Synergistic NGF/B27 Gradients Position Synapses Heterogeneously in 3D Micropatterned Neural Cultures

Native functional brain circuits show different numbers of synapses (synaptic densities) in the cerebral cortex. Until now, different synaptic densities could not be studied in vitro using current cell culture methods for primary neurons. Herein, we present a novel microfluidic based cell culture method that combines 3D micropatterning of hydrogel layers with linear chemical gradient formation. Micropatterned hydrogels were used to encapsulate dissociated cortical neurons in laminar cell layers and neurotrophic factors NGF and B27 were added to influence the formation of synapses. Neurotrophic gradients allowed for the positioning of distinguishable synaptic densities throughout a 3D micropatterned neural culture. NGF and B27 gradients were maintained in the microfluidic device for over two weeks without perfusion pumps by utilizing a refilling procedure. Spatial distribution of synapses was examined with a pre-synaptic marker to determine synaptic densities. From our experiments, we observed that (1) cortical neurons responded only to synergistic NGF/B27 gradients, (2) synaptic density increased proportionally to synergistic NGF/B27 gradients; (3) homogeneous distribution of B27 disturbed cortical neurons in sensing NGF gradients and (4) the cell layer position significantly impacted spatial distribution of synapses.

[1]  Claire Wyart,et al.  Colloid-guided assembly of oriented 3D neuronal networks , 2008, Nature Methods.

[2]  M. Shoichet,et al.  Immobilized concentration gradients of nerve growth factor guide neurite outgrowth. , 2004, Journal of biomedical materials research. Part A.

[3]  J. Chen,et al.  Synergistic effects of NGF, CNTF and GDNF on functional recovery following sciatic nerve injury in rats. , 2010, Advances in medical sciences.

[4]  I. Klejbor,et al.  Do two models of acute and chronic stress stimulation influence the amount of nerve growth factor (NGF) and its receptor TrkA in the hippocampal neurons of middle aged rats? , 2011, Brain Research.

[5]  A. Arnold,et al.  Sex differences in mouse cortical thickness are independent of the complement of sex chromosomes , 2003, Neuroscience.

[6]  Anders M. Dale,et al.  CORTICAL THICKNESS AND SUBCORTICAL VOLUMES IN SCHIZOPHRENIA AND BIPOLAR DISORDER , 2010, Schizophrenia Research.

[7]  J. Szentágothai,et al.  Brain Research , 2009, Experimental Neurology.

[8]  Jeremy S H Taylor,et al.  Synergistic effects of osteonectin and NGF in promoting survival and neurite outgrowth of superior cervical ganglion neurons , 2009, Brain Research.

[9]  D. Hoffman-Kim,et al.  Neurite outgrowth at the interface of 2D and 3D growth environments , 2009, Journal of neural engineering.

[10]  K. Kimpinski,et al.  Neurite growth promotion by nerve growth factor and insulin‐like growth factor‐1 in cultured adult sensory neurons: Role of phosphoinositide 3‐kinase and mitogen activated protein kinase , 2001, Journal of neuroscience research.

[11]  C. Petersen,et al.  The Excitatory Neuronal Network of the C2 Barrel Column in Mouse Primary Somatosensory Cortex , 2009, Neuron.

[12]  A. Cowey,et al.  The axo-axonic interneuron in the cerebral cortex of the rat, cat and monkey , 1982, Neuroscience.

[13]  Kozo Kaibuchi,et al.  [Neuronal polarity]. , 2008, Tanpakushitsu kakusan koso. Protein, nucleic acid, enzyme.

[14]  Ralph G Nuzzo,et al.  Guiding neuron development with planar surface gradients of substrate cues deposited using microfluidic devices. , 2010, Lab on a chip.

[15]  Tara Sankar Roy,et al.  Prenatal nicotine exposure evokes alterations of cell structure in hippocampus and somatosensory cortex. , 2002, The Journal of pharmacology and experimental therapeutics.

[16]  F. Polleux,et al.  Establishment of axon-dendrite polarity in developing neurons. , 2009, Annual review of neuroscience.

[17]  Michelle C. LaPlaca,et al.  Synapse-to-neuron ratio is inversely related to neuronal density in mature neuronal cultures , 2010, Brain Research.

[18]  Albert Folch,et al.  A new method for studying gradient-induced neutrophil desensitization based on an open microfluidic chamber. , 2010, Lab on a chip.

[19]  Michele Giugliano,et al.  Micropatterning neural cell cultures in 3D with a multi-layered scaffold. , 2011, Biomaterials.

[20]  M. Tuszynski,et al.  Neurotrophic Factor Therapy: NGF, BDNF and NT-3 , 2009 .

