Synaptic plasticity in micropatterned neuronal networks

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Biomaterials 26 (2005) 2549–2557 www.elsevier.com/locate/biomaterials

Synaptic plasticity in micropatterned neuronal networks Angela K Vogta, Gu¨nter Wrobela, Wolfgang Meyerb, Wolfgang Knollc, Andreas Offenha¨ussera, a Institute of Thin Films and Interfaces (ISG-2), Forschungszentrum Ju¨lich, D-52425 Ju¨lich, Germany Central Institute of Applied Mathematics (ZAM), Forschungszentrum Ju¨lich, D-52425 Ju¨lich, Germany c Max-Planck Institute for Polymer Research, Ackermannweg 10, D-55128 Mainz, Germany

b

Received 9 April 2004; accepted 19 July 2004 Available online 17 September 2004

Abstract Synaptic plasticity is thought to be of central importance for information processing by the nervous system. Additionally, specific neuronal connectivity patterns in the brain are implicated to play a role in the perception, processing and storage of incoming signals. Experimental control over connectivity within functional neuronal networks is therefore a promising approach in research on signal transduction and processing by the nervous system. A cell culture system is presented that allows experimental determination of neuronal connectivity patterns in an in vitro network. Rat embryonic cortical neurons were grown on patterns of extracellular matrix proteins applied to polystyrene substrates by microcontact printing. Cells comply well with the pattern and form synaptic connections along the experimentally defined pathways. Chemical synapses identified by double patch-clamp measurement showed paired pulse depression as well as frequency-dependent depression in response to trains of stimuli. This type of short-term plasticity has similarly been reported by others in brain slices. Thus, the system reproduces features central for neuronal information processing while the architecture of the network is experimentally manipulable. The ability to tailor the geometry of functional neuronal networks offers a valuable tool both for fundamental questions in neuroscientific research and a wide range of biotechnological applications. r 2004 Elsevier Ltd. All rights reserved. Keywords: Neural network; Micropatterning; ECM (extracellular matrix); Cell adhesion; SEM (scanning electron microscopy)

1. Introduction The ability to process and store information is one of the most striking properties of the central nervous system. Activity-dependent modifications of synaptic efficacy are thought to be of vital importance for this capability. While long-term changes in synaptic strength are believed to be involved in learning and the formation of memories [1–3], short-term plasticity has been implicated in the processing of incoming signals, e.g. in recognition of different input patterns [4,5]. Sensitivity to particular patterns of sensory input is additionally Corresponding author. Tel.: +49-2461-61-2330, fax: +49-2461-612333. E-mail address: [email protected] (A. Offenha¨usser).

0142-9612/$ - see front matter r 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.biomaterials.2004.07.031

attributed to features of cortical microcircuitry, which is characterized by extensive recurrent connections. These may—depending on the architecture of the particular network—serve to amplify or attenuate afferent signals and control the activity of the circuit [6]. The investigation of synaptic plasticity in vitro is mostly performed by patch-clamp recordings between two synaptically connected neurons in brain slices. However, the extreme complexity of neuronal connectivity within this organ renders the investigation of single synaptic contacts extremely difficult. Synaptic input from outside the constellation of interest interferes with the experimentally applied signals and impedes reproducibility. For the study of plasticity at single synaptic contacts, low-density neuronal cultures have proven a useful

