In vitro transduction of neurons and glia by lentiviral vectors pseudotyped with different lyssavirus glycoproteins. Primary cell cultures were transduced using pseudotyped vectors encoding β-Gal (103 vector particles per cell) or EGFP (104 vector particles per cell) and processed 3 days later for cell identification. A) Quantification of the percentage of transduced neurons in primary embryonic mixed spinal cord cultures demonstrated that the Rabies PV pseudotype transduced 70.35 ± 9.66% of the neurons. The VSV-G vector transduced 13.98 ± 1.39% of the neurons and the DuvSAF-pseudotyped vector transduced 4.68 ± 3.58% of the cells. No transduced neurons could be detected in the LagNGA and EBL1 conditions. The difference between vectors reached significance in all groups (#, ##, ### p < 0.05). B) Immunocytochemistry data revealing the neuronal pattern of transduction observed after treatment with the Rabies PV pseudotype in primary mixed spinal cord cultures. Cells were stained for Map-2 (red) for identification of EGFP-positive neurons. Arrowheads indicate transduced astrocytes (GFP-positive/Map-2 negative cells). Scale bar = 50 μm. C) Quantification of the percentage of transduced astrocytes in mixed spinal cord (dark gray bars) vs. pure astrocyte cultures (light gray bars) demonstrated that all pseudotypes have the ability to transduce astrocytes with no effect of the culture type (mixed or pure) in the Rabies PV, LAgNGA, and EBL1 conditions, but a statistically significant decrease effect in the VSV-G and DuvSAF groups (*, ** p < 0.05).