Neuronal pathfinding on mixed permissive-inhibitory substrates

• Mixed permissive-inhibitory substrate design by stamping + microfluidics: a) stamp design

Microfabrication steps for soft lithography: 1 - Exposure of 1^st mask with pattern generator, 2 - Development and etch of pattern, 3 - 5x Image reduction of 1^st pattern, 4 - Development and etch, 5 - UV exposure through 2^nd mask onto SU-8 spin coated on silicon wafer,  6 - SU-8 development,  7 - PDMS curing step, 8 - Removal of PDMS stamp, or alternatively 5a - PDMS curing step, and 6a - Removal of PDMS stamp. (from G.Hodgkinson, PhD Dissertation)

• Mixed permissive-inhibitory substrate design by stamping and microfluidics

Protein deposition by a combination of the micro contact printing and micro fluidics. (from G.Hodgkinson, PhD Dissertation)

• "Polka-dot" patterns of permissive-inhibitory signals for neuronal pathfinding experiments

Upper panels: Fluorescence images of stamped AF594-aggrecan patterns on uniform, unlabeled, LN field.  AG micro-island patterns in increasing coverages from left to right: 2%, 7% and 31%,  coverages, scale bar: 10 µm. Lower panels: AF488-AG patterns with various island density step functions: 0%, 0% to 11%, 3% to 11%, 5% to 11%, and 11% coverages, scale bar 10 µm. (G. Hodgkinson and V. Hlady, unpublished)

• Axon growth cone navigation on the "polka-dot" pattern of permissive-inhibitory: time-lapse images

Phase contrast time-lapse microscopy images showing that a narrow growth cone chooses an outgrowth pathway predominately between aggrecan islands (brighter dots) while filopodia appear to weave and sample the area to be advanced inbetween aggrecan islands. Substratum: laminin field covered by aggrecan µm-sized islands (31 % average AG surface coverage). Scale bar - 10 µm. (G. Hodgkinson and V. Hlady, unpublished)

***** Click here for the neuronal pathfinding movies*****