Adenoviral vectors (AVV) are widely used as tools for exploring gene function in studies of the central autonomic control, but the cellular specificity of the promoters commonly used in these vectors has not been studied. We evaluated AVV with four “wide-spectrum” promoters, human cytomegalovirus promoter (HCMV), synapsin-1 promoter (Syn1), tubulin-α1 promoter (Tα1), and neuron-specific enolase (NSE) for their ability to express enhanced green fluorescent protein (EGFP) within the dorsal vagal complex and the adjacent brain stem. They were compared with the PRSx8 promoter, specifically designed to target catecholaminergic neurons. AdHCMVEGFP, AdSyn1EGFP-WHE (woodchuck hepatitis enhancer element), AdTα1EGFP, and AdNSEEGFP were unable to drive expression of EGFP in dopamine β-hydroxylase-immunoreactive neurons of the A2 cell group, although the adjacent dorsal vagal motonucleus and especially hypoglossal motoneurons did express high levels of EGFP. AdPRSx8EGFP efficiently drove EGFP expression in the A2 cell group but also in choline acetyltransferase-positive vagal motoneurons. However, catecholaminergic neurons could be selectively and efficiently transduced via a retrograde route by injecting the vector into their target areas. Thus AVV with “wide-spectrum” promoters have strikingly different activity in the diverse cellular populations within brain stem cardiovascular control centers. The PRSx8 promoter is a valuable tool for the study of the role of catecholaminergic neurons.
Glucoprivation or hypoglycemia induces a range of counterregulatory responses, including glucose mobilization, reduced glucose utilization, and de novo glucose synthesis. These responses are mediated in part by the sympathetic nervous system. The aim of this study was to determine the chemical codes of sympathetic preganglionic neurons (SPN) activated by glucoprivation, induced by 2-deoxy-D-glucose (2DG). SPN controlling the adrenal glands and celiac ganglia, which ultimately can innervate the liver and pancreas, were targeted together with the superior cervical ganglia (control). 23.9% ± 1.3% of SPN in the T4-T11 region contained c-Fos immunoreactivity following 2DG; 70.3% ± 1.8% of SPN innervating the adrenal glands and 37.4% ± 3% of SPN innervating celiac ganglia were activated. 14.8% ± 3.5% of SPN (C8-T3) innervating superior cervical ganglia were activated. In the C8-T3 region 55% ± 10% of SPN activated contained PPCART, with only 12% ± 3% expressing PPE mRNA, whereas, in the T4-T11 region, 78% ± 4% contained PPE, with only 6.0% ± 0.6% expressing PPCART mRNA. Thus CART is not involved in glucose mobilization. Two chemically distinct populations of SPN (PPE⁺ 57.4% ± 5%, PPE⁻ ∼40%) were identified to regulate adrenaline release in response to glucoprivation. Multiple chemically distinct SPN populations innervating a specific target could suggest their graded recruitment. The two distinct populations of SPN (PPE⁺ 67.6% ± 9%, PPE⁻ ∼30%) projecting to celiac ganglia activated by glucoprivation could direct pancreatic and hepatic or other counterregulatory responses. Nearly all SPN that expressed PPE mRNA and projected to the adrenal glands or celiac ganglia were activated, suggesting a role for the inhibitory peptide enkephalin in responses evoked by glucoprivation.
We employ viral vectors to address questions related to the function of specific types of neurones in the central control of blood pressure. Adenoviral vectors (AVVs) or lentiviral vectors (LVVs) can be used to visualize specifically living GABAergic or noradrenergic (NAergic) neurones or to interfere with intracellular signalling within these cell types. Here, we review recent in vitro, in situ and in vivo applications of these vectors in the rat brainstem as performed in our laboratories. In organotypic slice cultures prepared from defined cardiovascular brainstem areas, viral vectors were used to study the electrophysiological properties, intracellular signalling and gene expression in selected neuronal phenotypes. In vivo, vectors were microinjected into brainstem nuclei to inhibit specific aspects of cell signalling by expression of dominant negative proteins, for example. Outcomes for cardiovascular control were measured either acutely in situ or chronically in vivo with radio telemetry in freely moving rats. We showed that AVVs and LVVs have distinct properties that need to be considered prior to their application. For example, LVVs can be manufactured very quickly, have no immunogenicity and can be pseudotyped to display higher tropism for neurones than glia. However, comparatively lower production yields of LVVs may limit their use for some types of applications. In contrast, AVVs require a lengthy construction period, are easy to amplify to high yields at moderate cost but may trigger an immune response when used at high titres in vivo. These features make AVVs particularly suitable for in vitro applications. As the two vector types complement each other in several ways we generated a shuttle system that simplifies transfer of transgene cassettes between the backbones of AVVs and LVVs. Thus, AVVs and LVVs are powerful experimental tools that can be used in a variety of experimental designs in vivo, in situ and in vitro.
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