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model system. Much insight in plastic modulations of synapses has been gained in ultra-low density cultures which allow investigation of cell–cell communication on the level of single monosynaptically connected cells [3,7]. In such cultures however, constellations of interconnected neurons form randomly and not reproducibly while their connectivity pattern is outside the control of the experimenter. It is therefore not possible to deliberately alter particular types of circuitry and systematically investigate their impact on the behavior of the constellation. A low-density culture in which neuronal interconnections can be manipulated experimentally is therefore a promising model system for the investigation of neuronal signal processing. Such a model is presented by neuronal networks on micropatterned substrates. These provide only a fraction of the surface for cell adhesion while the rest is cellrepellent. Individual cells are therefore forced to adhere to small, well-defined adhesive islands and can only connect along narrow permissive pathways provided by the pattern. Considering the multitude of interconnections that form in unpatterned neuronal cultures, the restraint of neuritic outgrowth to these pathways means a considerable decrease in network complexity and allows a large amount of control over network geometry. Surface patterning has been performed by a number of different techniques and using a wide range of different molecules for the permissive and unpermissive surface areas. One of the first examples of neuronal patterning was reported by Kleinfeld et al. in 1988 [8], who used photolithography to pattern different types of amines for neuronal attachment onto a background modified with cell-repellent alkane chains. Later on, a number of photochemical methods [9–12], laser ablation of self-assembled monolayers [13] and microcontact printing [14–17] were added as tools to create micronscale patterns of adhesive molecules against a nonadhesive background. The technique applied in the presented work, microcontact printing, is a cost effective and versatile method which can be used to print high-resolution patterns of a wide range of molecules onto a solid substrate using a high-relief elastomeric stamp. One of the major problems that has been encountered previously in neuronal patterning is that a strict compliance with the underlying geometry often had a negative impact on the physiological development of the neurons and on synapse formation [18,19]. In other cases, it has been reported that initial compliance with the high-resolution patterns was lost at the stage when the neurons gained excitability [10]. We recently reported networks of rat embryonic cortical neurons highly compliant to an underlying micropattern created by microcontact printing of extracellular matrix proteins onto polystyrene. These contained fully functional

chemical synapses as indicated by a similar average postsynaptic potential and a similar rate of synaptic failure when compared to control substrates [20]. The central issues to be addressed by patterned networks however do not concern the 1:1 relaying of signals but their processing and modulation within the network. Thus, evidence for synaptic plasticity similar to that seen in the intact tissue is of critical importance for the applicability of micropatterned networks as a model system. The presented work reports several forms of shortterm synaptic plasticity measured in neuronal networks highly compliant to a predefined pattern. Both pairedpulse depression and frequency-dependent depression in short stimulus trains were observed similarly as they have been described in slices [21,22]. As expected, these effects were not seen upon stimulation of electrical synapses, which were also encountered in the patterned networks. This study demonstrates that the presented culture system is suitable for the creation of simplified neuronal networks of manipulable geometry that reproduces basic features of signal processing by the nervous system.

2. Materials and methods 2.1. Substrate preparation by microcontact printing Microstamps were produced by photolithography and molding [23,24]. The grid pattern described below was transposed to a chromium-coated quartz wafer by electron beam writing. This wafer was used as a mask to create the master stamps by UV photolithography using spin coated 12.5 mm thick photoresist layers (AZ 4562, Clariant, Germany) on 0.6 mm thick silicon wafers (MEMC Electronic Materials, Germany). Polydimethylsiloxane (PDMS) stamps were then fabricated by pouring Sylgard 184 (Dow Corning, Midland, Michigan, USA) onto these masters (2 ml Eppendorf cups cut open at the bottom served as molds) followed by a 48 h curing step at 55 1C. After master stamp release, final curing was performed during 1 h at 110 1C. PDMS stamps were stored in Milli-Q water for 24 h before using, then decontaminated in a 70% ethanol bath for 1 min. Inking took place by immersing the stamp for 30 s in a 1:100 dilution of ECM–gel (Sigma, Germany) in Dulbecco’s Modified Eagle’s Medium (Invitrogen, Germany) containing 10 mg/ml Poly D-lysine, molecular weight 70.000–150.000 (Sigma, Germany). The inked stamp was dried in a stream of nitrogen and pressed to non-tissue culture polystyrene petri dishes of 35 mm diameter (Greiner, Germany) for 10 s. The stamps transferred grids of 4–6 mm wide lines and node points 12–14 mm in diameter to the surface. Meshes were 50 mm  100 mm [25]. Homogeneously

