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Identifying the efferent projections of leptin-responsive neurons in the dorsomedial hypothalamus using a novel conditional tracing approach.

The Journal of comparative neurology | 2010

Tracing the axonal projections of selected neurons is labor intensive and inherently limited by currently available neuroanatomical methods. We developed an adeno-associated virus (AAV) that can be used for efficiently tracing identified neuronal populations. The virus encodes a humanized Renilla green fluorescent protein (hrGFP) that is transcriptionally silenced by a neo cassette flanked by LoxH/LoxP sites (AAV-lox-Stop-hrGFP). Thus, hrGFP is expressed only in neurons with Cre recombinase activity. To demonstrate the utility of this approach, the virus was injected unilaterally into the dorsomedial hypothalamus (DMH) of mice that express Cre in neurons expressing the leptin receptor. Animals with DMH injections showed robust hrGFP expression in DMH neurons, as visualized by its endogenous fluorescence or following immunolabeling. We found that hrGFP was expressed in approximately one-third to one-half of Cre-expressing neurons at the site of injection, but not in non-Cre-expressing neurons. The expression of GFP allowed us to identify the projection fields of DMH leptin-responsive neurons. Our results show hrGFP-positive axonal projections and terminals in the paraventricular nucleus of the hypothalamus, arcuate nucleus, preoptic area, bed nucleus of the stria terminalis, paraventricular thalamus, periaqueductal gray, and precoeruleus. The aforementioned pattern of projections was similar to DMH projections determined by injections of biotinylated dextran amine in the mouse DMH. Interestingly, some hrGFP-positive terminals were seen contacting the ependymal layer of the third and fourth ventricles. In summary, this approach is an effective tool for tracing axonal projections of chemically identified neurons, including leptin-responsive neurons.

Pubmed ID: 20394060 RIS Download

Mesh terms: Animals | Dependovirus | Efferent Pathways | Gene Expression Regulation | Green Fluorescent Proteins | Humans | Hypothalamus | Integrases | Leptin | Mice | Neurons | Staining and Labeling

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Associated grants

  • Agency: NIMH NIH HHS, United States
    Id: MH61583
  • Agency: NINDS NIH HHS, United States
    Id: R01 NS033987
  • Agency: NIDDK NIH HHS, United States
    Id: TL1 DK081181
  • Agency: NIDDK NIH HHS, United States
    Id: R01 DK088423
  • Agency: NCRR NIH HHS, United States
    Id: 1UL1RR024923
  • Agency: NIDDK NIH HHS, United States
    Id: R37 DK053301
  • Agency: NIDDK NIH HHS, United States
    Id: R01 DK053301
  • Agency: NIDDK NIH HHS, United States
    Id: DK53301
  • Agency: NINDS NIH HHS, United States
    Id: NS33987
  • Agency: NIDDK NIH HHS, United States
    Id: 1PL1DK081182
  • Agency: NCRR NIH HHS, United States
    Id: UL1 RR024923
  • Agency: NIDDK NIH HHS, United States
    Id: PL1 DK081182
  • Agency: NIMH NIH HHS, United States
    Id: R01 MH061583

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