Surface-embedded mechanical and chemical cues modulate neuronal mechanoadaptation in primary hippocampal networks

The possibility of external modulation of neuronal electrical behavior is becoming increasingly valuable and captivating for modern (neuro)medical research. Indeed, exogenous neuromodulation could open to the development of innovative technological tools that, in the field of neuro-engineering, concretize in advanced implantable devices (e.g., active and passive neurostimulators), neuro-prosthesis (e.g., neuro-regenerative scaffolds or synthetic ganglia) or neural interfaces (e.g., brain-machine interfaces). In this framework, there is a rising expectation about the critical role that mechano-chemical artificial cues could play in triggering or temper neuronal activity. This belief is mainly supported by the natural role recognized to extracellular matrix (ECM) physicochemical properties (e.g., stiffness, micro- nano-topography and/or chemical composition) in governing different cellular processes as well as inducing physiological or pathological evolution in tissues. In reason of that, a deeper investigation of the impact of external mechanical and chemical stimuli in regulating neuronal behavior represents a critical necessity in the attempt to govern neuronal behavior in a controlled way. Therefore, in this study, I exploited functional substrates that aim to recapitulate in a in vitro model the complexity of ECM-mediated mechanochemical cues. Indeed, I studied the effect they have on primary murine neuronal cultures through a multidisciplinary approach. In the first study, I evaluated the impact of substrate stiffness on primary hippocampal cultures. Specifically, nerve cells were let to develop for 9-10 days-in-vitro interfaced with polyacrylamide (PAA) hydrogels characterized by different “physiological” stiffnesses (Young’s elastic modulus E in the range 1–50 kPa). I investigated the impact of PAA-coupling on neuronal network development and morphology via immunofluorescence experiments, and on its functional aspects exploiting electrophysiological techniques. I focused, in particular, on how the synaptic activity of neuronal cells changes in relation to environmental stiffness. Surprisingly, a drastic reduction in spontaneous network activity was detected when cells were cultured on soft substrates. By treating hippocampal cultures with specific compounds, we hypothesized that this electro-mechanical adaptation is ultimately associated with an altered homeostasis of plasma-membrane cholesterol in interfaced cells. In a second investigation, I mimicked the contribution of external chemical cues making use of substrates endowing chemical functionalities. For the purpose of the study, I let rat hippocampal neuronal networks develop above glass control, single-layer graphene, and carboxyl-modified graphene substrates. In this case, the morphological and functional network adaptation was also evaluated through immunofluorescence and electrophysiology experiments, respectively. Furthermore, the study has taken advantage of advanced nano-microscopy techniques (i.e., Atomic Force Microscopy, and Total Internal Reflection Microscopy) to investigate the different surface chemistry’s impact on focal adhesions organization and neuronal cell stiffness. My results demonstrated that, compared to glass controls, pristine graphene and functionalized graphene substrates induce an opposite effect on the spontaneous electrical activity of interfaced cells: an enhancement in the first case, a reduction in the latter. Finally, in the attempt to evaluate the synergic contribution of ECM mechanical and chemical cues, primary hippocampal neurons were grown on three-dimensional carbon nanotubes (CNTs) foams where specific chemical functionalizations were introduced with the intent to modify substrate stiffness and introduce neuronal-specific responses in the interfaced network. In these preliminary experiments, I evaluated the neuronal network morphology uncovering a significantly different adaptation induced by pristine and functionalized CNTs foams in the form of cell distribution, elongation and orientation. Furthermore, from a functional point of view, I monitored neuronal functionality in terms of spontaneous calcium oscillations. I highlighted an increased calcium activity in cells developed above the stiffer functionalized CNTs compared to the pristine ones.