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coated control substrates were created by wetting the entire surface with the inking solution, allowing it to adsorb for 30 min and removing it by aspiration. Substrates were dried overnight before use. 2.2. Cell culture Rat embryonic cortical neurons were obtained as described [26]. Briefly, embryos were recovered from pregnant CD rats at 18 days gestation (E18). Cortices were dissected from the embryonic brains; cells were mechanically dissociated by trituration in Hank’s Balanced Salt Solution (HBSS) (without calcium and magnesium), 0.035% NaHCO3, 1 mM Na-pyruvate, 10 mM HEPES, 20 mM glucose, pH 7.4 with a firepolished siliconized pasteur pipette. The cell suspension was diluted 1:2 in HBSS (with calcium and magnesium), 0.035% sodium bicarbonate, 1 mM pyruvate, 10 mM HEPES, 20 mM glucose, pH 7.4. For 3 min, nondispersed tissue was allowed to settle, the supernatant was centrifuged at 200 g for 5 min. The pellet was resuspended in 1 ml Neurobasal Medium, 1x B27s, 0.5 mM L-glutamine per hemisphere isolated. A sample was diluted 1:1 with trypan blue and dyeexcluding cells were counted in a Neubauer counting chamber. The remaining cells were diluted in Neurobasal medium with the above supplements and plated onto the substrates at a density of 16 000 cells per cm2. Half of the medium was changed every 3–4 days. All reagents were purchased from Invitrogen, Germany. 2.3. Sample preparation for SEM Cultured cells were fixed on the patterned substrates for 1 h at room temperature with 3.5% glutaraldehyde solution (Serva, Germany) (in 0.1 M phosphate-buffer [4 NaH2PO4: 1 Na2HPO4], 57 mM sucrose, pH 7.4). The samples were washed for 10 min with Milli-Q water. Then they were taken through a graded 2-propanol series to dehydrate the cells completely (30%: 10 min, 50%: 10 min, 70%: overnight, 90%: 10 min, 3  95% and 3  100%: 5 min). Finally the cells were dried using the critical point drying (CPD) technique. A 5 nm gold layer was deposited onto the samples by electron beam evaporation (ESV-4, Leybold-Heraeus GmbH, Germany). The fixed cells were studied using a LEO 1550 field emission scanning electron microscope (SEM) (LEO Elektronenmikroskopie GmbH, Germany). The images were further processed (contrast enhancement) using standard imaging software (Paint Shop Pro 7). 2.4. Electrophysiology Patch-clamp recordings were performed using a triple patch-clamp setup (EPC9/3, HEKA Elektronik,

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Germany) in current clamp mode, typically at 11–15 days in vitro (DIV). Cell pairs located on neighboring positions of the grid pattern were patched simultaneously. Borosilicate micropipettes (Hilgenberg, Germany) with a resistance of 6 MO were pulled using a micropipette puller (P–97, Sutter Instrument Company, CA, USA). For current-clamp recordings, membrane current was set to zero; action potentials were stimulated with 50 or 100 ms pulses of 130 or 200 pA. The extracellular solution contained (mM): 5 KCl, 150 NaCl, 1 MgCl2, 10 HEPES, 2.5 CaCl2, 10 Glucose, pH 7.4, adjusted with 1 M NaOH. The intracellular solution contained (mM): 125 K-gluconate, 20 KCl, 0.5 CaCl2, 2 MgCl2, 10 HEPES, 5 EGTA, 4 ATP, pH 7.4, adjusted with 1 M KOH. A liquid junction potential of 14.4 mV was calculated for the applied patch solutions using appropriate software (Junction Potential Calculator, Axon Instruments Inc., CA, USA) and subtracted from the measured membrane voltages. GABAergic and glutamatergic synapses were distinguished by determination of the reversal potential, analysis of the postsynaptic current and sensitivity to specific receptor antagonists. The reversal potential was determined by stimulating presynaptic action potentials while holding the postsynaptic cell in the voltage clamp mode at different potentials ranging from 60 to +20 mV and extrapolating the potential at which the postsynaptic current reversed. The decay time of the postsynaptic current was measured as the time between peak and half maximal current. Some synapses were in addition characterized chemically using specific antagonists. Bicuculline methiodide and Cyano-7-nitroquinoxaline2,3-dione (Sigma, Germany) were added to the external solutions to final concentrations of 50 and 20 mM for reversible chemical inhibition of GABAergic and glutamatergic synapses, respectively. The decay of the postsynaptic potential in response to stimulus trains was fit with a single exponential function 1 a(1 e (Stim# 1)/b). In this function, a is the maximal extent of depression such that the signal in the steady state is described by (1 a) while b describes the decay kinetics.

