Neocortical cells were obtained from rat fetuses at day 18 of gestation (E18). Pregnant CD rats were purchased from Charles River (Sulzfeld, Germany) and were sacrificed in accordance with the current German Animal Protection Law. All procedures were approved by the regional animal care and use committee (Landesuntersuchungsamt Rheinland-Pfalz, Germany). Fetuses were revealed by caesarean, decapitated, and transferred to ice-cold HBSS including 10 mM HEPES (Invitrogen, Germany) for removal of the brain. Meninges were peeled off, and frontal cortices were collected for cell extraction. Following incubation in medium containing cystein-activated papain (papain: 2 U/ml; l-cystein: 0.2 mg/ml; Sigma) and DNase I (300 U/ml; Roche) at 37°C for 30 min cortices were triturated using a set of Pasteur pipettes with progressively constricted opening diameters. The resulting cell suspension was then passed through a cell strainer (40 µm pore size; BD Biosciences, Heidelberg, Germany) and centrifuged (1,300 rpm, 4°C, 5 min). The pellet was re-suspended in serum-free B27/Neurobasal medium supplemented with 0.5 mM glutamine (Brewer et al. 1993; Brewer 1995), and density of living cells was determined by Trypan Blue exclusion.

Cells were seeded at a density of 200 cells mm⁻² on surface-modified MEAs or glass cover slips and cultivated in a humidified atmosphere containing 5% CO₂ at 37°C. For electrophysiological control experiments with random cultures cells were plated at a density of 1,000 cells mm⁻² to attain sufficient electrode coverage for recording of neuronal network activity. Medium renewal was performed twice a week. A culture dish lid made of a transparent and gas-tight FEP Teflon® (fluorinated ethylene propylene) membrane with selective permeability to oxygen and carbon dioxide served to reduce evaporation from the medium supply and at the same time prevented invasion of airborne contaminants (Potter and DeMarse 2001).

Patterned deposition of the cellular adhesion promoting agent poly-d-lysine (PDL) on MEA surfaces was performed by microcontact printing (Kane et al. 1999) using customized polydimethylsiloxane (PDMS) microstamps.

A master stamp providing the negative of the designated pattern was produced by photolithography using a chromium mask (Delta Mask, Enschede, the Netherlands) as template. Photoresist layers (AZ 9260; Clariant GmbH, Sulzbach, Germany) of 10 µm thickness were spin-coated on glass wafers and subsequently hard-baked at 100°C for 12 min. Structuring was done by exposure to UV irradiation while the mask was in close contact with the photoresist layer. From the resulting mold microstamps were cast after surface treatment with the anionic detergent sodium dodecyl sulfate (2% w/v, 5 min; Sigma) to facilitate later removal of the stamp. Casting was done by pouring the elastomeric polymer PDMS (Sylgard® 184; Dow Corning, Wiesbaden, Germany) into glass rings with a diameter of 5 mm on the master and subsequent overnight curing at 70°C. Stamps were then decontaminated in 70% ethanol for 30 s before they were soaked overnight in a sterile solution of 0.1 mg/ml PDL (70,000–150,000 MW; Sigma) in phosphate-buffered saline pH 8.0. Prior to use, microstamps were dried by a nitrogen airstream, optically adjusted to the electrode geometry of the MEA by means of a special stereomicroscope (Leica MS5, Leica Microsystems, Germany) combined with a fineplacer (Fineplacer-145 PICO, Finetech Electronics, Germany) and pressed to the MEA surface with a force of about 100 g/cm² for 15 min at room temperature.

Pattern geometry consisted of a grid of isosceles triangles with 4 or 6 µm line width, respectively, giving intersection points between three straight lines at a time and internodal distances of 100 µm at the base and 112 µm at the sides of the triangle. Nodes were arranged to match the electrode coordinates of the MEA leading to a situation with each third node being occupied by an electrode. This pattern was chosen to offer the neurons appropriate conditions for radial outgrowth and frequent branching of neurites in order to facilitate a cellular morphology similar to that obtained with unbiased conditions.

For control cultures, surfaces were homogenously treated with PDL to trigger accidental adhesion of cells leading to random network formation.

After 14 days in vitro, patterned and random cultures with a cell density of 200 cells mm⁻² on cover slips were fixed with 4% paraformaldehyde in phosphate buffer (pH 7.4) for 20 min at 4°C. Unspecific binding sites were blocked by incubation with 5% normal donkey serum (Dianova, Hamburg, Germany) in phosphate-buffered saline (PBS) for 30 min at room temperature before cells were permeabilized with Triton X-100 (0.6% in PBS, 20 min, 4°C). Primary antibodies (rabbit polyclonal anti-MAP2 (Chemicon, 1:200), mouse monoclonal anti-MAP2a/2b (Abcam, 1.5 µg/ml), mouse monoclonal anti-GFAP (Chemicon, 1:400), mouse monoclonal anti-neurofilament 160 (Abcam, 1:200), rabbit polyclonal anti-synapsin (Abcam, 1:500)) were diluted in 1% normal donkey serum and 0.15% Tween-20 in PBS and applied overnight at 4°C. After rinsing with PBS, secondary antibodies raised in donkey were applied for 3–12 h with a dilution of 1:100 (rhodamine anti-rabbit IgG, Fluorescein isothiocyanate anti-rabbit IgG, rhodamine anti-mouse IgG, Fluorescein isothiocyanate anti-mouse IgG (Chemicon)). Control experiments with omitted primary antibodies resulted in the complete absence of any staining indicating no unspecific binding of the secondary antibodies. In case of double-labeling, antibody treatments were performed serially for the given antigens.

