Synapsin-GFP Lentivirus

Stem cell-derived neurons are typically obtained in bulk culture lacking spatial organization and any meaningful connectivity. Here, researchers implemented a microfluidic system for the long-term culture of human neurons with patterned protrusions and synaptic terminals. Co-culturing human midbrain dopaminergic neurons and striatal medium spiny neurons on a microchip established a coordinated nigrostriatal circuit with functional dopaminergic synapses. The researchers used this platform to dissect mitochondrial dysfunction associated with genetic forms of Parkinson's disease (PD) with OPA1 mutations. Notably, axons of OPA1 mutant dopaminergic neurons exhibited a significant reduction in mitochondrial mass. This defect results in a significant loss of dopaminergic synapses, which is exacerbated by long-term culture. Therefore, the depletion of synaptic mitochondria associated with PD may precede the loss of neuronal connectivity and neurodegeneration. In vitro reconstitution of human circuitries by microfluidic technology offers a powerful system to study brain networks by establishing ordered neuronal compartments and correct synapse identity.

To demonstrate the presence of active monoaminergic synapses, the researchers used the fluorescent probe FFN206, which is a specific and sensitive substrate for VMAT2. FFN206 is taken up by VMAT2-expressing neurons and binds to transporters within acidic synaptic vesicles, maintaining a strong and sustained fluorescence intensity. Five weeks after neurons were seeded into the microdevice, FFN206 was mixed with artificial cerebrospinal fluid (ACSF) buffer and perfused within the central channel (Figure 1A). To specifically identify axonal terminals within the central chamber, the microwells containing DANs were exposed to lentivirus expressing Synapsin-GFP for 24 hours. Lentivirus infection in the microdevice transduced a large number of neurons and maintained them within the microwells without any noticeable diffusion between the microgrooves and other surrounding chambers. KCl-containing medium (56 mM) was perfused into the central channel and the side chambers containing DANs for 10 minutes before starting imaging. Live confocal imaging detected a large number of FFN206+ puncta, most of which colocalized with GFP+ fibers in the central compartment (Figure 1B-1D). It is possible that not all GFP+ fibers could be easily detected at low fluorescence signals in live imaging experiments, thus plausibly explaining why not all FFN206+ puncta could be mapped onto GFP+ axonal branches. These data suggest that a large number of monoaminergic synapses are established in the medial channel and show active recycling of VMAT2+ vesicles in DAT terminals.

Figure 1. DA-Specific Synaptic Identity and Functionality in the Human Nigro-striatal Pathway. (Iannielli A, et al., 2019)