3. Results 3.1. Investigation of patterned growth by light microscopy and SEM Substrates for the growth of patterned neuronal networks were prepared by microcontact printing as described earlier [20]. A blend of extracellular matrix proteins mixed with poly D-lysine was printed onto a background of polystyrene. Polystyrene is a highly hydrophobic and thus cell-repellent material that presents a sharp contrast to the protein pattern in terms of

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adhesive properties. The cells are therefore forced to adhere to the node points of the grid. These represent the largest continuous adhesive surfaces and allow for the attachment of a cell body while the narrower lines permit the outgrowth of neurites. Fig. 1 shows a typical image of a micropatterned substrate after 1 week in culture. The cells comply well with the pattern and form extensive networks interconnecting along the predefined pathways. Although cell adhesion to the polystyrene background was observed only very rarely, cells sometimes adhered to the lines rather than the node points of the pattern. At 11 days in culture, 7176.5% of the cells complied with the adhesion sites while the remainder was found on the lines (n=499; seven cultures were evaluated). To characterize the morphology of the patterned networks in more detail, scanning electron microscopy (SEM) micrographs were taken. Fig. 2A shows part of a neuronal network grown on a patterned surface. In addition to the adherent cells, nodes and lines of the stamped pattern can be observed as elevations from the surface. Except for slight defects at the edges of some lines, pattern transfer had been successful and the cells adhered faithfully to the protein-coated areas of the surface. Three multipolar neurons attaching to the pattern and forming connections with cells on neighboring positions of the grid are seen in the chosen area. The somata (S) are clearly discernible from the neurites and adhere exactly to the node points of the pattern. The neurites can further be subdivided into presumed axons (A) and dendrites (D) by their dimensions; both types of neurites grew accurately along the lines defined by the

Fig. 2. SEM micrograph of a neuron fixed at DIV 4 and sputtered with a 5 nm gold layer. (A) Three cells attaching to the node points of the grid pattern in the process of forming an interconnected network. Somata are labelled ‘‘S’’, presumed axons ‘‘A’’ and dendrites ‘‘D’’. A growth cone G can be seen in the bottom right corner. (B) Enlarged portion of A, showing a single neuron with three neurites growing onto three of the lines emerging from the adhesion spot.

Fig. 1. Typical image of a patterned neuronal network at DIV 7. Cell bodies adhere to the node points of the grid and connect along the pathways defined by the micropattern.

pattern. The sample had been prepared and fixed after 4 days in culture, thus the neurons are still in an early stage of differentiation. Therefore, in addition to more differentiated neurites, an axonal growth cone (G) is visible in Fig. 2A. Fig. 2B displays a patterned neuron in enlarged detail. Small appendages sprout from both the cell body and the neurites, which may represent filipodia. These extend from the stamped structures onto the uncoated area next to the pattern. However, as the overall outgrowth of the neurites remains on the pattern line with a high fidelity, it can be imagined that these filipodia serve to explore the area surrounding the neuron and will be retracted from the unadhesive surface areas. Consistent with this

