Cellular adhesion and neuronal excitability on functionalised diamond surfaces

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1 Cellular adhesion and neuronal excitability on functionalised diamond surfaces P. Ariano 1,2, P. Baldelli 1,3, E. Carbone1,3, A. Gilardino1,2, A. Lo Giudice1,4, D. Lovisolo1,2, C. Manfredotti1,4, M. Novara1,3, H. Sternschulte5, E. Vittone*1, INFM (National Institute for Matter Physics), UdR Torino-University, Torino, (I) Department of Animal and Human Biology and Nanostructured interfaces and surfaces (NIS) Centre of Excellence, University of Torino, (I) Department of Neuroscience and NIS, University of Torino (I) Experimental Physics Department and NIS, University of Torino (I) Institut für Physik, Universität Augsburg (D) Abstract The resting or evoked activity of neuronal networks can be effectively monitored by using multielectrode arrays (MEA) which allow non-invasive extracellular stimulation and recording of electrical signals in parallel from multiple cells. Diamond possesses unique properties (biocompatibility, optical transparency, possibility of possibility of modifying the electronic and hydrophilic/hydrophobic properties at the nanoscale) which makes it a promising material to fabricate stable MEAs for long-term extracellular recordings of electrical and optical signals in living neurons. In order to explore the capability of diamond for fabricating MEAs as cell-based biosensors, we report here the first study on the adhesion and excitability of cells on hydrogen (HTD) and oxygen (OTD) terminated diamond surfaces. Adhesion and functional properties of cultured rat hippocampal neurons and chick ciliary ganglia have been quantitatively evaluated using well established biophysical techniques. Cells survive, adhere and maintain their electrical properties for days provided that mixtures of adhesion molecules (poly-d-lysine, poly-dl-ornithine, laminin) are used to favour cell anchoring on diamond surface. 1. Introduction Neuronal activity is responsible for much of the complex behaviour of organisms. Voltage-gated ion channels are membrane proteins involved in maintaining the cellular membrane electrical potential and generating action potential firings. Their modulation via pharmacological manipulation can be detected by changes in cell excitability. Thus, the use of cultured neurons as sensor elements provides the opportunity for studying in vitro brain activity and for undertaking high-sensitivity pharmaceutical screening. P. Ariano and P. Baldelli contributed equally to this work. * Corresponding author: Ettore Vittone, vittone@ph.unito.it 2

2 Neuronal activity can be directly measured using extracellular microelectrodes which provide a stable, non invasive interface for monitoring the functioning of a neuronal network. Planar microelectrode arrays interfaced with cultured neurons generally consist of glass or silicon over which a conductor is deposited and patterned 6. The cell/sensor interface is created as neurons adhere directly to the planar electrode structure. Alternatively, neurons can be directly coupled to the bare gate of a silicon field effect transistors to improve the signal/noise ratio 6. However, the opacity of suitable, biocompatible and stable electrodes makes difficult the simultaneous recording of neuronal activity and optical signals generated by fluorescent indicators. Full biocompatibility of diamond makes this material an ideal candidate for the development of new bio-sensors for detecting chemical, optical and electrical signals during excitable cell activity. Moreover, the diamond surface can be made hydrophobic or hydrophilic and electrically conductive or insulating according to hydrogen (HTD) or oxygen (OTD) termination, respectively 6,6. The nanoscale functionalisation of the diamond surface can be performed using Local Anodic Oxidation by atomic force microscope (AFM) 6. These features allow the realisation of micro or nanoelectrodes which are stable in air and perfectly transparent in a wide spectral range. To explore the possibility of exploiting the above mentioned properties of diamond for the realisation of bio-sensors for simultaneous recording of electrical and optical signals from the same cell, it is essential to investigate adhesion and excitability of neurons on HTD or OTD surfaces. The first evidence of selective attachment of mammalian neurons and ordered outgrowth of neurites on patterned diamond surfaces has been recently reported by Specht et al 6. However, to our best knowledge, no report about the functional viability of neurons on functionalised diamond surfaces has been published to date. Here, using well established biophysical techniques, we show that cultured rat hippocampal neurons and chick ciliary ganglia adhere and survive for days on OTD and HTD surfaces preserving their morphology and electrical properties provided that mixtures of adhesion molecules (poly-dlysine, poly-dl-ornithine, laminin) are used as organic substrates to anchor the cells to the diamond surface. Survival ratios, synaptic transmission, Ca2+ channel properties and Ca2+ signalling measured with the patch-clamp technique and confocal fluorescence microscopy were fully compatible with those of control neurons grown on plastic surfaces. 2. Experimental 2.1 Diamond samples The studies described in this paper were performed using two 5 µ m thick homoepitaxial diamond films deposited on highly resistive (boron free) HPHT Ib diamond substrate, (100) 3

