High-resolution ex-vivo structural and functional analysis of sympathetic innervation using a novel confocal fluorescence technique

Neurotransmitter Transporter Uptake Assay (NTUA, Molecular Devices) is a fluorescent sympathomimetic that accumulates in sympathetic terminals by uptake via the noradrenaline transporter (NAT). We have utilised NTUA to visualise sympathetic innervation ex-vivo by confocal microscopy with single-terminal resolution. As proof-of-principle, we applied this technique to investigate inter-tissue differences in sympathetic innervation structure and NAT kinetics, as well as examine sympathetic remodelling in the heart
in response to chronic intermittent hypoxia (CIH). Tissues were obtained from male adult Wistar rats (200-300g) under non-recovery terminal inhalation isoflurane (3-5% in O2, 1.5L min-1). To investigate sympathetic nerve remodelling in response to CIH, left
atrial appendages (LAAs) were obtained from normoxic rats (N; n=4) and CIH (FiO2=0.06-0.21, 15 cycles hour-1, 8 hours day-1, 21-24 days; n=3). In an additional group of N rats, LAAs (n=9) and vasa deferentia (VD; n=6) were excised to investigate inter-tissue differences in sympathetic innervation. Tissues were pinned flat, transferred to a confocal microscope and superfused with NTUA. Image stacks were recorded over 15 minutes of NTUA delivery. Innervation structure and NAT kinetics were quantified using FIJI (v1.53). VD and LAA tissues showed similar sympathetic innervation structure in terms of overall terminal density,
terminals per nerve fibre length, overall nerve fibre length and nerve branch density (p>0.05 ).
In the LAA, CIH exposure was associated with a significant decrease in overall terminal density (p=0.014), as well as a modest but statistically insignificant decrease in terminals per nerve fibre length (p=0.072), overall nerve fibre length (p=0.078) and nerve branch density (p=0.10). NTUA uptake rates were not statistically different between the VD and LAA (p=0.24), nor following CIH exposure in the LAA (p=0.73). Overall, this study provides a basis for application of the NTUA assay for rapid, ex-vivo investigation of sympathetic innervation. This may represent a useful tool for studying sympathetic remodelling.