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assumption, most filipodia are seen to extend within the borders of the adhesive regions. Single neurites extend down two of the provided pathways, while two neurites twisting round each other are seen along the pathway in front of the image. The second neurite is possibly extended by the neighboring neuron seen in Fig. 2A and may provide a reciprocal connection between the two cells. The small number of neurites on each pathway shows that the cell density in the network is low enough for the investigation of single synaptic contacts. The stringent compliance of neuritic outgrowth with the provided pathways further indicates that connectivity between the patterned cells obey the experimentally chosen geometry. 3.2. Formation of chemical and electrical synapses To investigate the functional properties of the patterned network, cell–cell connections were studied electrophysiologically by double patch-clamp measurements. To identify synaptic connections, both cells were recorded in the current-clamp mode and action potentials (APs) were induced in one cell at a time by a depolarizing current pulse. Chemical as well as electrical synapses had formed on the patterned substrates. Chemical synapses were identified when a presynaptic AP but not subthreshold depolarizations consistently induced a postsynaptic potential. Only monosynaptic connections were evaluated, these were characterized by a synaptic delay of p6 ms. A typical recording is shown in Fig. 3. Chemical synapses were further subdivided into excitatory (glutamatergic) and inhibitory (GABA-ergic). As a high chloride intracellular patch solution was used,

Fig. 3. Typical chemical synapse. Presynaptic action potentials but not subthreshold depolarizations consistently result in a postsynaptic potential in the unstimulated cell (lower trace). Both cells were held in the current clamp mode.

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both types of synapses caused a depolarization of the postsynaptic cell or an inward current in current or voltage clamp mode, respectively. Fig. 4 displays typical recordings of the postsynaptic current measured at an excitatory (4A) and an inhibitory (4B) synapse. The two synapse types were distinguished by their reversal potentials and the decay time of the postsynaptic current. Reversal potentials of inhibitory synapses were close to that of chloride as calculated using the Nernst equation ( 43.274.8 mV), while excitatory synapses reversed near 0 mV (1.876.5 mV) (Fig. 5). The decay time of the postsynaptic current was 2–6 ms for excitatory and X16 ms for inhibitory synapses, which is in agreement with values found in the literature [27–29]. The average amplitudes for both excitatory and inhibitory currents varied from synapse to synapse. Typically, currents with peak amplitudes between 20 and 150 pA were encountered. In contrast to the all-or-none-signal transmission seen in chemical synapses, electrical synapses (gap junctions) characteristically transmitted both action potentials and subthreshold depolarizations. This can clearly be seen in Fig. 6, which displays a recording from two cells coupled by an electrical synapse. As in Fig. 3, one of the two cells is stimulated to fire an action potential. Both the AP and the following plateau, during which the cell is still depolarized experimentally but does not fire as the Na+channels are refractory, are reflected in the membrane potential of the second cell. Signal transmission

Fig. 4. Postsynaptic current at (A) an excitatory and (B) an inhibitory synapse. Both recordings were performed holding the postsynaptic cell at 60 mV in the voltage clamp mode while the presynaptic cell was stimulated to fire an action potential.

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Fig. 7. Synapse exhibiting paired pulse depression. Two successive PSPs were elicited by two presynaptic APs fired at 0.8 Hz. Fig. 5. Excitatory and inhibitory synapses were distinguished by the reversal potential of the postsynaptic current. To determine the reversal potential, the postsynaptic cell was held in the voltage-clamp mode at different holding potentials while the presynaptic cell was stimulated to fire action potentials. Plotting the postsynaptic current against the respective holding potential yielded the reversal potential as the x-axis intersection. The synapse determined as inhibitory (filled circles) reverses at ca. 41 mV while the excitatory synapse (open circles) can be extrapolated to reverse at about 5 mV.

