Flexible Gold Nanocone Array Surfaces as a Tool for Regulating Neuronal Behavior.

Accelerated neurite outgrowth of rat cortical neurons on a flexible and inexpensive substrate functionalized with gold nanocone arrays is reported. The gold nanocone arrays are fabricated on Teflon films by a bottom-up approach based on colloidal lithography followed by deposition of a thin gold layer. The geometry of nanocone arrays including height and pitch is controlled by the overall etching time and template polystyrene beads size. Fluorescence microscopy studies reveal high viability and significant morphological changes of the neurons on the structured surfaces. The elongation degree of neurite is maximized on the nanocone arrays created with 1 µm polystyrene beads by a factor of two with respect to the control. Furthermore, the interface between the neurons and the nanocones is investigated by scanning electron microscopy and focused ion beam cross-sectioning. The detailed observation of the neuron/nanocone interfaces reveals the morphological similarity between the nanocone tips and the neuronal processes, the existence of interspace at the interface between the cell body and the nanocones, and neurite bridging among the neighboring structures, which may induce the acceleration of neurite outgrowth. The flexible gold nanocone arrays can be a good supporting substrate of neuron culture with noble electrical and optical properties.

Emergence of nociceptive functionality and opioid signaling in human induced pluripotent stem cell-derived sensory neurons.

ABSTRACT: Induced pluripotent stem cells (iPSCs) have enabled the generation of various difficult-to-access cell types such as human nociceptors. A key challenge associated with human iPSC-derived nociceptors (hiPSCdNs) is their prolonged functional maturation. While numerous studies have addressed the expression of classic neuronal markers and ion channels in hiPSCdNs, the temporal development of key signaling cascades regulating nociceptor activity has remained largely unexplored. In this study, we used an immunocytochemical high-content imaging approach alongside electrophysiological staging to assess metabotropic and ionotropic signaling of large scale-generated hiPSCdNs across 70 days of in vitro differentiation. During this period, the resting membrane potential became more hyperpolarized, while rheobase, action potential peak amplitude, and membrane capacitance increased. After 70 days, hiPSCdNs exhibited robust physiological responses induced by GABA, pH shift, ATP, and capsaicin. Direct activation of protein kinase A type II (PKA-II) through adenylyl cyclase stimulation with forskolin resulted in PKA-II activation at all time points. Depolarization-induced activation of PKA-II emerged after 35 days of differentiation. However, effective inhibition of forskolin-induced PKA-II activation by opioid receptor agonists required 70 days of in vitro differentiation. Our results identify a pronounced time difference between early expression of functionally important ion channels and emergence of regulatory metabotropic sensitizing and desensitizing signaling only at advanced stages of in vitro cultivation, suggesting an independent regulation of ionotropic and metabotropic signaling. These data are relevant for devising future studies into the development and regulation of human nociceptor function and for defining time windows suitable for hiPSCdN-based drug discovery.

Anthrax toxins regulate pain signaling and can deliver molecular cargoes into ANTXR2+ DRG sensory neurons.

Bacterial products can act on neurons to alter signaling and function. In the present study, we found that dorsal root ganglion (DRG) sensory neurons are enriched for ANTXR2, the high-affinity receptor for anthrax toxins. Anthrax toxins are composed of protective antigen (PA), which binds to ANTXR2, and the protein cargoes edema factor (EF) and lethal factor (LF). Intrathecal administration of edema toxin (ET (PA + EF)) targeted DRG neurons and induced analgesia in mice. ET inhibited mechanical and thermal sensation, and pain caused by formalin, carrageenan or nerve injury. Analgesia depended on ANTXR2 expressed by Nav1.8+ or Advillin+ neurons. ET modulated protein kinase A signaling in mouse sensory and human induced pluripotent stem cell-derived sensory neurons, and attenuated spinal cord neurotransmission. We further engineered anthrax toxins to introduce exogenous protein cargoes, including botulinum toxin, into DRG neurons to silence pain. Our study highlights interactions between a bacterial toxin and nociceptors, which may lead to the development of new pain therapeutics.

Asymmetric, nano-textured surfaces influence neuron viability and polarity.

Three dimensional, nanostructured surfaces have attracted considerable attention in biomedical research since they have proven to represent a powerful platform to influence cell fate. In particular, nanorods and nanopillars possess great potential for the control of cell adhesion and differentiation, gene and biomolecule delivery, optical and electrical stimulation and recording, as well as cell patterning. Here, we investigate the influence of asymmetric poly(dichloro-p-xylene) (PPX) columnar films on the adhesion and maturation of cortical neurons. We show that nanostructured films with dense, inclined polymer columns can support viable primary neuronal culture. The cell-nanostructure interface is characterized showing a minimal cell penetration but strong adhesion on the surface. Moreover, we quantify the influence of the nano-textured surface on the neural development (soma size, neuritogenesis, and polarity) in comparison to a planar PPX sample. We demonstrate that the nanostructures facilitates an enhancement in neurite branching as well as elongation of axons and growth cones. Furthermore, we show for the first time that the asymmetric orientation of polymeric nanocolumns strongly influences the initiation direction of the axon formation. These results evidence that 3D nano-topographies can significantly change neural development and can be used to engineer axon elongation. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 1634-1645, 2018.