3 oriented (Sumitomo Electric Co. Ltd.). Two homoepitaxial IIa films were used: sample A ( 9 mm2 surface area) was directly supplied from Sumitomo; sample B (16mm2 surface area) was grown (deposition time = 1080 min) by micro wave plasma assisted CVD (total power = 1100 W) 6: the substrate temperature was 800 C and the total gas flow was 200 sccm with 1% of methane in hydrogen. In order to remove surface contaminants and to leave a clean oxygen-terminated surface, the samples were first oxidized using a sulfochromic acid solution at 170 C, rinsed in deionized (DI) water, cleaned in a boiling solution of H2O2:NH4OH and then dipped again several times in DI water 6. Surface hydrogenation was carried out in a hot filament CVD reactor, locating the samples for about 30 minutes at a distance of about 1.5 cm from Ta hot (2100 C) filaments and using purified hydrogen gas (750 sccm) at a pressure of about 3000 Pa. After the exposure to air at room temperature, the HTD surface showed an electrical conductance of S, as measured using silver paste electrodes placed at a distance of about 1 mm, six order of magnitude higher than the conductance measured on the OTD surface. 2.2 Cell cultures We used cultured embryonic chick ciliary ganglia and hippocampal neurons of rat embryos for two main reasons: 1) they possess different roles on the control of animal activity and cover a wide range of central and peripheral neurophysiological functions and 2) they are suitable model systems for testing cell survival and neuronal activity in vitro. Chick ciliary ganglion neurons cultures were prepared from 7-8 days embryos, plated at a density of 200 cells/mm2 and maintained for 1-4 days in chemically defined N2 medium in the presence of basic fibroblast growth factor (bfgf; 20 ng/ml), on diamond surfaces with or without adsorbed poly-d-lysine (PL, 100 µ g/ml) and laminin (LN, 2 µ g/cm2). Hippocampal neurons from rat embryos (E18) were grown in culture as previously described 6. For the present studies, the isolated neurons were plated at a low density (200 cells/mm 2) and used after 2 days in vitro (DIV). To promote cell adhesion, poly-dl-ornithine (PO, 0.5 mg/ml) and LN (5 µ g/ml) were used. 3. Results and discussion 3.1 Ciliary ganglion neurons. When plated in the absence of adhesion molecules (poly-d-lysine, laminin, collagen), neurons grown on either OTD or HTD surfaces showed very low survival ratios (less than 10%) for the first 4

4 2 days in culture. The few surviving cells quickly aggregated, formed clusters, and emitted very few neurites (data not shown). The treatment of diamond surfaces with poly-d-lysine (PL, 100 µ g/ml) and laminin (LN, 2 µ g/cm2) allowed cells to attach, survive and emit long neuritic processes. No significant difference either in morphology (Fig.1) or in survival ratios (Fig. 2a) could be detected between cells grown on the two types of diamond surface and between them and control cultures (plastic dishes). Survival ratios remained high for several days, but, due to the tendency of neurons to aggregate 6 cell counts were not reliable after 2 days. To test the functional viability (up to 4 days) of neurons from E7/E8 chick embryos grown on HTD treated with PL and LN, Ca2+ signals in response to activation of voltage-gated Ca2+ channels were observed in Ca2+-imaging experiments. Ca2+ influx through voltage-gated channels was activated by depolarising the membrane potential with a solution containing 40 mm KCl, thus shifting the K+ equilibrium potential to more positive values. Cytofluorimetric recordings were performed by means of confocal microscopy, using the fluorescent Ca2+ indicator Fluo-3. Upon repeated stimulations with KCl, neurons responded with reversible Ca2+ transients (Fig. 2b), as observed in control conditions. 3.2 Hippocampal neurons Cultured hippocampal neurons from rat embryos of 18 days (E18) could easily grow on plastic and OTD surfaces provided that mixtures of proteic adhesion molecules (0.5 mg/ml PO and 5 µ g/ ml LN) were used as substrate for cell anchoring. Under these conditions, hippocampal neurons maintained the same morphology, size and neurite extension (Fig. 3). Survival ratios were approximately identical up to 8 DIV in both conditions and decreased partially between 8 to 14 DIV when neurons were plated on OTD (Fig. 4a). Voltage gated Ca2+ currents recorded with a patch electrode placed at the soma of a neuron had comparable amplitude, voltage-dependence and activation-deactivation kinetics (Fig. 4b), suggesting that the Ca2+ channels controlling somatic activity and neurotransmitter release preserved the same biophysical properties of the Ca2+ channels recorded from neurons plated on plastic. It is worth noticing the overlapping of the two Ca 2+ current densities recorded at 0 mv and the good correspondence of current density amplitudes at different voltages (black vs. grey lines in fig. 4c). Neurons grown on OTD surfaces were also tested for the ability to preserve their synaptic activity. To do this, we compared the inhibitory GABAergic response of control and OTD-plated neurons evoked in pairs of neurons forming monosynaptic contacts (Fig. 5a). Presynaptic stimulation in the presence of kynurenic acid (a specific antagonist of excitatory glutamatergic synapses) gave rise to the typical inhibitory postsynaptic current (eipsc) with transient time course 5

