Figure 3

Cortical Aβ-peptide exposures induce early synaptic loss in a reconstructed cortical-hippocampal network. a. Phase contrast picture of the microfluidic device comprising funnel-shaped micro-channels allowing unidirectional axonal growth. Cortical neurons (Cx) were seeded in the left chamber, hippocampal neurons (Hi) in the right chamber, and were cultured for 14 days to reconstruct a cortical-hippocampal network. A cartoon representing one cortical neuron connected to one hippocampal neuron is inserted for clarity. b-e. Effect of Aβ42 oligomers and Aβ25-35 peptides on synaptic connections. Cortical and hippocampal neurons were cultured in μFD chambers as shown in a. b,c) Representative fluorescence micrographs from somato-dendritic compartment of Cx neurons (left panels) and from Hi neurons in the distal chamber receiving cortical fibers (right panels). Cx chambers were treated with sham (b, Ø/ Ø) or Aβ25-35 (c, Aβ /Ø). Neurons, axons and synapses were immunodetected using anti-MAP2 (blue), anti-α-tubulin (green), and anti-α-synuclein (red) respectively. Scale bar: 20 μM. Similar presynaptic patterns were observed with anti-synaptophysin labeling (not shown). d) Representative higher magnification showing presynaptic clusters (anti-α-synuclein, red) affixed to hippocampal dendrites (anti-MAP2, blue) in hippocampal neurons cultured alone (Hi) connected as in b (Hi-Cx) and connected plus treated with Aβ as in c (Hi-Cx + Aβ). e) Quantification of presynaptic structures affixed to hippocampal dendrites after cortical exposure to oligomeric Aβ1-42 (10 nM), fibrillar Aβ1-42 (10 nM), Aβ25-35 peptides (10 μM), and Aβ35-25 peptides (10 μM). (n = 3; **P < 0.01; ***P < 0.001, ANOVA).