Topologically Defined Neuronal Networks Controlled by Silicon Chips

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CHAPTER 2. NETWORKS OF DEFINED TOPOGRAPHY Figure 2.1: Four types of mechanisms contribute to guiding growth-cones: contact attraction, chemoattraction, contact repulsion and chemorepulsion. Individual growthcones might be ‘pushed’ from behind by a chemorepellant (red), ’pulled’ from afar by a chemoattractant (green), and ’hemmed’ in by attractive (grey) and repulsive (yellow) local cues; from [110]. Directional cues are either attractive or repulsive, long-range or short-range. The long-range guidance mechanism, called chemotaxis, is based on gradients of diffusible, soluble molecules, whereas the short-range contact-mediated mechanism is mediated by tissue-bound, non-diffusible molecules. Fig. 2.1 depicts the four forces guiding growth-cones: contact attraction, chemoattraction, contact repulsion and chemorepulsion, and lists examples of ligands governing these mechanisms. Some guidance molecules are not exclusively attractive or repulsive but rather bifunctional, depending on their concentration, type of receptor and presence of other molecules. In vivo, neuronal outgrowth is generally controlled by several cues acting simultaneously and synergistically, e.g. the growth-cone is repelled from its origin by one ligand and attracted to the target tissue by another. Besides these ligand-mediated cues, axonal outgrowth is further influenced by trophic factors that are needed for survival and growth as well as by adhesion molecules providing adhesive surfaces for the growth-cone. Although many directional signals are present in vivo, in vitro studies revealed that a single ligand is often sufficient for controlling neuronal outgrowth. These studies set the stage for the design of defined neuronal nets. 2.1.2 Chemical patterns Tracks of substrate bound molecules that are either attractive or repulsive to growth-cones (haptotaxis) are the most widely used technique for growth-cone guidance in vitro. In contrast to gradients of soluble molecules, they are easy to produce and can be kept in culture medium for a week or more without loosing their potency. Two major issues are raised with chemical patterns: what molecules are best and how to deposit them on the substrate? The vast body of literature on this subject is summarized by several comprehensive reviews [5, 22, 30, 52, 54]. In an early experiment, P.C. Letourneau deposited areas of palladium onto different substrates by evaporation through EM-grids as masks [61]. Growth-cones from chick sensory ganglia neurons preferentially elongated on the more adhesive areas; for Pd on glass petri dishes they stayed on the Pd, while if polyornithine-coated dishes were used they preferred the Pd-free areas. Since then, many chemicals have been tested for patterns controlling cell adhesion and neuronal outgrowth. Among them are adhesive proteins like extracellular matrix protein (ECM) [35], laminin [42], growth factors [81] and nonadhesive proteins like albumin [21]. Artificial growth-promoting molecules such as poly-L-lysine and poly-D-lysine [8], and inhibitors such as poly ethylene glycol (PEG) are also common [121]. All these substances are only physisorbed to the substrate. Due to the weak binding 6
2.1. HOW TO CONTROL NEURONAL OUTGROWTH - AN OVERVIEW Figure 2.2: Techniques for patterning chemicals onto surfaces. A, B: Photolithography with selective deposition (A) or removal (B) of molecules. C: Resist free lithography. D: Microcontact printing. E: Microfluidic networks. forces involved, this may result in gradual desorption and release to the cell culture medium with a subsequent loss of directional information. Conversely, self-assembled monolayers of silanes and thiols are tightly bound to silicon or gold surfaces by covalent bonds, making them very resistant to desorption. Silane patterns on Si-wafers have been cleaned and reused in hippocampal cell culture several times without loosing their directional information [23]. Above all, their adhesive properties can be tailored by appropriate terminal groups. Fluorinated, chlorinated and alkyl chains [56, 87, 100] inhibit protein binding and neuronal outgrowth, while amino groups promote it [87]. Complex molecules that are not compatible with silane or thiol chemistry, like proteins and peptides, can be covalently bound to substrates by heterobifunctional crosslinkers [8, 95, 121]. Molecules are patterned onto the substrate with techniques adapted from microelectronic device fabrication. These are summarized in fig. 2.2. D. Kleinfeld et al. were the first to use photolithography for producing pathways of adhesive and nonadhesive silanes [56]. Their technique has been adapted by many researchers since then [16, 126]. Photoresist is spin-coated on a substrate, usually a silicon wafer, silica or glass, exposed through a lithographic mask, and developed. The substrate is then incubated with a solution containing growth mediating molecules; the molecules bind to the resist as well as to uncovered areas. Stripping the resist leaves patterns of guidance molecules surrounded by areas of blank substrate, see fig. 2.2A. In fig. 2.2B, a similar technique is illustrated, with the