Comparison of bNOS and chat immunohistochemistry in the laterodorsal tegmentum (LDT) and the pedunculopontine tegmentum (PPT) of the mouse from brain slices prepared for electrophysiology

• bNOS and CHAT are colocalized in the LDT and PPT of the mouse.
• bNOS antibody labels more neurites and deeper somas than CHAT.
• PV is virtually absent from the LDT and PPT.
• bNOS serves as a reliable marker of cholinergic neurons of the LDT and PPT.

BackgroundIdentification of cell phenotype from brain slices upon which in vitro electrophysiological recordings have been performed often relies on conducting post hoc immunohistochemistry on tissue that necessarily has not been ideally prepared for immunohistochemical procedures. In such studies, antibody labeling against neuronal nitric oxide synthase (bNOS) has been used to identify cholinergic neurons of the laterodorsal tegmental nucleus (LDT) and the pedunculopontine tegmental nuclei (PPT), two brainstem nuclei importantly involved in arousal. However, a widespread perception maintains that antibody staining for enzymes involved in synthesis or transport, of acetylcholine would be a more definitive marker and hence, preferable.New methodColocalization of bNOS and CHAT in the LDT/PPT, and presence of parvalbumin (PV), was examined in non-ideally prepared mouse brain slices using currently available antibodies.ResultsUsing fluorescent-based immunohistochemistry in LDT/PPT slices prepared for in vitro recordings, a near 100% colocalization of bNOS and CHAT was observed.Comparison with existing methodWe confirm in the mouse, findings of near 100% colocalization of bNOS and CHAT in the LDT/PPT, and we expand upon data from rat studies using optimally prepared tissue, that for dendritic visualization, bNOS staining exceeded the quality of CHAT staining for visualization of a higher degree of detail of fine processes. PV is not highly present in the mouse LDT/PPT.ConclusionCHAT and bNOS are equally useful target proteins for immunofluorescent identification of cholinergic LDT/PPT cells in mouse brain slices prepared for in vitro recordings, however, antibody targeting of bNOS allows for a superior appreciation of structural detail.