Cholinergic septal neurons do not undergo apoptosis after axotomy. A, Triple-immunofluorescence against Neu-N (neuronal marker), GFAP (astroglial marker) and activated Caspase-3 shows no colocalization of the apoptotic marker with neurons at different time points (1, 3, 7, 14 days) after axotomy. There is an increase in the number of cells that are immunopositive for activated caspase-3 with time. The correlation between GFAP and activated Caspase-3 suggests that astrocytes are undergoing apoptosis (scale bar: 50 μm). B, Triple-immunofluorescence against ChAT, p75 and p53, shows no colocalization of p53 (an early apoptotic marker) with p75- or ChAT-immunopositive neurons in the brains of axotomized rats 14 days after the axotomy. Confocal microscopy (scale bar: 50 μm). Triple-immunofluorescence against ChAT, p75 and activated caspase-3 shows no colocalization of activated Caspase-3 and p75 or ChAT immunopositive neurons after 14 days of axotomy. Confocal microscopy (scale bar 50 μm). C, Axotomized septal neurons are not labeled with Fluorojade C (a specific staining for degenerating neurons). Superior panel, a brain section from a rat injected in medial septum with 100 mM H2O2 was stained with Neurotrace and Fluorojade C as a positive control. The arrow indicates a degenerating neuron. Center panel, double-labeling with Neurotrace and Fluorojade in a brain section from an untreated rat. Inferior panel shows no colocalization of Neurotrace and Fluorojade C in medial septal neurons 14 days after axotomy (scale bar 50 μm).