A–B , confocal immunofluorescence (IF) analysis of 7‐day cardiac sympathetic neurons (cSNs), isolated from the superior cervical and stellate ganglia of neonatal rats, cultured in the presence (+NGF) or the absence (‐NGF) of nerve growth factor (NGF). Cells were stained with an antibody against tyrosine hydroxylase (TH). Nuclei were counterstained with DAPI. C, confocal IF analysis of 7‐day (left panels) vs. 14‐day (right panels) SN/ cardiomyocyte (CM) co‐cultures, maintained in the presence or in the absence of NGF. Cells were co‐stained with antibodies against α‐actinin and TH. Nuclei were counterstained with DAPI. D–E, quantification of SN varicosity area (D) and interindividual distance between varicosities (E) in cSNs co‐cultured with CMs, in the absence (‐) vs. the presence (+) of NGF in the culture medium. Data distribution is represented by the individual values. Mean and error bars, representing 95% confidence intervals, are shown. Differences among groups were determined using the Mann–Whitney test. (**, P < 0.01; **
**, P < 0.0001; (+) 7 days n = 101 and n = 44; (+) 14 days n = 60 and n = 85; (‐) 7 days n = 67 and n = 39; (‐) 14 days n = 102 and n = 34 varicosities for each group. Three independent cell preparations were analysed). [Colour figure can be viewed at wileyonlinelibrary.com]