Targeting neurons of rat nucleus tractus solitarii with the gene transfer vector adeno-associated virus type 2 to up-regulate neuronal nitric oxide synthase

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.

Fig. 1

Pseudo-colored confocal images of the rat NTS at the injection level 7 days after AAV2eGPF had been injected unilaterally into this nucleus, which then was subjected to immunofluorescent staining for PGP9.5 (c, d) to show the structure of the NTS. Numerous cells and fibers expressing eGFP (green), the fluorescent protein, are observed in the injected side (b, d). Transfected cells in the adjacent areas, such as the dorsal motor nucleus of vagus (DMV), area postrema (AP), and gracilus nucleus (Gr) are also noted. A few cells and fibers expressing eGFP are observed in the contralateral NTS (a, c). Panels c and d are merged confocal images of eGFP and PGP9.5-IR of the same section as a and b, respectively. Tr tractus solitarius. Scale bar = 100 μm

Fig. 2

Time course of eGFP expression in NTS (upper panel) and NG (lower panel) after unilateral injection of AAV2eGFP into the NTS. The number of eGFP expressing cells (average per section) increased from day 1 to day 28, and then decreased on day 60 in both the injected and contralateral NTS. The number of eGFP expressing cells first appeared on day 4, increased until day 28, and then decreased on day 60 in both ipsilateral and contralateral NG

Fig. 3

Confocal images of rostral-caudal levels of the NTS 7 days after AAV2eGFP had been injected into this nucleus. Neurons and fibers expressing eGFP are seen throughout the length of NTS. a Rostral level at Bregma −12.7 mm, b intermediate level at Bregma −13.3 mm, c subpostremal level at Bregma −13.7 mm, d caudal level at Bregma −14.2 mm. 4V 4th ventricle, Cu cuneate nucleus, DMV dorsal motor neuron of vagus, Gr gracilus nucleus, Tr tractus solitarius. Scale bar = 100 μm

Fig. 4

Confocal images of rat NTS 7 days after injection of AAV2eGFP into the nucleus, which was then subjected to double immunofluorescent staining for PGP9.5 and GFAP. Panel d is a merged confocal image of panels a (eGFP, green), b (PGP9.5, red) and c (GFAP, blue). White arrows in a, b and d indicate representative cells that are positive for both eGFP and PGP9.5-IR and thus appear yellow in the merged image in panel d. Note that the majority of AAV2eGFP transduced NTS cells also contain PGP9.5-IR, and none of them contains GFAP-IR. Empty arrows in c and d indicate cells and processes that contain only GFAP-IR. Scale bar = 20 μm

Fig. 5

Confocal images with immunofluorescent stain for PGP9.5 of the ipsilateral NG, NA and CVLM 14 days after unilateral injection of AAV2eGFP into the NTS. Numerous cells and fibers expressing eGFP are seen in the NG, as shown in a. Panel b, a merged confocal image of the same section as panel a, shows both eGFP (green) and PGP9.5-IR (red). Arrows in a and b indicate NG neurons that are double-labeled for eGFP and PGP9.5-IR and appear yellow. Numerous eGFP expressing fibers are noted in the NA (c) and CVLM (e). Panels d and f, merged confocal images of the same sections b and c, respectively, show both eGFP expression and PGP9.5-IR. Fibers expressing eGFP in the NA and CVLM often surround and are in close apposition to cell bodies (arrows in d and f) that do not express eGFP but are positive for PGP9.5-IR. Scale bar = 100 μm in a–b, 50 μm in c–f

Fig. 6

Representative Western blots showing increased expression of nNOS protein in HEK cells (left panel) and rat NTS (right panel) after transduction with AAVp-nNOS plasmid or AAV2nNOS, respectively. HEK cells that were not treated with AAVp-nNOS and rat NTS that were not injected with AAV2nNOS served as control. There was no change in GAPDH expression in rat NTS after transduction (not shown). All lanes were loaded with 10 μg of protein. Positions of molecular weight markers are indicated on the left side of each panel

Fig. 7

Real time RT-PCR results showing injection of AAV2nNOS significantly (left panel, **P < 0.005) increased mRNA levels of nNOS in the NTS of rats (n = 5), as compared with that of un-injected control rats (n = 4) NTS. Data were normalized by β actin expression. Analyses of the same group of rats showed that levels of eNOS mRNA were not changed (right panel). Values were the average of 2 sets of real time RT-PCR, with nearly identical results for each NTS sample

Fig. 8

Confocal images showing increased nNOS-IR in the NTS after AAV2nNOS injection into the area (panel b) as compared with that in the un-injected control rat (a). This increase was also observed in adjacent areas such as the dorsal motor nucleus of vagus (DMV) and hypoglossal nucleus (XII) in AAV2nNOS injected rats (panel d) as compared with that of the control rat (c). Scale bar = 50 μm