Proceedings of The Physiological Society
University of Cambridge (2004) J Physiol 555P, C27
Cellular phenotype specificity of viral vectors delivered into cardiovascular control centres in the brainstem
T. Lonergan*, A.G. Teschemacher, J.F.R. Paton* and S. Kasparov*
* Department of Physiology, School of Medical Sciences, University of Bristol, Bristol, BS8 1TD. . Department of Pharmacology, School of Medical Sciences, University of Bristol, Bristol BS8 1TD, UK
Viral vectors are widely used as tools for exploring gene function. Despite this, very little is known about the cellular phenotype specificity of the promoters used in these vectors within the central nervous system. It is often assumed that all neuronal populations will express a transgene if it is driven by either the human cytomegalovirus (hCMV) promoter, the most commonly used, or the synapsin1 promoter that is active only in neurones. With our interest in the role of central catecholaminergic neurones in cardiovascular control, we have compared the expression of green fluorescent protein in the dorsal vagal complex when driven by three different promoters operating within an adenoviral backbone. The promoters were: hCMV, the synapsin1 promoter and PRSx8 (a synthetic promoter based on Phox2 binding motifs, which determine the catecholaminergic phenotype; see Hwang et al. 2001).
Male Wistar rats (75-150g) were anaesthetised (ketamine, 60 mg kg-1 and medetomidine, 250 µg/kg, ip) and 2 bilateral injections (1 µl each, 0.5 µl/min) of adenoviral vectors were made into the dorsomedial medulla, including the A2 region (AdhCMVeGFP: 2.7 X 1010 pfu/ml, n = 3; AdPRSx8eGFP: 2.2 X 1010 pfu/ml, n = 3; AdSYN1eGFP-WHE: 4.4 X 107 pfu/ml, n = 3), or A1 region (AdPRSx8eGFP, n = 3) or A6 region (bilateral, AdPRSx8eGFP, n = 3). The rats were allowed to recover for 5 days and were then deeply anaesthetised (pentobarbitone 100 mg kg-1, ip), perfused and brainstem sections processed for dopamine β-hydroxylase (DBH, an enzyme characteristic for catecholaminergic neurones).
Following injection of AdhCMVeGFP or AdSYN1eGFP-WHE no expression of eGFP was observed in DBH-immunoreactive neurones but adjacent dorsal vagal-and hypoglossal motoneurones did exhibit green fluorescence. When the PRSx8 promoter was used in the A2 and A1 regions 26 ± 3 % and 56 ± 11 % of DBH-immunoreactive neurones expressed eGFP, respectively (M ± S.E.M.). The morphology of the remaining eGFP expressing neurons was consistent with that of the cholinergic motoneurons of the dorsal motonucleus of the vagus and nucleus ambiguus, which are also known to express Phox2. In contrast, 96 ± 3 % GFP neurons in the A6 were DBH-immunoreactive, suggesting high selectivity in this region.
Thus, it appears that while the hCMV and synapsin promoters do express in some neurones they are not useful for targeting catecholaminergic neurones. On the other hand, using PRSx8 promoter we achieved transgene expression in all catecholaminergic groups tested and therefore believe this particular vector will be a useful tool for studying the role of these neurones in cardiovascular control. However, further refinements of this vector may be necessary to avoid expression in other phenotypes.
Financial support: WT (AL/069061), Royal Society (23697), BBSRC (7/JE616459), BHF (RG/02/011).AdhCMVeGFP or AdSYN1eGFP-WHE vectors were kindly supplied by Prof. J. Uney (UOB)
Where applicable, experiments conform with Society ethical requirements