main difference being that surface-bound molecules are selectively removed rather than deposited [16]. Before spinning photoresist, the substrate is uniformly coated with the desired molecules. Again, substrates are exposed and the resist is developed. A plasma etch removes guidance molecules in regions not protected by the resist, leaving a pattern complementary to the one in fig.2.2A after resist stripping. Photolithography is an excellent tool for functionalizing surfaces with down to sub-micrometer resolution in any desired geometry. However, the harsh solvents (e.g. acetone) involved in the lithographic process are not compatible with many guidance molecules such as proteins. This problem is overcome by recently developed techniques illustrated in fig.2.2C-E. Photoresist and solvents are completely omitted if UV light is used directly to selectively destroy, ablate or photocleave a homogeneous layer of growth mediating molecules. Washing away the residues in the exposed areas leaves intact chemicals in the areas shielded from the UV light by the mask. The only requirement here is UV light with a wavelength short enough to destroy or photocleave molecules, or with sufficient power for ablation (e.g. ArF laser λ=193nm). Several chemicals have been patterned 7
Page 1: Max-Planck-Institut für Biochemie
Page 5 and 6: Contents 1 Introduction 1 1.1 Why b
Page 7 and 8: Chapter 1 Introduction This first c
Page 9 and 10: 1.3. OUTLINE OF THIS THESIS displac
Page 11: Chapter 2 Networks of defined topog
Page 15 and 16: 2.1. HOW TO CONTROL NEURONAL OUTGRO
Page 17 and 18: 2.2. CELL CULTURE Figure 2.4: A: Po
Page 19 and 20: 2.2. CELL CULTURE Figure 2.5: A: Do
Page 21 and 22: 2.3. BUILDING DEFINED NETWORKS - TE
Page 23 and 24: 2.3. BUILDING DEFINED NETWORKS - TE
Page 25 and 26: 2.4. THEORY PART I: NEURONS, SYNAPS
Page 33 and 34: y voltage-gated channels, as outlin
Page 35 and 36: 2.5. RESULTS Figure 2.14: A: Neuron
Page 37 and 38: 2.5. RESULTS DM (without diluting i
Page 39 and 40: 2.5. RESULTS Based on the assumptio
Page 41 and 42: 2.5. RESULTS Figure 2.21: Electrica
Page 43 and 44: 2.5. RESULTS Figure 2.22: A: Histog
Page 45 and 46: 2.5. RESULTS Figure 2.24: Three neu
Page 47 and 48: 2.5. RESULTS Figure 2.26: Statistic
Page 49 and 50: Chapter 3 Single neurons on chips A
Page 51 and 52: 3.2 Theory part II: Field-effect tr
Page 53 and 54: 3.2. THEORY PART II: FIELD-EFFECT T
Page 55 and 56: A C 3.2. THEORY PART II: FIELD-EFFE
Page 57 and 58: 3.3. THE TRANSISTOR-STIMULATOR CHIP
Page 59 and 60: 3.3. THE TRANSISTOR-STIMULATOR CHIP
Page 61 and 62: Silicon dioxide 3.3. THE TRANSISTOR
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A C B D 3.3. THE TRANSISTOR-STIMULA
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3.3. THE TRANSISTOR-STIMULATOR CHIP
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3.3. THE TRANSISTOR-STIMULATOR CHIP
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3.4. MEASUREMENT SETUP Figure 3.11:
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3.4. MEASUREMENT SETUP onboard memo
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3.5. THEORY PART III: NEURON-SILICO
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characterizing the dynamics of the
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3.5. THEORY PART III: NEURON-SILICO
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3.6. NEURON-SILICON INTERFACE: EXPE
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demonstrated by the examples in fig
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Chapter 4 Defined networks on chips
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4.2. RESULTS Figure 4.2: A: Microgr
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4.2. RESULTS 2 postsynaptic action
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4.2. RESULTS Since neuron 1 could a
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4.2. RESULTS Figure 4.5: A: Transis
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4.3. FINAL DISCUSSION Data in diagr
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4.3. FINAL DISCUSSION Some very har
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Chapter 5 Conclusion and outlook Th
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Appendix A Cell culture protocols a
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ethanol and 30% water during UV-ste
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For two-layer structures, steps 3 t
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The flatband voltage VF B and elect
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Appendix D Abbreviations ABS : anti
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Bibliography [1] R. W. Aldrich, P.
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BIBLIOGRAPHY [32] P. Fromherz. Neur
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BIBLIOGRAPHY [69] C. D. McCaig, A.
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BIBLIOGRAPHY [105] N. I. Syed, A. G
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Ich danke PROF. DR. P. FROMHERZ fü
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Topologically Defined Neuronal Networks Controlled by Silicon Chips

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