5 (Fig. 5b). The eipsc was inward at 70 mv holding potential and reversed at 0 mv, as expected from the Nernst equilibrium potential for a Cl permeable channel with internal and external equimolar Cl concentration 6. Neurons plated on OTD surfaces displayed eipscs of similar amplitude and time course to those grown on plastic, with mean amplitudes and SEM as shown in Fig. 5c. Given that OTD had no significant effects on GABAergic inhibitory responses we tested also whether the probability of neurotransmitter release (Pr) was not affected by OTD treatments. Pr is a critical parameter of presynaptic activity and can be estimated from the percent of paired-pulse depression (PPD) at very short interpulse intervals ( t = 25 ms; Fig. 6a) 6. As shown in fig. 6b, the percentage of PPD versus time was well preserved at short and long t, suggesting that Pr was not affected in hippocampal inhibitory synapses formed on OTD surfaces. 4. Conclusions In the presence of adhesion molecules, rat hippocampal neurons plated on OTD surface survive and preserve their synaptic activity and somatic Ca2+ current densities. Chick ciliary ganglion neurons adhere, survive and are functional on HTD surface as well. The tested biocompatibility of central and peripheral neurons with diamond surfaces indicates that patterned OTD and HTD planar electrodes are suitable for interfacing with different types of neuronal networks and can be used for wide-spectrum pharmacological screenings. Our results highlight the potentiality of functionalised diamond surfaces as substrates for constructing microelectrode arrays for multiparametric recording of electrical activity and optical signals in neuronal networks. 5. Acknowledgements This work is supported by the PAIS-INFM project DIBIOREX and by the Nanostructured Interfaces and Surfaces (NIS) Centre of Excellence of the University of Torino, (Italy) 6. References [1] M.. Maher, J. Pine, J. Wright, Yu-Chong Tai, Journal of Neuroscience Methods 87 (1999) 45 [2] R. A. Kaul, N. I. Syed, P. Fromherz, Phys. Rev. Lett. 92, (2004), [3] H. Kawarada, Surface Science Reports, 26, (1996), 205. [4] C.G.Specht, O.A. Williams, R.B.Jackman, R.Schoepfer, Biomaterials 25 (2004), 4073 [5] C. Manfredotti, E. Vittone, C. Paolini, L. Bianco, F. Fizzotti, A. Lo Giudice, P. Olivero, Surface Engineering, 19 (6) (2003) [6] H. Sternschulte, M. Schreck, B. Stritzker, A. Bergmaier, G. Dollinger, Diamond and Related Materials 12 (2003), 318, and references therein. 6

6 [7] P. Baldelli, M. Novara, V. Carabelli, J.M. Hernandez-Guijo, E. Carbone, European Journal of Neuroscience, 16 (2002), 2297 [8] C. Distasi, M. Torre, S. Antoniotti, L. Munaron, D. Lovisolo, European Journal of Neuroscience, 10, (1998) [9] W.G.Regehr, C.F. Stevens, Synapses, Eds. M. Cowan, T.C. Südhof, C.F. Stevens, The John Hopkins University Press, Baltimore (USA), (2001) Figure Captions Fig. 1 - Morphological appearance and neurite outgrowth of chick ciliary neurons grown on non- hydrogenated (a) and hydrogenated (b) diamond, in the presence of PL and LN at 48 h of culture, as compared to control conditions (c) Fig. 2 (a) Survival ratios at 24 and 48 h of neurons cultured on OTD and HTD surfaces, in the presence of bfgf, PL and LN, as compared to cells grown on plastic dishes in the same conditions. Percentages relative to cell counts made 2 hours after plating. Data are means of 2-6 experiments. (b) Cytofluorimetric recordings of intracellular [Ca2+]. The [Ca2+] increase from baseline ( F/F) was induced by perfusion of a solution containing 40 mm KCl (grey bars at the bottom). Trace is the average of 17 neurons. Fig. 3 - Photomicrographs of hippocampal neurons plated on a standard plastic dish and on an OTD surface after 2 DIV. Fig. 4 - a) Time course of survival in control and OTD-plated neurons from 2 to 14 DIV. (b, c) Voltage-gated Ca2+ currents at 0 mv and I-V relationships recorded in control (black traces) and OTD-plated (grey traces) neurons. Neurons were patch-clamped in the whole-cell configuration at the soma [7]. Ca2+ current densities (pa/pf) versus voltage were determined in 10 mv step depolarisations from 50 to +50 mv (holding potential 90mV). Fig. 5 - (a) Photomicrograph of the experimental apparatus for measuring synaptic activity. Current pulses were injected through an extracellular stimulating electrode in contact with the presynaptic neuron. Postsynaptic current responses were recorded with a patch-clamp electrode. (b) Electrically evoked inhibitory post-synaptic currents (eipscs) recorded after from neurons of 14 DIV. (c) Mean amplitude (± SEM) of eipscs calculated from n = 8 control and OTD plated neurons. 7

7 Fig. 6 - (a) Two eipscs recorded from a whole-cell clamped hippocampal neuron in response to paired stimuli separated by 400 ms ( t). A1 and A2 indicate the amplitude of eipscs. (b) Percentage of paired-pulse depression (% PPD = 1-A2/A1) versus t. The PPD at short t (25 ms) varies between 0.52 and 0.63 and approximates Pr. 8

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