Introduction: The release of noradrenaline (NA) from the sympathetic nervous system (SNS) exerts powerful chronotropic and inotropic effects (Zaika et al., 2011) and can be pro-arrhythmogenic. Many cardiac arrhythmias have been attributed to formation of macroscopic excitation patterns, but the influence of the SNS on wave dynamics is not well understood. In the current work, we describe new methodologies for dye free mapping of waves in myocyte monocultures and neuron/myocte co-cultures and report the effect of nicotine and nerve growth factor (NGF) on macroscopic wave dynamics. Methods: Hearts were isolated from neonatal SD rat pups (P1-P3) and killed by Schedule 1 in accordance to UK Home Office regulations. Briefly, ventricles were isolated, enzymatically digested, and enriched for myocyte content before plating on poly-L-lysine coated petri-dishes. After 24 hours, stellate neurons were isolated from litter mates and plated on top of the myocytes in 1:20 ratio. We used dye-free imaging techniques to record macroscopic wave patterns and beat rate in myocyte-only and co-culture preparations. Experiments were carried out in an Okolab stage incubator controlled for heat (37°), CO2 (5%) and humidity. Results: (i) We find that co-culture wave speed is significantly faster than myocyte-only cultures (52%±14%, p<0.001, n=7); (ii) Addition of nicotine to co-cultures systems causes an altered behaviour by change in initiation sites of wave patterns, but no significant changes in conduction velocity (n=7); (iii) Application of 10 µM nicotine to co-cultures produced a transient increase in contraction rate of up to 37%, and this was significantly sustained in 3 of 5 experiments; (iv) DAPI-stained images of both myocyte and co-culture plates grown either in the presence or absence of NGF were analysed in terms of a clumping factor (CF), a newly developed statistical measure of cell-cell spacing. CF were significantly higher (indicative of more clumped cells) for cultures grown in the absence of NGF compared to those grown in the presence of NGF (50ng/ml), for both myocyte-only (8.02±0.99, n=6 vs. 5.48±0.51, n=5) and co-culture (9.33±1.17, n=5 vs. 5.99±0.74, n=4) plates (all mean ± S.E.M, comparisons were performed with a t-test, p<0.05). Discussion: Our observations may be relevant to understanding the onset and evolution of complex waves during life threatening cardiac arrhythmias, which can be attributed to large scale spiral waves (Bub et al., 2002; Bub et al., 2005). The finding that NGF modulates the patterning of myocytes raises the question of how NGF and its receptors interact to generate changes in cardiac and synaptic function.