Cover slips were mounted on microscopic slides using fluorescence mounting medium (DakoCytomation, Carpinteria, CA, USA), and samples were analyzed by confocal microscopy using a Zeiss LSM 510 (Zeiss, Goettingen, Germany) equipped with an argon and helium/neon laser.

Fluorescent labeling was quantified using MetaMorph® (Molecular Devices, Downingtown, PA, USA). Labeled morphological features were extracted from the micrographs by intensity thresholding, and respective pixel number or labeled area was determined. Photographs were taken with a 40fold objective (pixel size: 0.23 µm × 0.23 µm).

Electrical activity was extracellularly recorded using a MEA60 setup (Multi Channel Systems, Reutlingen, Germany) consisting of a 60 channel amplifier and data acquisition card for conversion of analog signals to digital data streams. Data sampling was performed with a rate of 10 kHz. A thermal element maintained a culture medium temperature of 37°C during the experiments. Multielectrode arrays (MEA; Multi Channel Systems) consisted of 60 electrodes made of titanium nitride embedded in a glass substrate in an 8 × 8 array. Electrode diameters were 30 µm and inter-electrode spacing was 200 µm. Recorded signals (spikes) were displayed in real time using the software package MC_Rack (Multi Channel Systems) and saved for offline analysis covering signal detection, waveform sorting, and signal rate determination which was performed with the same software.

Occurrence of bursts in the spike pattern was detected by a custom-made application written in Java^® based on an analysis of consecutive interspike intervals (Tam 2002; Chiappalone et al. 2005). For burst detection the following criteria were chosen: (i) Interspike intervals at burst onset should not exceed 10 ms; (ii) a burst should consist of at least 3 spikes; (iii) after the third spike, interspike intervals of maximally 20 ms were tolerated reflecting the observation of increasing interspike intervals with burst duration; (iv) intervals between consecutive bursts should last for more than 500 ms. Otherwise, respective spike barrages were considered a single burst.

Test substances (Bicuculline methiodide, GABA; Sigma) were dissolved in culture medium and were manually applied by bulk exchange.

Data (given as mean ± standard deviation of n samples) was collected from different cell cultures. For quantitative analysis of the immunostaining results 6 micrographs were taken from every culture. Mean values of the samples were compared statistically by using either Student’s t-test for normally distributed values or Mann–Whitney U test as a non-parametric test. Differences were considered statistically significant for P < 0.05.

Data points of dose-response curves for electrophysiological recordings were fitted by using the logistic function y = A₂ + [A₁–A₂ / (1 + (c/c₀)^(n_H))] where c donates the concentration of the test substance, c₀ the half maximal effective concentration (designated as EC₅₀ for excitation and IC₅₀ for inhibition), n_H the Hill coefficient, and A₁ and A₂ the burst rate under control conditions and following a saturating concentration of the test substance.

Surface patterning of poly-D-lysine (PDL) resulted in a clear triangular network structure, and cell adhesion as well as growth followed the pattern reliably. The PDL-coating promoted adhesion of cell bodies and stellate neurite outgrowth, whereas uncoated areas were not overgrown by neurites and were almost devoid of cell bodies.

For comparison of cell growth on electrodes in structured and in non-manipulated networks, cells were seeded with a density of 200 cells mm⁻² on a MEA surface that was either homogeneously coated with PDL or printed with a triangular pattern of 6 µm wide PDL lines.

Neurons cultured on patterned surfaces assembled into an ordered network with cell bodies confined to the nodes of the meshwork and neurites extending along the predefined internodal lines. As the nodal intersection points were arranged to match the electrode coordinates of the MEA, the triangular PDL pattern promoted cell adhesion at the electrode positions while the homogeneously coated surfaces resulted in random electrode coverage (Fig. 1(a) and (b)). Fig. 1

Neuronal cell cultures on microelectrode arrays (a, b) Labeling of neuronal cell bodies and dendrites using an antibody directed against MAP-2 for random (a) and patterned (b) networks (line width: 6 µm) at 7 div. Some electrodes are highlighted by arrows. Scale bars, 50 µm. (c) Average number of electrodes covered by neuronal cell bodies for random and patterned cell growth at 7 div (n = 12; *** P < 0.001)

To determine whether guided cell growth was associated with major alterations in cellular organization, parameters as astroglial growth, neurite elongation, and synaptic density were analyzed by immunostaining.

Growth of astrocytes, which are known to promote synaptogenesis and synaptic efficiency (Pfrieger and Barres 1997), was assessed by immunocytochemical staining of glial fibrillary acidic protein (GFAP), an intermediate filament of the astroglial cytoskeleton. Due to the thin lines forming the pattern, astrocytes showed a rather slim morphology of the cell body in comparison to cells on homogeneously coated surfaces (Fig. 2(a) and (b)). The area labeled by antibody staining of GFAP was analyzed to compare astrocyte growth on patterned and homogeneously coated substrate surfaces. Some astrocytes were detected after 7 days in vitro (div), whereas the number increased substantially within the second week in vitro but remained at a constant level thereafter (Fig. 2(c)). The average GFAP-labeled area of random networks after 14 div is slightly higher in comparison to structured networks with 6 µm wide lines, though this difference was not significant. But cultures showed a very low number of GFAP positive cells when a pattern with 4 µm line width was applied resulting in a significantly (P < 0.001) reduced GFAP-labeled area compared to random cultures and networks on 6 µm wide lines. Fig. 2

Astrocyte growth for different culture conditions (a, b) Astrocytes were labeled by antibody staining of GFAP in random (a) and patterned (b) networks at 14 div. Staining of astrocytes in networks with 4 µm wide lines is not shown as almost no staining was present in these cultures. Scale bars, 20 µm. (c) Average labeled area per picture of GFAP-positive structures at 14 div (n = 60; *** P < 0.001)