Adeno-Associated Virus Technologies and Methods for Targeted Neuronal Manipulation

Abstract

Cell-type-specific expression of molecular tools and sensors is critical to construct circuit diagrams and to investigate the activity and function of neurons within the nervous system. Strategies for targeted manipulation include combinations of classical genetic tools such as Cre/loxP and Flp/FRT, use of cis-regulatory elements, targeted knock-in transgenic mice, and gene delivery by AAV and other viral vectors. The combination of these complex technologies with the goal of precise neuronal targeting is a challenge in the lab. This report will discuss the theoretical and practical aspects of combining current technologies and establish best practices for achieving targeted manipulation of specific cell types. Novel applications and tools, as well as areas for development, will be envisioned and discussed.

Keywords: AAV; cell-type specificity; gene delivery; intersectional methods; neuroscience; targeted neuronal manipulation; viral vectors; virus technologies.

Figure 1

Several aspects of experimental design affect neuronal targeting and manipulation including (A) viral delivery method, (B) composition of viral capsid proteins, (C) promoters and/or enhancers driving transgene expression, (D) IRES or 2A elements for multicistronic expression coupled with fluorescent proteins (FP) or protein epitopes, (E) post-translational regulatory elements such as WPRE or 3′-UTR, and (F) Recombinase (Cre, CreER, Flp, or FlpER) expression from transgenic driver lines (inserted genomically via targeted or random integration) and ligand-dependent or recombinase-dependent expression elements such as TRE or lox sites, respectively. Abbreviations: TRE, tetracycline-response element; lox, LoxP sequence; IRES, internal ribosomal entry site; 2A, 2A sequence for self-cleavage; FP, fluorescent protein; WPRE, woodchuck hepatitis virus posttranscriptional regulatory element; 3′-UTR, 3′-untranslated sequence.

Figure 2

Various strategies for neuronal targeting using AAV. Delivery of neuronal effectors via AAV labels (green) axons and terminals with cell bodies at the injection site. (A) Effectors under a general promoter express in all transduced neurons with cell bodies at the injection site. Specific regions can be optically stimulated (red beam). (B) Effectors under cell-type promoters express only within a cell type. (C) Effectors delivered via a retrograde AAV express in all transduced neurons with axons that project into the injection site. Cell bodies in regions of interest can be optogenetically stimulated (red beam). (D) Delivery of a retrograde AAV expressing Cre recombinase (Retrograde Cre) to the projection site coupled with local delivery of a Cre-dependent effector limits expression to neurons within specific circuits.