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Additional supporting information can be found in the online version of this article.

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ANA_23747_sm_SuppFig1.tif665KSupporting Information Figure 1. Supplementary Figure 1. Microfluidic device to separate soma from axons and second order neurons. (A) Diagram of microfluidic devices. The device consists of four open cylindrical chambers each with a volume of 200μl. Each pair of chambers is connected by a 100μm tall channel, with a total volume of 5μl into which neurons are plated. The two channels are connected by 450μm long microgrooves. (B) Cortical neurons were cultured in one chamber for one week, stained with Calcein AM, and visualized by fluorescence microscopy. Axons can be observed to have extended from the somal chamber through the microgrooves and into the axonal chamber. (C) Test for passive diffusion of a 30nm virus between the chambers in the absence of neurons. 106 plaque-forming units of Theiler's murine encephalomyelitis virus were introduced into one chamber of a microfluidic device that did not contain neurons. The other chamber was kept with a slightly larger volume of medium throughout the experiment. At each indicated time post introduction (hpi), 50μl of medium were removed from the second chamber and the virus titer was determined by plaque assay. The limit of detection of the plaque assay was 3 plaque-forming units. No virus above this limit was detected in the second chamber at any time point. At 32 hours, 50μl were removed from the chamber containing the virus (32 hpi, inoc.) and the titer was measured.
ANA_23747_sm_SuppFig2.tif288KSupporting Information Figure 2. Supplementary Figure 2. Axons in the axonal chamber. Neurons were cultured for one week in a microfluidic device according to the modified protocol (see Material and Methods). The culture was fixed with 4% paraformaldehyde and stained with the axon specific monoclonal SMI 31. The goat secondary antibody was Alexa-555 labeled.
ANA_23747_sm_SuppFig3.tif16194KSupporting Information Figure 3. Supplementary Figure 3. Alexa 555-labeled Aβ42 fibrils and BSA in neurons grown in microfluidic devices. Neurons were cultured for one week in the first neuron channel to allow them to extend their axons to the opposite channel. At that time, Alexa 555-labeled Aβ42 fibrils were added to the chamber containing these neuron and freshly isolated neurons were added to the opposite channel. The cells in both channels were fixed 24h later and stained with an anti-ßIII-tubulin antibody. (A) Aß42 fibrils in the first-neuron channel. Original magnification: 20x. (B) Aß42 fibrils in axons in the microgrooves. Original magnification 45x. (C) Aß42 fibrils in axons in the axon channel. Original magnification 45x. (D) Alexa 555-labeled BSA in the first neuron channel. Original magnification 45x.
ANA_23747_sm_SuppFig4.tif327KSupporting Information Figure 4. Supplementary Figure 4. Respective positions of the soma of recipient neurons and of the axons of first neurons in a microfluidic device during the transfer of ?-synuclein fibrils from the axons of first neurons to the soma of second neurons. Neurons were seeded and cultured for one week in one channel. Fresh neurons were then seeded in the other channel and cultured for 24 h. The channel wih fresh neurons was scanned from bottom to top with a 45x objective. Pictures were taken every 10μm. The figure shows the series of 11 pictures obtained. The soma of the second neurons are in focuss at the bottom of the channel whereas the axons from the first neurons are in focuss at the top. The distance between recipient neurons and axons of first neurons is of the order of 60μm.
ANA_23747_sm_SuppInfo.doc28KSupplementary Figure Caption
ANA_23747_sm_SuppMov1.mp41765KSupplementary Movie 1. Anterograde axonal transport of ?-synuclein fibrils in axons in a microgroove of a microfluidic device. The neurons were culture for one week. ATTO-550 labeled fibrils were added to the soma channel while keeping an excess volume of 150μl of medium in the axon compartment. Punctae were imaged in the axons 5h after adding fibrils using a 60x oil immersion lens. During imaging, cells were kept at 37°C in 10% CO2. Images were acquired every 6 seconds.
ANA_23747_sm_SuppMov2.mp418104KSupplementary Movie 2. Axonal transport of ?-synuclein fibrils in axons in the axon channel of a microfluidic device. Neurons were seeded in the soma channel and incubated for 7 days. ATTO-550 labeled fibrils were then added to the soma channel while keeping an excess volume of 150μl of medium in the axon compartment. During imaging, cells were kept at 37°C in 10% CO2. Punctae were imaged in the axons of the axon channel 5hr later using a 60x oil immersion lens. Images were acquired every 8 seconds.

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