Abstract
While Cre-dependent viral systems permit the manipulation of many neuron types, some cell populations cannot be targeted by a single DNA recombinase. Although the combined use of Flp and Cre recombinases can overcome this limitation, insufficient recombinase activity can reduce the efficacy of existing Cre+Flp-dependent viral systems. We developed a sensitive dual recombinase-activated viral approach: tTA-driven Recombinase-Guided Intersectional Targeting (tTARGIT) adeno-associated viruses (AAVs). tTARGIT AAVs utilize a Flp-dependent tetracycline transactivator (tTA) 'Driver' AAV and a tetracycline response element-driven, Cre-dependent 'Payload' AAV to express the transgene of interest. We employed this system in Slc17a6FlpO;LeprCre mice to manipulate LepRb neurons of the ventromedial hypothalamus (VMH; LepRbVMH neurons) while omitting neighboring LepRb populations. We defined the circuitry of LepRbVMH neurons and roles for these cells in the control of food intake and energy expenditure. Thus, the tTARGIT system mediates robust recombinase-sensitive transgene expression, permitting the precise manipulation of previously intractable neural populations.
Highlights
The molecular heterogeneity of the nervous system requires a rich toolkit for precise study of distinct cell populations
The density of LepRb neurons in the adjacent arcuate nucleus (ARC), dorsomedial hypothalamus, and lateral hypothalamic area complicated our initial attempts to study LepRbVMH neurons using LeprCre mice and Cre-dependent vectors; viruses targeted to the ventromedial hypothalamus (VMH) spread to other nearby LeprCre neurons, confounding the interpretation of our results
Because Slc17a6, encoding the vesicular glutamate transporter 2 protein, expression is largely restricted to LepRbVMH neurons and absent from most surrounding LepRb cells (Vong et al, 2011), we generated a Slc17a6FlpO strain and crossed it with LeprCre and a novel Flp- and Cre-dependent reporter (Rosa26RCFL-eGFP-L10a)
Summary
The molecular heterogeneity of the nervous system requires a rich toolkit for precise study of distinct cell populations. By introducing a viral vector into mice that produce either Cre or Flp only in one particular type of neuron, researchers can limit the activity of the cargo gene to that neuronal type. Sometimes even this approach is not selective enough. Sabatini et al have overcome this limitation by developing and testing a new set of viral vectors that are active only in neurons that produce both Cre and Flp. The vectors are called tTARGIT AAVs and allow researchers to target cells more precisely than was possible with the previous version of the toolkit. To overcome these challenges and reveal a specific role for LepRbVMH neurons in suppressing food intake and increasing energy expenditure to promote weight loss
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