[21]  M. Sofroniew,et al.  Nerve growth factor signaling, neuroprotection, and neural repair. , 2001, Annual review of neuroscience.

[22]  Sang Won Seo,et al.  Variations in cortical thickness with dementia severity in Alzheimer's disease , 2008, Neuroscience Letters.

[23]  Grace N Li,et al.  Tissue-engineered platforms of axon guidance. , 2008, Tissue engineering. Part B, Reviews.

[24]  Kristina D. Micheva,et al.  Single-Synapse Analysis of a Diverse Synapse Population: Proteomic Imaging Methods and Markers , 2010, Neuron.

[25]  X. Cao,et al.  Defining the concentration gradient of nerve growth factor for guided neurite outgrowth , 2001, Neuroscience.

[26]  F. Hefti,et al.  Response of embryonic rat hippocampal neurons in culture to neurotrophin-3, brain-derived neurotrophic factor and basic fibroblast growth factor , 1993, Neuroscience.

[27]  J. Conner,et al.  Nerve growth factor (NGF) content in adult rat brain tissues is several-fold higher than generally reported and is largely associated with sedimentable fractions , 1996, Brain Research.

[28]  A. Windebank,et al.  Insulin-like growth factor-I prevents apoptosis in neurons after nerve growth factor withdrawal. , 1998, Journal of neurobiology.

[29]  G. Whitesides,et al.  Gradients of substrate-bound laminin orient axonal specification of neurons , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[30]  Roger D Kamm,et al.  A high-throughput microfluidic assay to study neurite response to growth factor gradients. , 2011, Lab on a chip.

[31]  G. Brewer,et al.  Optimized survival of hippocampal neurons in B27‐supplemented neurobasal™, a new serum‐free medium combination , 1993, Journal of neuroscience research.

[32]  M. Shoichet,et al.  The use of immobilized neurotrophins to support neuron survival and guide nerve fiber growth in compartmentalized chambers. , 2010, Biomaterials.

[33]  Jean-Louis Viovy,et al.  Wallerian-Like Degeneration of Central Neurons After Synchronized and Geometrically Registered Mass Axotomy in a Three-Compartmental Microfluidic Chip , 2010, Neurotoxicity Research.

[34]  P. Fernyhough,et al.  Insulin and insulin-like growth factor I enhance regeneration in cultured adult rat sensory neurones , 1993, Brain Research.

[35]  A. Granholm,et al.  Nerve growth factor in treatment and pathogenesis of Alzheimer's disease , 2006, Progress in Neurobiology.

[36]  Molly S. Shoichet,et al.  Miniaturized system of neurotrophin patterning for guided regeneration , 2008, Journal of Neuroscience Methods.

[37]  Erin M. Schuman,et al.  Microfluidic Local Perfusion Chambers for the Visualization and Manipulation of Synapses , 2010, Neuron.

[38]  George Adelman,et al.  Encyclopedia of neuroscience , 2004 .

[39]  Anja Kunze,et al.  Co-pathological connected primary neurons in a microfluidic device for Alzheimer studies. , 2011, Biotechnology and bioengineering.

[40]  G. Brewer,et al.  Serum-Free Media for Neural Cell Cultures , 2001 .

[41]  S. Saker,et al.  Histopathological and biochemical changes of morphine sulphate administration on the cerebellum of albino rats. , 2010, Tissue & cell.

[42]  S. Weinbaum,et al.  A diffusion wake model for tracer ultrastructure-permeability studies in microvessels. , 1995, The American journal of physiology.

[43]  K. Shen,et al.  Guidance molecules in synapse formation and plasticity. , 2010, Cold Spring Harbor perspectives in biology.

[44]  Watt W Webb,et al.  Diffusion of nerve growth factor in rat striatum as determined by multiphoton microscopy. , 2003, Biophysical journal.

[45]  E. Recio-Pinto,et al.  Insulin and related growth factors: effects on the nervous system and mechanism for neurite growth and regeneration , 1988, Neurochemistry International.

[46]  M. Shoichet,et al.  Immobilized concentration gradients of neurotrophic factors guide neurite outgrowth of primary neurons in macroporous scaffolds. , 2006, Tissue engineering.

[47]  K. Stroka,et al.  Guiding axons in the central nervous system: a tissue engineering approach. , 2009, Tissue engineering. Part B, Reviews.

[48]  B. Tucker,et al.  The synergistic effects of NGF and IGF‐1 on neurite growth in adult sensory neurons: convergence on the PI 3‐kinase signaling pathway , 2003, Journal of neurochemistry.

[49]  J. Hell,et al.  NS21: Re-defined and modified supplement B27 for neuronal cultures , 2008, Journal of Neuroscience Methods.