Paired pulse depression was quantified by dividing the second PSP by the first. This was done for the synapses encountered in a total of 14 networks recovered in nine independent preparations. Paired pulse depression was found to be more pronounced in excitatory synapses, which were depressed to 0.6370.06 the initial value (n=13), while inhibitory synapses were only depressed to 0.7370.03 (n=25). Paired pulse facilitation was never observed. 3.4. Synaptic depression in short trains of stimuli

Fig. 6. Typical recording of two cells coupled through a gap junction. Both action potentials and subthreshold depolarizations (represented by the plateau after the AP) are transmitted. Both cells are recorded in the current-clamp mode while one is stimulated by an experimentally applied current pulse to fire an AP. The scale on the y-axis applies to the stimulated cell while the trace of the unstimulated cell is shifted vertically and displayed at an enlarged scale corresponding to the scale bar on the right.

occurred with similar conductances in both directions. Conductances for the electrical synapse shown in Fig. 6 e.g. were determined to be 0.4 and 0.6 nS (data not shown). 3.3. Paired pulse depression To determine whether synapses in patterned networks exhibit short term plasticity similar to that described in slices, responses to consecutive presynaptic APs fired at 0.8 Hz were recorded. The second postsynaptic potential (PSP) was smaller than the first in all synapses tested (n=38), consistent with paired pulse depression. Fig. 7 displays an examplary recording of two successive PSPs.

In the next step, the development of synaptic strength in response to trains of APs was investigated. Series of 10 pulses were applied while the development of the postsynaptic response was recorded. As for the paired pulse protocol, the relative strength of each PSP in the train was determined by dividing it by the first. Fig. 8A shows an excitatory and an inhibitory neuron reciprocally coupled by two synapses (reversal potentials 4 and 43 mV, decay time 5.3 and 25.6 ms, respectively). The two cells were stimulated in alternation with series of 10 pulses at 0.8 Hz. The relative synaptic strength of each stimulus within the train was averaged over 11 trains each (Fig. 8B). The decay of the relative postsynaptic signal within the stimulus train is most prominent in the first half of the stimulation protocol and appears to reach a steady state after 3–5 pulses. This decay can be fit with a mono-exponential function. As in paired pulse depression, depression in response to 10 APs was more pronounced for the excitatory synapse (open circles) for which the fit revealed a steady state of 0.50. The steady state of the inhibitory synapse (filled circles) was 0.79. A corresponding effect was not seen at lower frequencies (Fig. 8C). The average steady state of all investigated excitatory synapses (n=8) was 0.6270.08, that of inhibitory synapses (n=16) 0.7070.03, confirming a trend for stronger depression in excitatory synapses although the effect is less pronounced than that seen in paired pulse depression. Data were recorded from 14 different substrates.

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3.5. Frequency-dependent synaptic depression To investigate short-term synaptic depression over a wider scale of frequencies, an inhibitory synapse was stimulated with pulse series at 1, 5 and 10 Hz. A phase contrast image of the cellular constellation that was recorded is shown in Fig. 9A. The arrow points from the pre- to the postsynaptic cell. The height of the postsynaptic potential was normalized with respect to the first PSP in the stimulus train and plotted in Fig. 9B. Short-term synaptic depression was notable in all three cases and augments with the frequency at which the presynaptic cell was stimulated. The steady state was 0.65 for 1 Hz, 0.50 for 5 Hz and 0.32 for 10 Hz. No depression was observed when the presynaptic cell was stimulated with 0.1 Hz. In contrast to chemical synapses, signal transmission through gap junctions did not decay during pulse series

Fig. 8. Reciprocally connected pair of one excitatory and one inhibitory neuron. (A) Phase contrast picture of the two cells. (B) Development of the relative PSP height in response to trains of 10 presynaptic APs delivered at 0.8 Hz. Open circles represent the excitatory, filled circles the inhibitory synapse. Both traces represent an average over 11 trains and were fit with a mono-exponential function 1 a(1 e (Stim#–1)/b). In this function, a is the maximal extent of depression such that the signal in the steady state is described by (1 a). (C) Relative PSP height in response to presynaptic APs delivered at 0.4 Hz, average over three trains each.

