Efficient retrograde connectivity tracing by rAAV9-Retrovariant
Previous report had shown that inserting the selected sequences from the AAV9 libraries into the capsid of AAV2 could not increase transduction efficiency compared with AAV9 variants [52], and the 7-mer PHP.B peptide (TLAVPFK) from the AAV9 libraries can improve transduction efficiency of AAV1 in vivo and in vitro when inserted in between S588 and T589 of AAV1 capsid [57]. Therefore, whether the peptide segments from AAV2-Retro (AAV2 libraries) can be integrated into the capsid of AAV9 to achieve high-efficiency retrograde labeling remains to be verified. To verify this, the 10-mer peptide sequence (LADQDYTKTA) from AAV2-Retro (AAV2 libraries) was inserted in between Q588 and A589 of the AAV9 capsid to produce AAV9-Retro (Fig 1A). rAAV-CaMKIIa-EGFP and rAAV-CaMKIIa-mCherry was packaged by using this AAV9-Retro (Fig 1B). In order to evaluate whether AAV9-Retro can achieve high-efficiency retrograde labeling input neurons, 300 nl of rAAV9-Retro-CaMKIIa-EGFP virus was infused into the VTA region, then local infection and cortical region projecting to VTA were imaged by confocal microscopy at 21 days post-injection (DPI). A large number of green fluorescent signals were found in VTA and cortex (Fig 1C), this result indicated that AAV9-Retro has high-efficiency retrograde access to input neurons.
rAAV9-Retro shows a similar retrograde gene transduction efficiency compared with rAAV2-Retro
rAAV2-Retro has higher retrograde transduction efficiency than AAV9 and plays an important role in the analysis of nerve circuits [5]. In order to compare the retrograde infection efficiency of rAAV9-Retro virus (based on AAV9 modification) with rAAV2-retro, the two viruses were injected into VTA sites (Fig 2A-2B), respectively. We found that the two viruses could infect the same upstream brain regions (Fig 2C-2J), including prefrontal cortex (PFC), somatomotor areas (MO), medial septal complex (MSC) and Midbrain reticular nucleus (MRN), etc, and there was no statistical difference in the total number of neurons projection to VTA (Fig 2K, 10770 ± 1394 for AAV2-Retro; 10278 ± 1957 for AAV9-Retro. P = 0.8477). These results indicate that rAAV9-Retro and rAAV2-Retro have the similar retrograde infection efficiency.
rAAV9-Retro and rAAV2-Retro can retrogradely label the same projection neurons through mixed injection
In order to verify whether rAAV9-Retro and rAAV2-Retro can retrogradely infect the same brain regions that project to the injection site and avoid the difference caused by injection site deviation or individual variation of animals, rAAV2-Retro-CaMKIIa-mCherry and rAAV9-Retro-CaMKIIa-EGFP viruses were mixed at the particles ratio of 1:1 and injected into VTA at 300 nl (Fig 3A), local infection and projection regions were imaged by the Olympus VS120 Slide Scanner microscope at 21 days post-injection (DPI). Both green and red fluorescent signals appeared in VTA (Fig 3B-3D) and in the same brain areas that projection to VTA (Fig 3E-3P), including anterior cingulate area (ACA), somatomotor areas (MO), prefrontal cortex (PFC), agranular insular area (AI) and medial septal complex (MSC), etc, and most of them overlapped. These results showed that rAAV9-Retro and rAAV2-Retro can retrogradely label the same projection neurons, when mixed injection to the same brain area.
rAAV9-Retro and rAAV2-Retro exhibit similar retrograde infection tropism and efficiency at different brain regions
Viruses may have different infective characteristics and efficiency when injected into different brain areas. Therefore, we injected the two viruses into the brain area of Caudate putamen (CPu, Fig 4A and Fig 4E), which had been reported to mainly receive input from cerebral cortex (CTX), basolateral amygdalar nucleus (BLA) and thalamus (TH) via rAAV2-Retro [5]. We found that, like rAAV2-Retro (Fig 4B-4D), rAAV9-Retro could effectively infect these brain regions (Fig 4F-4H), and the efficiency was equivalent between them through quantitative analysis (Fig 4I, from CTX: 6495±479.8 for AAV2-Retro, 7119±507.6 for AAV9-Retro, P = 0.4219; from BLA: 737.3±19.19 for AAV2-Retro, 871±203.1 for AAV9-Retro, P = 0.5481; from TH: 1281±250.0 for AAV2-Retro, 945.0±256.6 for AAV9-Retro, P = 0.4010). Also, there was no statistical difference in the total number of neurons projection to CPu (Fig 4J, 8888 ± 672.8 for AAV2-Retro, 9366 ± 633.6 for AAV9-Retro, P = 0.6318). These results indicate that rAAV9-Retro and rAAV2-Retro have the similar retrograde infection tropism and efficiency at different brain regions.
rAAV9-Retro mediates efficient transduction across the central nervous systems after intravenous administration
Compared with AAV2, AAV9 has the advantage of transduction across the blood-brain barrier. In order to verify whether rAAV9-Retro can mediate efficient transduction across the central nervous systems, GFP-expressing rAAV9-Retro or rAAV2-Retro (5 × 1011 vg/mouse) were intravenously injected into adult mice, fluorescence signal in the brains of mice were imaged after 4 weeks of expression. We found that rAAV9-Retro allowed efficient transduction across the blood-brain barrier after intravenous administration, and eGFP signals were observed in several important brain regions (including cortex, striatum, pallidum, hippocampus, thalamus, hypothalamus and periaqueductal gray), which were not shown in rAAV2-Retro.