Manipulating gene expression in projection-specific neuronal populations using combinatorial viral approaches - PubMed (original) (raw)

Manipulating gene expression in projection-specific neuronal populations using combinatorial viral approaches

Bryan B Gore et al. Curr Protoc Neurosci. 2013.

Abstract

The mammalian brain contains tremendous structural and genetic complexity that is vital for its function. The elucidation of gene expression profiles in the brain, coupled with the development of large-scale connectivity maps and emerging viral vector-based approaches for target-selective gene manipulation, now allow for detailed dissection of gene-circuit interfaces. This protocol details how to perform combinatorial viral injections to manipulate gene expression in subsets of neurons interconnecting two brain regions. This method utilizes stereotaxic injection of a retrograde transducing CAV2-Cre virus into one brain region, combined with injection of a locally transducing Cre-dependent AAV virus into another brain region. This technique is widely applicable to the genetic dissection of neural circuitry, as it enables selective expression of candidate genes, dominant-negatives, fluorescent reporters, or genetic tools within heterogeneous populations of neurons based upon their projection targets.

Keywords: AAV; CAV; neural circuit; stereotaxic surgery; viral vector.

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Figures

Figure 1

Figure 1

Combinatorial viral delivery to study pathway specific gene function. (A) Illustration of combined viral injections of a conditional AAV-FLEX construct (red) into a region containing a neuronal population of interest and a CAV2-Cre viral vector (blue) into a projection target of the neurons of interest. (B) Illustration of local (red) AAV-FLEX viral vector transduction and retrograde (blue) CAV2-Cre transduction. (C) Demonstration of combinatorial viral-mediated gene expression. Injection of CAV2-Cre into a target region of interest (blue) of a Cre-dependent reporter line (Ai14) illustrates coverage area of viral vector injection and the number of neurons retrogradely transduced. Injection of AAV-FLEX containing conditional expression cassette of interest locally transduces neurons at the sight of injection; however, only neurons with combined CAV2-Cre and AAV-FLEX will express the transgene (red).

Figure 2

Figure 2

Illustration demonstrating the identification of Bregma and Lambda on a mouse skull. Following incision, the scalp is held open using skin clamps, allowing access to the top of the skull. Bregma can be seen as the intersection of the midline rostral-caudal fissure and the second major medial-lateral fissure. Lambda can be identified as the most rostral medial-lateral fissure. Stereotaxic×and y coordinates are set to zero at Bregma. Moving from midline to the left is in the negative×direction and positive from midline to the right. Similarly, y coordinates are negative caudal to Bregma and positive rostral to Bregma. Stereotaxic targeting of the y coordinate can be improved using a Bregma-Lambda correction factor (F), taken as the distance between Bregma and Lambda and dividing by 4.21 (the distance from Bregma to Lambda in a commonly used mouse brain atlas).

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