Position along the nasal/temporal plane affects synaptic development by adult photoreceptors, revealed by micropatterning

Abstract

In retinal degeneration, death of photoreceptors causes blindness. Repair of the retina by transplanting photoreceptors has resulted in limited functional connectivity between transplanted and host neurons. We hypothesize that absence of appropriate biological cues, specifically positional (retinotopographic) cues, reduces synaptogenesis. Here we use micropatterning to test whether regional origin affects the early synaptic development of photoreceptors. Right and left retinas from salamanders were first labelled with dextran tetramethyl-rhodamine and fluorescein, respectively, bisected into nasal (N)-temporal (T) or dorsal (D)-ventral (V) halves, individually dissociated, mixed, and cultured for 1 week. Origin of cells was identified by the fluorescent label. Interactions between photoreceptors and neighboring (target) cells were assessed by the number of neuritic contacts with a presynaptic swelling (varicosity). Randomly-plated photoreceptors showed no preference for cellular origin, likely due to multiple potential interactions available to each cell. To reduce cell-cell interactions, culture substrate was patterned using a microfluidic device with 10 μm-wide channels separated by 200 μm, thus allowing only 1–2 targets per photoreceptor. In patterned cultures, 36.89% of N rod cells contacted T targets but only 27.42% of N rod cells contacted N targets; similarly 35.05% of T rod cells contacted N cells but only 17.08% contacted T cells. Thus, opposite regions were more permissive of contact. However, neither cone nor D/V rod cells showed preferences for positional origin of targets. In conclusion, micropatterning demonstrated that neuritic differentiation by rod cells depends on retinotopographic cues along the nasal/temporal plane, suggesting that transplanting rod cells of known positional origin will increase transplant success.

Microfluidic device‐assisted etching of p‐HEMA for cell or protein patterning

“…We anticipate that this technique may be modified to be utilized for the study of other cell types, such as fibroblasts or neurons. Similar techniques have been used with cells, such as retinal neurons, PC12 cells, and fibroblasts, and thus, it is likely that p‐HEMA patterning is compatible with these and other cell types. Viability of C2C12 myoblasts after patterning was not directly examined in this manuscript; however, the high number of adherent cells after 7 DIV suggests that p‐HEMA patterning does not have adverse effects on cell viability (Figure ).…”

“…Microfluidic devices were fabricated as previously described . Briefly, a 50 µm thin layer of photoresist SU‐8 50 (MicroChem, USA) was spin‐coated onto a 3 inch silicon wafer (University wafers, USA) and exposed to 365 nm UV light through a transparency photo‐mask after pre‐exposure bake on hotplate.…”

“…For example, nanoimprint lithography of water‐stable and patterned protein films and hybrid multilayer films produced by layer‐by‐layer technology allow for regions on biomaterials where cells adhere and grow and other areas devoid of cells. For distinct biological applications, polydimethylsiloxane (PDMS) microfluidic devices have been used to pattern 2‐methacryloyloxyethyl phosphorylcholine for examining C2C12 myoblast fusion and calcium transients, and more recently, amphibian retinal neurons have been patterned to study regional effects on photoreceptor varicosity formation . Although a number of techniques can create regions of cell adhesion and repulsion with very high fidelity to the designed patterns, complex materials and methods to create these regions limit their usefulness.…”

“…For distinct biological applications, polydimethylsiloxane (PDMS) microfluidic devices have been used to pattern 2-methacryloyloxyethyl phosphorylcholine for examining C2C12 myoblast fusion and calcium transients, 8 and more recently, amphibian retinal neurons have been patterned to study regional effects on photoreceptor varicosity formation. 9 Although a number of techniques can create regions of cell adhesion and repulsion with very high fidelity to the designed patterns, complex materials and methods to create these regions limit their usefulness. Therefore, the creation of a method to simply and easily pattern cells and proteins is of great importance to advancing biological studies of cellular function, responses, and interactions.…”

Compartmentalized Microfluidic Platforms: The Unrivaled Breakthrough ofIn VitroTools for Neurobiological Research

“…Over the last decade, several studies took advantage of microfluidic technology to further understand the mechanisms underlying axonal behavior, such as axonal transport, local protein synthesis, and synaptogenesis (Wu et al, 2005;Cox et al, 2008;Taylor et al, 2009;Gumy et al, 2011;Lu et al, 2012;Park et al, 2014a;Kung et al, 2015). In a recent study, Lu et al (2012) slightly modified an already described CMD, including a larger compartment to avoid neuronal death in the closed compartment, as a way to investigate the role of mitochondrial transport in axonal degeneration during Parkinson disease.…”