Loss of transmitter-associated enzyme staining following axotomy does not indicate death of brainstem cholinergic neurons.

To assess the validity of using loss of transmitter-associated enzyme staining to document the death of injured cholinergic neurons, vagal and hypoglossal cholinergic neurons were quantitatively identified at 7 and 28 days following unilateral axotomy using staining procedures for choline acetyltransferase (ChAT) or acetylcholinesterase (AChE), using Nissl stains, and using the retrogradely transported dye, True blue, as an intracellular marker. At 7 days post-axotomy, the transmitter-associated enzymes, ChAT and AChE, had disappeared from over 95% of vagal neurons (P greater than 0.001) and from over 50% of hypoglossal neurons (P greater than 0.001) as compared with the unlesioned control side. At 28 days post-axotomy, ChAT and AChE were still absent from over 70% of vagal neurons (P greater than 0.001) but only from about 15% of hypoglossal neurons as compared with unlesioned control sides. In contrast, no statistically significant losses of either vagal or hypoglossal neurons were found at 7 or 28 days post-axotomy using Nissl stains or detection of retrogradely transported True blue. These findings indicate that transmitter-associated enzyme expression can be regulated independently of neuronal survival following injury. We conclude that absence of transmitter-associated enzyme staining is not an absolute indicator of death of cholinergic neurons.