamatergic and GABAergic neurotransmission in networks of NT2 neurons that were cultured without glial cells.
The human Ntera2/D1 cell line (NT2) was obtained from ATCC (American Type Culture Collection, VA, USA). We generated NT2 neuronal cultures using the differentiation protocol in free -floatin g aggregates (Paquet-Durand et al. 2003). The post mitotic NT2 neurons were plated on poly-D-lysine and matrigel (Becton-Dickinson, Bedford, MA, USA) coated cover glasses at a density of 20,000 cells per well and cultured for 3-5 weeks.
To test for GABAergic and glutamatergic neurotransmitter markers in these cultures, we initially performed immunocytochemical staining against synapsin, the GABA synthesizing e nzyme GAD, GABA and the vesicular glutamate transporter 1. For immunocytochemistry, cells were washed with PBS and fixed for 30 min at room temperature with 4% paraformaldehyde (PFA). Cells were rinsed after each step three times for 5 minutes in PBS containing 0.2% Triton X-100 (PBS-T). Blockin g solution containing PBS-T and 5% normal horse serum was applied for one hour. Primary antibodies used in experiment: monoclonal mouse anti-synapsin 1 (106001, Synaptic Systems, Goettingen, Germany, 1:500), polyclonal rabbit anti-gamma-amino-butyric acid (GABA; A2052, Sigma, 1:5000), polyclonal rabbit anti-glutamic acid decarboxylase (GAD; G5163, Sigma, 1:1000), polyclonal rabbit anti-vesicular glutamate transporter 1 (VGluT1; 135302, Synaptic Systems, 1:1000).
All antibodies were diluted in blocking solution and applied overnight at 4°C. Secondary biotinylated antibodies (Vector, Burlingame, MA, USA), diluted 1:250 in blocking solution, were added for one hour incubation at room temperature. Immunofluorescence was detected by applying streptavidin-Alexa Fluor 488 (Mobitec, Goettingen, Germany) or streptavidin-CY3 (Sigma, Taufkirchen, Germany) for one hour at room temperature, dilution 1:250. Finally, cells were incubated for five minutes with 2
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µM DAPI (4',6-diamidino-2'-phenylindol- dihydrochloride) as a nuclear counterstain.
Synapsin-immunoreactivity could be found as discrete, punctate staining on the neurites and more homogeneously in the cell bodies of the NT2 neurons (Fig. 1A). Immunocytochemical stainings aga inst glutamate decarboxylase (GAD, Fig. 1B) and against GABA (Fig. 1D) suggest that the differentiation process generates ca. 14 % GABAergic neurons. The presence of immunoreactivity to the vesicular glutamate transporter (VGlut, Fig. 1C) provided evidence for glutamate as likely neurotransmitter in about 40% of the neurons.
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Fig.1 Inverted immunofluorescence staining of NT2 neurons for markers related to neurotransmission.
(A) Punctuate staining to for synapsin on the neurites of NT2 neurons with an increase in density in regions of connecting neurites (arrow). (B) Expression of glutamate decarboxylase (GAD) in a subpopulation of NT2 neurons (arrows) while absence of this enzyme is shown in other neurons (arrowhead). (C) Immunoreactivity for vesicular glutamate transporter (VGlut- 1) (arrows) together with immuno-negative neuron (arrowhead). (D) GABA immunoreactive (arrows) together with immno-negative neurons (arrowhead). Scale bars: 100 µm.
To record spontaneous activity in the cultured neural network we performed whole-cell voltage-clamp experiments using the cell bodies of the NT2 neurons. Patch pipettes were pulled out from borosilicate glass tubes with a DMZ-Universal puller (Zeitz Instruments, Augsburg, Germany). The open tip
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resistance with intracellular solution filled in was between 7 and 10 MΩ. The intracellular solution contained in mM: 140 KCl, 11 EGTA, 10 Hepes, 10 glucose, 2 MgCl2. The extracellular solution contained in mM: 162 NaCl, 5.3 KCl, 2 CaCl2, 0.67 NaHPO4, 0.22 KH2PO4, 15 Hepes, 5.6 glucose.
The osmolarity was adjusted to 340 mOsm/l with mannitol and pH was adjusted with KOH or NaOH to 7.4. Cells were constantly perfused with extracellular solution with flow rate 2 ml/min. All solutions were freshly prepared and filtered before each experiment.
Data were recorded with an Axopatch 200B patch-clamp amplifier (Axon Instruments, Union City, CA, USA). Ensemble currents were sampled with 20kHz using a Digidata 1200 Interface and pCLAMP6 software suit on a PC (Axon Instruments, Union City, CA, USA). The holding potential was -70 mV.
Signals with amplitudes of at least two times above the background noise were selected (>4 pA).
