Evidence for the cholinergic nature of C-terminals associated with subsurface cisterns in alpha-motoneurons of rat.

C-terminals can be distinguished at the ultrastructural level from other types of nerve endings on motoneurons by their prominent and regularly occurring postsynaptic specializations termed subsurface cisterns (SSC). We have previously shown (Yamamoto et al., 1991) that an antibody directed against a sequence within the gap junction protein connexin32 immunolabels these motoneuronal SSCs and can therefore serve as a immunohistochemical tool to visualize indirectly the location of C-terminals on motoneurons at the light microscope level. Here we have used this anti-SSC antibody in combination with antibodies against choline acetyltransferase (ChAT) and acetylcholinesterase (AChE) to determine whether C-terminals on motoneurons contain these cholinergic enzyme markers. In sections at all major spinal cord levels and in several cranial motor nuclei examined, motoneuronal cell bodies and their proximal dendrites were studded with large ChAT-immunoreactive (ChAT-IR) boutons. Boutons having a similar distribution and appearance on motoneurons were also immunolabeled for AChE. In addition, motoneurons were surrounded by a dense plexus of AChE-immunoreactive (AChE-IR) varicose fibers and fine preterminal axons. In double-labeled sections, AChE-IR boutons corresponded to those immunolabeled for ChAT. In sections processed for simultaneous immunofluorescence detection of ChAT and SSCs, ChAT-IR boutons were very often found in apposition to immunolabeled SSCs. In sections processed for simultaneous labeling of AChE and SSCs. AChE-IR boutons were again frequently seen abutting labeled SSCs. These results provide the first strong evidence at the LM level that a large proportion, if not the entirety, of C-terminals are cholinergic and show that these terminals consist in part of relatively large varicosities along highly varicose axons that form en passant type contacts on motoneurons. At the same time, our results substantially narrow possibilities regarding the as yet undetermined source of C-terminals, which can now be considered to originate from cholinergic neurons, such as those located in the brainstem and/or the spinal cord.