Regulation of Dendrite Arborization by Substrate Stiffness is Mediated by Glutamate Receptors

Abstract

Brain injury or disease can initiate changes in local or global stiffness of brain tissue. While stiffness of the extracellular environment is known to affect the morphology and function of many cell types, little is known about how the dendrites of neurons respond to changes in brain stiffness. To assess how extracellular stiffness affects dendrite morphology, we took biomaterial and biomedical engineering approaches. We cultured mixed and pure hippocampal neurons on hydrogels composed of polyacrylamide (PA) of varying stiffnesses to mimic the effects of extracellular matrix stiffness on dendrite morphology. The majority of investigations of cortical and spinal cord neurons on soft hydrogels examined branching at early time points (days in vitro (DIV) 2-7), an important distinction from our study, where we include later time points that encompass the peak of branching (DIV 10-12). At DIV 12, dendrite branching was altered by stiffness for both pure and mixed neuronal cultures. Furthermore, we treated hippocampal cultures with glutamate receptor antagonists and with astrocyte-conditioned media. Blocking AMPA and NMDA receptors affected the changes in dendrite branching seen at varying rigidities. Moreover, extracellular factors secreted by astrocytes also change dendrite branching seen at varying rigidities. Thus, astrocytes and ionotropic glutamate receptors contribute to mechanosensing.

Matrix stiffness modulates formation and activity of neuronal networks of controlled architectures

“…It is worth pointing out that contrary to other reports our cortical neuronal preparation contains a negligible amount of glial cells that permit to exclude their role in the mechanosensing mechanism of cortical neurons. Despite recent investigations on transmembrane integrin proteins [19,32], RhoA [33], glutamate receptors [4] and specific ion channels [34], mechanotransduction in neurons is still poorly understood and further investigations will be required to understand this complex phenomenon. By using neuronal networks on compliant substrates, future work will focus on elucidating the role of the matrix stiffness on the balance of excitatory versus inhibitory receptors and the possible role for tension forces in neuronal and network function [35].…”

“…Soft matrices have been found to promote neurite outgrowth and axon specification [2,3], whereas more rigid substrates lead to increased dendrite number and branching [4]. In a pathological context, degenerative disease or brain injury may imply significant modifications of the local or global stiffness of brain tissues.…”

Fibroblast Morphology on Dynamic Softening of Hydrogels

“…3,25,29,34,46 Cells sense and react to various ECM stimuli, including chemical and physical. [27][28][29]39,44,45 ECM-cell models composed of PDMS, 4 polyacrylamide, 26,39,44,45 collagen, 51 alginate, 19,35 Matrigel, 58 and other materials have been used to examine the influences of mechanical cues on the cell's behavior and physiology. Previous in vitro studies, including those in our laboratory, have used static matrices (where the mechanical properties remain unchanged throughout the culture period) to examine the effects of matrix stiffness on neuron and fibroblast morphology.…”

Preparation of DNA-crosslinked Polyacrylamide Hydrogels

“…Second, standard cell and tissue culturing techniques are used for these experiments and can be found in numerous original articles, technical articles, and reference books. Techniques for specifically plating fibroblasts and neurons onto DNA gels can be found in previous publications [9][10][11][19][20][21] .…”

“…Plate cells. For protocols regarding dissection and/or culturing of neurons or fibroblasts mentioned in this article, refer to one of the following publications [9][10][11][19][20][21] . 2.…”