Off-target expression of Cre-dependent adeno-associated viruses in wild type C57BL/6J mice

Adeno-associated viruses (AAVs) are a commonly used tool in neuroscience to efficiently label, trace, and/or manipulate neuronal populations. Highly specific targeting can be achieved through recombinase-dependent AAVs in combination with transgenic rodent lines that express Cre-recombinase in specific cell types. Visualization of viral expression is typically achieved through fluorescent reporter proteins (e.g., GFP or mCherry) packaged within the AAV genome. Although non-amplified fluorescence is usually sufficient to observe viral expression, immunohistochemical amplification of the fluorescent reporter is routinely used to improve viral visualization. In the present study, Cre-dependent AAVs were injected into the hippocampus and cortex of wild-type C57BL/6J mice. While we observed weak but consistent non-amplified off-target DIO expression in C57BL/6J mice, antibody amplification of the GFP or mCherry reporter revealed extensive Cre-independent viral expression. Off-target expression of DIO constructs in wild-type C57BL/6J mice occurred independent of vendor, AAV serotype or promoter. We also evaluated whether Cre-independent expression had functional effects via Designer Receptors Exclusively Activated by Designer Drugs (DREADDs). The DREADD agonist C21 had no effect on contextual fear conditioning or cFos expression in DIO-hM3Dq-mCherry+ cells of C57BL/6J mice. Taken together, our results indicate that DIO constructs have considerable off-target expression in wild type subjects. Our findings are particularly important for the design of experiments featuring sensitive systems and/or quantitative measurements that could be negatively impacted by off-target expression. Significance Statement Adeno-associated viruses (AAV) are widely used in neuroscience because of their safety and ease of use. Combined with specific promoters, Cre/loxP, and stereotaxic injections, highly specific targeting of cells and circuits within the brain can be achieved. In the present study we injected Cre-dependent AAVs into wild-type C57BL/6J mice and found considerable Cre-independent viral expression of AAVs encoding mCherry, GFP, or hM3Dq following immunohistochemical amplification of the fluorescent reporter protein. Importantly, we observed no functional effects of the Cre-independent expression in the hippocampus, as C21 had no detectable effect on DIO-hM3Dq-mCherry infected neurons in C57BL/6J mice. Given the widespread use of DIO rAAVs by the neuroscience community, our data supports careful consideration when using DIO constructs in control animals.