There is no difference between the number of septal neurons when comparing the ipsilateral and contralateral regions of brains 21 days after axotomy. A, Left, confocal microscopy of triple labeling for Neurotrace (red), NeuN (green) and GFAP (blue) from the septal region of a lesioned brain. In order to assure that we were counting only neurons, we used two neuronal markers and an astrocytic marker (GFAP). Right, inset magnification of a neuron (scale bar: 80 μm) showing the Neurotrace and NeuN labeling profiles. B, Diagram illustrating the area of the medial septum (MS) that was considered for the quantification of cholinergic or total number of neurons (adapted from Rat Brain Atlas ). Each side of the total area was divided to 4 fields and then photographed and manually quantified. The anatomical landmarks used to define the MS are also indicated (see Experimental Methods). cc, corpus callosum; LV, lateral ventricle; aca, anterior commissure. C, Quantification of p75-positive, ChAT-positive and total septal neurons. Comparison of the number of septal neurons on contralateral and ipsilateral sides shows differences in the numbers of p75- and ChAT-immunopositive neurons, but no significant difference in total number of neurons (n = 5; Student's t-test, p > 0.001, ± SD) 21 days after axotomy.