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Targeting Neurons of Rat Nucleus Tractus Solitarii with the Gene Transfer Vector Adeno-Associated Virus Type 2 to Up-Regulate Neuronal Nitric Oxide Synthase

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Abstract

Adeno-associated virus (AAV) has distinct advantages over other viral vectors in delivering genes of interest to the brain. AAV mainly transfects neurons, produces no toxicity or inflammatory responses, and yields long-term transgene expression. In this study, we first tested the hypothesis that AAV serotype 2 (AAV2) selectively transfects neurons but not glial cells in the nucleus tractus solitarii (NTS) by examining expression of the reporter gene, enhanced green fluorescent protein (eGFP), in the rat NTS after unilateral microinjection of AAV2eGFP into NTS. Expression of eGFP was observed in 1–2 cells in the NTS 1 day after injection. The number of transduced cells and the intensity of eGFP fluorescence increased from day 1 to day 28 and decreased on day 60. The majority (92.9 ± 7.0%) of eGFP expressing NTS cells contained immunoreactivity for the neuronal marker, protein gene product 9.5, but not that for the glial marker, glial fibrillary acidic protein. We observed eGFP expressing neurons and fibers in the nodose ganglia (NG) both ipsilateral and contralateral to the injection. In addition, eGFP expressing fibers were present in both ipsilateral and contralateral nucleus ambiguus (NA), caudal ventrolateral medulla (CVLM) and rostral ventrolateral medulla (RVLM). Having established that AAV2 was able to transduce a gene into NTS neurons, we constructed AAV2 vectors that contained cDNA for neuronal nitric oxide synthase (nNOS) and examined nNOS expression in the rat NTS after injection of this vector into the area. Results from RT-PCR, Western analysis, and immunofluorescent histochemistry indicated that nNOS expression was elevated in rat NTS that had been injected with AAV2nNOS vectors. Therefore, we conclude that AAV2 is an effective viral vector in chronically transducing NTS neurons and that AAV2nNOS can be used as a specific gene transfer tool to study the role of nNOS in CNS neurons.

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Abbreviations

AAV:

Adeno-associated virus

AAV2:

Adeno-associated virus type 2

AAV2eGFP:

Adeno-associated virus type 2 encoding enhanced green fluorescent protein

AAV2nNOS:

Adeno-associated virus type 2 encoding cDNA of nNOS

AP:

Area postrema

CMV promotor:

Cytomegalovirus promotor

CVLM:

Caudal ventrolateral medulla

DMV:

Dorsal motor nucleus of vagus

eGFP:

Enhanced green fluorescent protein

eNOS:

Endothelial nitric oxide synthase

GAPDH:

Glyceraldehyde-3-phosphate

Gr:

Gracilus nucleus

GFAP:

Glial fibrillary acidic protein

NA:

Nucleus ambiguus

NG:

Nodose ganglion

nNOS:

Neuronal nitric oxide synthase

NO:

Nitric oxide

RT-PCR:

Reverse transcription polymerase chain reaction

RVLM:

Rostral ventrolateral medulla

PGP9.5:

Protein gene product 9.5

NTS:

Nucleus tractus solitarii

PBS:

Phosphate buffered saline

RRX:

Rhodamine red X

Tr:

Tractus solitarius

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Acknowledgments

We thank Dr. Yi Chu for his help with quantitative real time RT-PCR. This work was funded in part by NIH RO1 HL 59593 (to W. T. Talman) and NIH RO1 HL 088090 (to L. H. Lin and W. T. Talman).

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Authors and Affiliations

  1. Department of Neurology, University of Iowa, 1191 ML, Iowa City, IA, 52242, USA

    Li-Hsien Lin, Deidre Nitschke Dragon, Jingwen Jin & William T. Talman

  2. Neurology Service, Veterans Affairs Medical Center, Iowa City, IA, 52246, USA

    William T. Talman

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  1. Li-Hsien Lin
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  2. Deidre Nitschke Dragon
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  3. Jingwen Jin
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  4. William T. Talman
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Correspondence to Li-Hsien Lin.

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Lin, LH., Dragon, D.N., Jin, J. et al. Targeting Neurons of Rat Nucleus Tractus Solitarii with the Gene Transfer Vector Adeno-Associated Virus Type 2 to Up-Regulate Neuronal Nitric Oxide Synthase. Cell Mol Neurobiol 31, 847–859 (2011). https://doi.org/10.1007/s10571-011-9674-5

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