Fig. 9. Inhibitory synapse challenged with presynaptic APs at different frequencies. (A) Phase contrast picture of the two cells just before patching; the white arrow indicates the electrophysiologically identified synapse. (B) Development of the relative PSP height elicited by trains of APs at different frequencies fit with a single exponential.

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Fig. 10. Signal transmission through a gap junction at different stimulation frequencies. (A) Phase contrast picture. (B) Development of the relative amplitude of the transmitted signal at 10 Hz (triangles) or 0.1 Hz (squares). Filled symbols: Stimulation of cell 1, open signals: Stimulation of cell 2. Traces were shifted vertically for clarity.

irrespective of the applied stimulation frequency. This can be seen in a cellular constellation depicted in Fig. 10A. Two cells coupled by an electrical synapse were stimulated alternatingly with pulse series at 0.1 and 10 Hz. The signal transmitted to the unstimulated cell did not decay within the stimulus train at either frequency and in either direction (Fig. 10B). This behavior was expected, since gap junctions are not known to exhibit plastic modulations.

4. Discussion Micropatterned substrates have long been considered a promising approach for the creation of defined neuronal circuits in vitro [30–35]. A number of approaches has been taken to grow neurons in experimentally defined circuits on modified surfaces. A major obstacle for the applicability of patterned net-

works as a model system so far lied in a frequently observed impairment of physiological development of cells grown on the highly restrictive surfaces. The presented results establish for the first time synaptic plasticity in micropatterned networks and thus demonstrate their applicability as a model for signal processing by neuronal networks. The patterned networks reproduce different forms of synaptic plasticity that had been reported with brain slices. Paired pulse depression was observed as well as an exponential decay of consecutive postsynaptic potentials in response to trains of presynaptic APs. Consistent with the literature, both types of depression were more pronounced at excitatory than at inhibitory synapses. This effect has been implicated in the control of network activity and the prevention of runaway excitation upon repetitive stimulation [6,21]. In addition, synaptic depression in response to stimulus trains was shown to depend on the stimulation frequency. Such behavior has been described to a similar extent in slices and is thought to be important for the amplification of afferent signals with an adjustable gain [22]. Taken together, these results indicate that synapses forming on the highly confining geometry of the micropattern are physiologically normal and capable of plastic modulations. These have been implicated in signal perception, filtering and processing by neuronal networks. Neither the low number of synaptic interaction partners nor the imposition of an experimentally defined connectivity pattern had a negative impact on these features. As microcontact printing is a very versatile method, networks of almost any two-dimensional connectivity pattern can easily be realized. These may either mimic microcircuitry as it is encountered in the intact tissue and to deliberately alter well-defined aspects of the network to determine their impact on network behavior. Such aspects may be the number of neurons in a feedback circuit, the number of branch points offered to each neuron or the convergence of different neuronal pathways onto a single cell. Since not only the connectivity pattern but also the adhesion sites for neuronal cell bodies can strictly be controlled, the technique is also attractive in the context of extracellular recording devices. It allows for an accurate placing of neurons onto the sensitive spots of a multi-electrode array, substantially improving the signal-to-noise ratio such that long-term recordings of defined neuronal circuits become feasible. Apart from applications in basic research, this system will also be valuable in biotechnology, e.g. in the context of cellbased biosensors. Interesting possibilities can further be envisioned in the development of neuronal prostheses, where micropatterned surfaces may be applied to define the interaction of a neurological implant with the surrounding nervous system.

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5. Conclusions Geometrically confined neuronal networks were realized on surfaces patterned by microcontact printing. Neurons cultured under these conditions were found to form chemical and electrical synapses. Chemical synapses exhibit short-term modulations similar to those reported in brain slices. Thus, a high degree of control over the network architecture was achieved without an obvious impairment of cellular physiology. A future challenge will be the demonstration of long-term modulations in patterned networks.

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