Microinjections and Stimulation

All animal procedures were approved by the Institutional Animal Care and Use Committee at the Marine Biological Laboratory in Woods Hole, MA following standards set by the National Institutes of Health. Lampreys (Petromyzon marinus; 11–13 cm; 5–7 years old of either sex) were anesthetized in 0.1 g/L MS-222 (tricaine methanesulfonate; Syndel, Ferndale, WA, United States). Next, 2–3 cm segments of spinal cord were dissected and pinned ventral side up in a Sylgard-lined dish containing fresh, oxygenated Lamprey Ringer (100 mM NaCl, 2.1 mM KCl, 1.8 mM MgCl₂, 4 mM glucose, 2 mM HEPES, 0.5 mM L-glutamine, 2.6 mM CaCl₂, pH 7.4). Axonal microinjections were performed as previously described (Medeiros et al., 2017; Walsh et al., 2018; Banks et al., 2020; Soll et al., 2020). Briefly, SEC fraction 12 containing human α-synuclein was diluted in lamprey internal solution (180 mM KCl and 10 mM HEPES K+; pH 7.4) to a pipet concentration of 800 nM and subsequently loaded into glass microelectrodes (20–25 MΩ) for microinjection into giant RS axons. Microinjections were performed using small pulses of nitrogen (4–20 ms, 40 psi, 0.2 Hz) delivered through a picospritzer. Co-injection with a fluorescent dye (70 KDa fluorescein dextran; Thermo Fisher) approximating the molecular weight of brain-derived α-synuclein multimers allowed us to determine the spread of the protein along the axon with respect to the injection site. After injection, the proteins are normally diluted ∼10–20 times near the injection site and up to 200 times farther away. We therefore estimated that the human α-synuclein was diluted to a final axonal concentration between 40 and 80 nM at distances near to the injection site (30–125 μm), which we refer to as the “high concentration”; we also estimated the α-synuclein concentration to be ∼4–30 nM at distances farther away from the injection site (140–400 μm), which we refer to as the “low concentration” (Figure 1A). Control synapses were collected from the same axon at distances >430 μm from the injection site where no protein had diffused, providing an internal control. Immunodepleted samples were injected using the same methods. After injections, the axons were stimulated at 20 Hz for 5 min, as in our prior studies (Busch et al., 2014; Medeiros et al., 2017; Banks et al., 2020; Soll et al., 2020).

Lamprey reticulospinal (RS) synapses and microinjection strategy. (A) Diagram of a lamprey spinal cord showing axonal microinjection and stimulation strategy. Brain-derived human α-synuclein was co-injected into RS axons along with a fluorescent dye of similar molecular weight. After stimulation and fixation, synapses were evaluated within three regions of the axon: those treated with higher concentrations of the protein (estimated 40–80 nM based on the fluorescence of co-injected dye), lower concentrations of the protein (4–39 nM), or no protein (0 nM; Controls). (B) Diagram of an injected axon showing the locations of RS synapses along the axonal perimeter. (C) Electron micrograph of an unstimulated, control RS synapse showing the large size of the synaptic vesicle cluster.