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Introduction: Norepinephrine transporter (NET) rate is regulated by changes in NET trafficking, activity and expression. While chronic sympathetic neuron depolarisation has been consistently shown to increase NET rate, the effect of acute changes in action potential (AP) frequency is been less clear. Here, we optically measure dynamic changes of murine cardiac single-terminal NET activity ex vivo in response to firing rates over the physiological range. Methods: Left atrial appendages (LAA) of C57BL/6 mice were isolated, loaded with fluorescent NET substrate (Neurotransmitter Transporter Uptake Assay; NTUA) and imaged with confocal microscopy, as we recently described (Cao et al., 2020). Electrical field stimulation (EFS) was delivered via two parallel electrodes (0.1-0.2 ms pulse width, 80% of cardiomyocyte contraction threshold, 0.2-10 Hz) in square wave pulses to initiate APs. Competition by NE at NET was assessed by exogenous NE application during the NTUA wash-out phase. ‘nt’: individual nerve terminals; data were compared by Kruskal-Wallis test and post-hoc analysis. Results: In the first 3 minutes of EFS, NTUA uptake was progressively suppressed increasing EFS frequency in the mouse LAA. Thus, median (IQR) NET reuptake rate in the absence of stimulation [15.8 (8.0-23.5)%.min-1 (n = 7, nt = 90)] was reduced by 61% at 2 Hz [6.2 (0.6-20.3)%.min-1], 66% at 5 Hz [5.3 (-1.6 to 15.3)%.min-1] and 72% at 10 Hz [4.4 (-4.7 to 14.0)%.min-1] (all stimulation groups: n = 4, nt = 60; p < 0.01). In the absence of EFS, exogenously applied NE (1-100 µM) during wash-out significantly accelerated NTUA displacement median (IQR) rate in a concentration-dependent manner compared to control [6.6 (6.1-16.7)%.min-1], for example, by over two-fold in the presence of 1 µM [24.9 (16.8-45.0)%.min-1] (all groups: n = 4, nt = 60; p < 0.0001). Conclusion: In the heart, acute increases in sympathetic firing rate progressively saturate intra-terminal NET reuptake rate. This implies a supralinear relationship between firing frequencies and extracellular NE concentrations, with implications for cardiac overexcitability and arrhythmogenesis. Furthermore, substrate competition of exogenous NE at NET also has implications for the clinical interpretation of 123I-MIBG cardiac scans under conditions of varying sympathetic tone.