Classification of spontaneous activities record from NT2 neurons with respect to the time constant of decay (ms) of the postsynaptic current events was done using Origin software (OriginLab, Northampton, MA). All data are presented as means± S.E.M.
When NT2 neurons were cultured for three weeks, spontaneous inward currents were observed under voltage clamp conditions (Fig. 2A). The amplitude of such currents varied between 4 and 25 pA. The inset of Fig. 2 A shows two examples of spontaneous currents that are characterized by a fast risin g phase but rather different time constants for decay. The samples of spontaneous activity can be divided into two major populations which were fitted with a single exponential. The statistical analysis over n=7 recorded neurons showed one group of events with 14.73±0.78 ms as time constant of decay and the other with 33.3±1.03 ms (Fig. 2C). The group with faster decay may correspond to EPSCs (excitatory postsynaptic currents) whereas the slower decaying events may reflect IPSCs (inhibitory postsynaptic currents) (Smith et al. 2000; Pérez-León et al. 2003).
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Fig. 2 Patch clamp recordings from NT2 neurons. (A) Spontaneous activity measured in a NT2 neuron cultured for more than 3 weeks in the whole cell mode. The trace contains characteristic inhibitory postsynaptic currents (IPSC) and excitatory postsynaptic current (EPSC). Inset shows currents at higher time resolution. (B) Stimulation of NT2 neurons with 200 ms pulses of 1mM GABA and 10 mM glutamate recorded in an outside out mode. (C) Classification of spontaneous activities recorded from NT2 neurons clamped at –70 mV (n=7), with respect to the time constant of decay (ms) of the postsynaptic current events in the whole cell mode.
To test whether the cultured NT2 neurons were responsive to the inhibitory transmitter GABA and the excitatory transmitter glutamate, we performed outside out patch-clamp experiments according to Hamill et al. (1981). A piezo-driven, double-barreled tube element was used in ultrafast solution
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exchange of test substances within less than 100 μs (Krampfl et al. 2002). As shown by the two characteristic traces of Fig. 2 B, the GABA induced inward currents lead after a desensitisation phase to steady state amplitude, whereas the glutamate induced current desensitized with close-to-zero steady state amplitude.
A quantitative evaluation of the kinetic parameters of the currents is provided by Table 1. The GABA-induced currents of human NT2 neurons desensitize in a bi-exponential way, with fast and slow time constant 2= 8.86±0.86 ms, 1=38.03±4.78 ms, respectively. The value of slow time constant 1 is smaller compared to the recordings from HEK293 cells transfected with or GABAA
receptors (P icton and Fisher 2007). One of the reasons can be the outside out configuration (Krampfl et al. 2005) which produced faster responses of the receptors than whole cell configuration. Another indication is the expression of various subtypes of GABA receptors on the NT2 neurons, which contribute to different current-decay kinetics (Bosman et al. 2002; Lagrange et al. 2007).
The currents in response to 10mM glutamate desensitize in a mono-exponential way, with the time constant of 12.64±1.52ms, which was comparable with the recordings from the rod amacrine cells in the rat retina (Veruki et al. 2003) and from parasagittal slicesof the rat midbrain (Shi et al. 2001). In the case of the glutamatergic currents we have observed no deactivation phase due to a fast desensitization time (12.64±1.52ms).
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Tab. 1 Stimulation of NT2 neurons with 1mM GABA and 10mM glutamate using the fast application system. 1 represents the slow component of time constant for desensitization, 2 represents the fast component of time constant for desensitization, deac represents the time constant for deactivation, rft : time constant for rise time fit, rCdes: relative current amplitude of steady state, n represent number of neurons in the single experiment.
The cellular responses to amino acid neurotransmitter induced currents (Fig. 2B), the immunocytochemical detection of cell markers related to GABAergic and glutamatergic neurotransmission (Fig. 1) and the two types of characteristic spontaneous postsynaptic currents (Fig.
1A,C) argue for the formation of chemical synapses between the cultured NT2 neurons. Similar to NT2 neurons (Gao and Greenfield 2005, Wegner et al. 2007) generated according to the classica l differentiation method of Pleasure et al. (1992), the cell aggregate differentiated NT2 neurons do also display GABA induced currents. A glutamate mediated extracellular calcium influx has already been detected by microfluorometric calcium imaging (Paquet-Durand and Bicker 2004). Moreover, the present work demonstrates that synaptic networks of cultured NT2 neurons can form in the complete absence of glial cells. It will be interesting to examine, how the co-culture with glial cells and addition of glial derived factors will influence the formation of functional synapses. In summary, the cultured NT2 neurons are a promising model for electrophysiological investigations of electrically active human neuronal networks.
53 Acknowledgments
G. Podrygajlo is a Marie Curie Actions fellow and and G. Bicker was supported by the Federal Ministry for Education and Research (BMBF) PTJ-BIO 0313732B.
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